Update on fertility preservation from the Barcelona International Society for Fertility PreservationESHREASRM 2015 expert meeting: indications, results and future perspectives



# Introduction
Reproductive capacity may be seriously affected by age, different conditions, including genetic syndromes, and also by treatments, especially those with gonadal toxicity. Fertility preservation (FP) is a fundamental issue for individuals of reproductive age, both male and female, or prepubescent boys and girls whose future fertility may be compromised. There is general consensus on the need to provide counselling about currently available FP options to all individuals wishing to preserve their fertility. Timely referral to a fertility specialist for informed FP decisions becomes essential.
Several techniques for FP are nowadays well established while others are still considered experimental. These techniques have been the subject of continuous review by experts with the aim of providing physicians involved in FP with up-to-date knowledge and counselling. Given the particular nature of FP, recommendations are largely based on cohort studies, case series, small non-randomized clinical trials, or case reports, which further makes FP a challenging but rather controversial field.
Reviews have been mostly focused on cancer, which is probably the main indication for FP given its high incidence and impact on reproductive health [bib_ref] Recommendations for fertility preservation in patients with lymphoma, leukemia, and breast cancer, Isfp Practice Committee [/bib_ref] [bib_ref] of Clinical Oncology clinical practice guideline update, Loren [/bib_ref] [bib_ref] Fertility preservation during cancer treatment: clinical guidelines, Oktay [/bib_ref] [bib_ref] Fertility preservation in female cancer patients: an overview, Mahajan [/bib_ref] [bib_ref] Cancer and fertility preservation: international recommendations from an expert meeting, Lambertini [/bib_ref]. However, the need for FP in other pathologic situations, either due to the disease itself or to gonadotoxic treatment, and even in non-medical indications, is on the rise. Moreover, new perspectives to tackle FP are being developed, and evidence about the results of spontaneous pregnancy and ART after current FP procedures is growing, which may further help clinicians provide adequate counselling. With the aim of reviewing all these aspects and drawing recommendations, an international meeting with representatives from expert scientific societies involved in FP was held in in June 2015. This paper summarizes the topics debated, with a special focus on indications for FP, current outcomes and future perspectives. A condensed version of this summary has been included in the print issue of Human Reproduction.

# Materials and methods
Twenty international FP experts belonging to the American Society of Reproductive Medicine (ASRM), ESHRE and the International Society of Fertility Preservation (ISFP) attended the meeting. Experts conducted a review of the literature and evidence presented in scientific meetings up to June 2015 to be discussed at the meeting. At the time this manuscript was being written, new evidence considered relevant for the debated topics was published, and has consequently been included. Given the lack of studies in large cohorts or with a randomized design, the quality of evidence according to the European Society for Medical Oncology Clinical Practice Guidelines for fertility preservation in cancer patients [bib_ref] Cancer and fertility preservation: international recommendations from an expert meeting, Lambertini [/bib_ref] for most of the evidence reviewed was three or below.

## Indications for fp

## Cancer
Many forms of cancer are associated with impaired semen quality or ovarian function at the time of cancer diagnosis. However, the main effect on fertility arises from commonly used treatments such as chemotherapy with alkylating agents and pelvic radiation that present well-known gonadotoxic side effects. Gonadal failure resulting from these treatments may affect different aspects of reproductive health, including pubertal development, hormone production, and sexual function in adults [bib_ref] Fertility preservation during cancer treatment: clinical guidelines, Oktay [/bib_ref] [bib_ref] A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal..., Picton [/bib_ref]. The fact that more than 80% of children and adolescents with cancer become long-term survivors has raised an increased interest in the long-term effects of cancer treatment on fertility.

## Male
Spermatogonia are especially sensitive to chemotherapy and radiotherapy. The effect, which is dose-dependent, may not be permanent if the spermatogonial stem cell (SSC) population is not fully depleted [bib_ref] Fertility preservation during cancer treatment: clinical guidelines, Oktay [/bib_ref]. Data about the impact of recent biological or targeted cancer therapies on male fertility are limited [bib_ref] of Clinical Oncology clinical practice guideline update, Loren [/bib_ref]. For most of these therapies, the effects seem to be mild, mostly involving reproductive endocrinology [bib_ref] Effects of chemotherapy and radiotherapy on spermatogenesis in humans, Meistrich [/bib_ref]. Finally, surgical pelvic interventions for malignant or benign disease may affect the anatomy or normal functioning of reproductive organs [bib_ref] Preserving fertility in children and adolescents with cancer, Levine [/bib_ref].

## Female
Chemotherapy and radiotherapy may induce premature ovarian insufficiency (POI) in women [bib_ref] Fertility preservation in women, Donnez [/bib_ref]. Ovarian damage is drug-and dose-dependent and increases as the patient ages (Ethics Committee of ASRM, 2013). Radiotherapy may also affect the uterus, leading to reduced vascularity, myometrium damage (fibrosis) and hormone-dependent insufficiency [bib_ref] Fertility preservation in female cancer patients: an overview, Mahajan [/bib_ref]. Recent evidence in female survivors of childhood cancer shows that chemotherapy without radiotherapy to the brain or the pelvis has few effects on future pregnancy or live births [bib_ref] Pregnancy after chemotherapy in male and female survivors of childhood cancer treated..., Chow [/bib_ref]. In any case, FP should be considered prior to chemotherapy to maximize future reproductive potential. Data about the impact of recent biological or targeted cancer female fertility therapies is also limited except for bevacizumab, with a 34% rate of POI reported [bib_ref] of Clinical Oncology clinical practice guideline update, Loren [/bib_ref]. Fertility may also be impaired by surgical removal or damage to reproductive organs.

## Non-oncological medical indications
FP options should also be discussed with adult and younger women and men affected by several non-oncological medical conditions. Nononcological systemic diseases, such as haematological and autoimmune conditions, usually require chemotherapy or radiotherapy, especially for those in need of a bone marrow or haematopoietic stem cell transplantation (HSCT) [bib_ref] Fertility preservation in women, Donnez [/bib_ref]. In other conditions reproductive function may be compromised by genetic causes or by surgical interventions. Finally, FP may be offered to patients at high risk of fertility loss as a result of severe body trauma requiring surgical intervention. [fig_ref] Table I: Non-oncological conditions requiring fertility preservation [/fig_ref] summarizes the most common non-oncological conditions requiring FP. [fig_ref] Table I: Non-oncological conditions requiring fertility preservation [/fig_ref] summarizes autoimmune diseases reported to benefit from immunosuppressive therapy with alkylating agents (cyclophosphamide) [bib_ref] Fertility preservation in women, Donnez [/bib_ref]. POI in these women is also affected by disease duration and presence of anti-Ro and anti-U1RNP (ribonucleoprotein) antibodies [bib_ref] The impact of cyclophosphamide on menstruation and pregnancy in women with rheumatologic..., Harward [/bib_ref]. Continuous POI in women with chronic autoimmune diseases also increases the risk of hypoestrogenism-related comorbidities (including cardiovascular disease and osteoporosis) [bib_ref] Ovarian damage during chemotherapy in autoimmune diseases: broad health implications beyond fertility, Marder [/bib_ref]. Males with systemic lupus erythematosus show a high frequency of testicular Sertoli cell dysfunction associated with semen abnormalities [bib_ref] Testicular Sertoli cell function in male systemic lupus erythematosus, Suehiro [/bib_ref]. New treatment approaches are changing the prognosis of patients with autoimmune diseases, although information about toxicity for reproduction is still limited.

## Autoimmune diseases

## Hematopoietic stem cell transplantation
HSCT (autologous or allogeneic) has been an important therapeutic tool for some oncological and non-oncological systemic diseases. Patients undergoing HSCT are at particularly high risk of developing ovarian (64-85%) or testicular (50-90%) failure since aggressive chemotherapy and radiotherapy is needed to destroy pre-existing bone marrow [bib_ref] Clinical guide to fertility preservation in hematopoietic cell transplant recipients, Joshi [/bib_ref].The most common non-oncological diseases benefiting from HSTC are autoimmune diseases that are unresponsive to immunosuppressive therapy or benign haematological diseases [fig_ref] Table I: Non-oncological conditions requiring fertility preservation [/fig_ref] [bib_ref] Fertility preservation in women, Donnez [/bib_ref].

## Medical conditions causing poi
POI may also result from several other causes, including an altered hypothalamic-pituitary-gonadal axis, ovarian oophoritis, benign ovarian tumours, either due to their extensive or progressive nature, or bilateral adnexectomy [bib_ref] The impact of cyclophosphamide on menstruation and pregnancy in women with rheumatologic..., Harward [/bib_ref]. POI is also common in Turner's syndrome. However, FP may not be feasible for most patients with this disease since by the time they reach puberty their primordial follicle reserve may already be depleted. Hematopoietic stem cell transplantation Autoimmune diseases unresponsive to immunosuppressive therapy Haematological diseases (sickle cell anaemia, thalassaemia major, plastic anaemia)

## Medical conditions causing poi (eshre poi guideline development group, 2015)
Altered hypothalamic-pituitary-gonadal axis Ovarian oophoritis Benign ovarian tumours Mosaic Turner's syndrome Fragile X Mental Retardation 1 [bib_ref] Early decline in functional ovarian reserve in young women with low (CGGn..., Gleicher [/bib_ref] Galactosemia [bib_ref] Ovarian function in girls and women with GALT-deficiency galactosemia, Fridovich-Keil [/bib_ref] Beta-thalassaemia [bib_ref] Beta-thalassemia major and female fertility: the role of iron and ironinduced oxidative..., Roussou [/bib_ref] Endometriosis [bib_ref] Fertility preservation in women with endometriosis: for all, for some, for none?, Somigliana [/bib_ref] Male genetic disorders Klinefelter's syndromeTesticular damage [bib_ref] Indications and strategies for fertility preservation in men, Stahl [/bib_ref] Gender reassignment proceduresSevere body trauma requiring surgical intervention POI, premature ovarian insufficiency.
after careful consideration of increased pregnancy-associated risks [bib_ref] Feasibility of fertility preservation in young females with Turner syndrome, Lau [/bib_ref] [bib_ref] Guideline on the management of premature ovarian insufficiency, Eshre Poi Guideline Development [/bib_ref]. Other conditions associated with POI include FMR1 gene mutations (Fragile X Mental Retardation 1) as a result of premature ovarian aging [bib_ref] Early decline in functional ovarian reserve in young women with low (CGGn..., Gleicher [/bib_ref] , classic galactosemia [bib_ref] Ovarian function in girls and women with GALT-deficiency galactosemia, Fridovich-Keil [/bib_ref] or beta-thalassaemia in female patients who suffer from hypogonadotrophic hypogonadism associated with amenorrhoea, anovulation and infertility [bib_ref] Beta-thalassemia major and female fertility: the role of iron and ironinduced oxidative..., Roussou [/bib_ref].
It is well documented that women with endometriosis are at increased risk of POI and that about half of them will experience infertility [bib_ref] Fertility preservation in women with endometriosis: for all, for some, for none?, Somigliana [/bib_ref]. The causal relationship between endometriosis and infertility is unclear (Practice Committee of the ASRM, 2012). FP may be of interest for reproductive-age women at risk of impaired fertility due to progression or surgical treatment of this condition . Nevertheless, some authors restrict FP to women with bilateral unoperated endometriomas and those with previous unilateral endometriomaremoval requiring surgery for a contralateral recurrence [bib_ref] Fertility preservation in women with endometriosis: for all, for some, for none?, Somigliana [/bib_ref].

## Male genetic disorders and testicular tissue damage
Klinefelter's syndrome is the most common sex chromosomal disorder in humans. This syndrome causes hypogonadism and azoospermia in >90% cases . Testicular injury may also result in irreparable damage to the testicular tissue leading to infertility [bib_ref] Indications and strategies for fertility preservation in men, Stahl [/bib_ref]. A recent study has highlighted the relevance of attempting salvage, even in cases of subjectively dead testicle, and to offer the patient FP options .

## Gender reassignment procedures
Removal of testicles or ovaries destroys the ability to have geneticallyrelated children, while feminizing/masculinizing medications used in gender reassignment procedures may lead to diminished fertility. The World Professional Association for Transgender Health has emphasized the need to discuss and provide counselling about FP and fertility treatment 'before starting hormone therapy or undergoing surgery to remove/alter their reproductive organs' even at a younger age [bib_ref] Standards of care for the health of transsexual, transgender, and gender-nonconforming people,..., Coleman [/bib_ref]. Further, the Endocrine Society recommends providing counsel about FP 'prior to initiation of puberty suppression in adolescents and before treatment with sex hormones of the desired sex in both adolescents and adults' [bib_ref] Endocrine treatment of transsexual persons: an Endocrine Society clinical practice guideline, Hembree [/bib_ref]. Evidence regarding reproductive health issues in individuals receiving treatments for gender dysphoria is scarce. Currently, there are no established techniques for preserving gonadal function in prepubertal or pubertal adolescents, who will never develop reproductive function in their natal sex owing to blockers or cross-gender hormones [bib_ref] Standards of care for the health of transsexual, transgender, and gender-nonconforming people,..., Coleman [/bib_ref]. There is an ongoing ethical debate on whether FP should or should not be offered to transgender individuals [bib_ref] ESHRE Task Force on Ethics and Law 23: medically assisted reproduction in..., De Wert [/bib_ref].

## Delayed childbearing
Female fertility decreases gradually but significantly after age 32 years, and faster after 37 years, which compromises fertility when delaying childbearing (The ACOG Committee on Gynecologic Practice, 2014). This is important, since an increasing proportion of couples in developed countries choose to have children later in life (35 years). Given that delaying childbearing is considered a non-medical indication for FP, the term 'AGE banking' (oocyte banking for anticipated gamete exhaustion) has been proposed for oocyte cryopreservation in these cases [bib_ref] Oocyte banking for anticipated gamete exhaustion (AGE) is a preventive intervention, neither..., Stoop [/bib_ref].

## Available procedures for fp
The most recent practice guidelines issued for cancer patients by the ASRM (Ethics Committee of ASRM, 2013) and the American Society of Clinical Oncology [bib_ref] of Clinical Oncology clinical practice guideline update, Loren [/bib_ref] provide an exhaustive review of all evidence supporting currently available FP procedures, which may be adaptable to all scenarios where fertility may be compromised.

## Women
Both, embryo and oocyte cryopreservation (slow freezing or vitrification) are first-line FP methods . However, oocyte cryopreservation is increasingly preferred in adolescent girls or young women without a life partner given that it overcomes religious, ethical or practical issues related to embryo storage [bib_ref] of Clinical Oncology clinical practice guideline update, Loren [/bib_ref]. Mature oocyte vitrification is preferred in post-pubertal women when gonadotoxic treatment can be delayed to allow time for controlled ovarian stimulation (COS) [bib_ref] Is vitrification of oocytes useful for fertility preservation for age-related fertility decline..., Cobo [/bib_ref]. Harvesting of immature oocytes by aspiration would be an option for patients unable to undergo COS such as pre-pubertal girls, women with aggressive or hormonesensitive cancers, or those with polycystic ovary syndrome [bib_ref] Toward precision medicine for preserving fertility in cancer patients: existing and emerging..., Kim [/bib_ref]. The benefits of adding tamoxifen or letrolozole to COS regimens administered to women with breast cancer are still unclear [bib_ref] Reproductive choices and outcomes after freezing oocytes for medical reasons: a follow-up..., Dahhan [/bib_ref]. IVM has been shown to improve outcomes in breast cancer patients undergoing COS for FP [bib_ref] In vitro maturation improves oocyte or embryo cryopreservation outcome in breast cancer..., Oktay [/bib_ref].
Ovarian tissue cryopreservation (OTC) is a COS-independent experimental technique which also allows immediate cancer treatment, and iscurrently the only FP option in paediatric patients [bib_ref] Fertility preservation in women, Donnez [/bib_ref] and in hormone-dependent diseases [bib_ref] Toward precision medicine for preserving fertility in cancer patients: existing and emerging..., Kim [/bib_ref]. Reimplantation of this tissue either in the pelvic cavity (orthotopic) or elsewhere (heterotopic) has the potential of restoring fertility and ovarian hormone secretion. Reimplantation of frozen-thawed ovarian tissue in the pelvic cavity is usually carried out by laparoscopy, but the surgical technique is contingent on the presence (or not) of at least one ovary. If an ovary is present, the remaining atrophic cortex is removed using scissors, thereby creating a grafting bed onto which the thawed ovarian fragments are placed. If no ovaries remain, the ovarian pieces are placed in a peritoneal window created in the peritoneum of the broad ligament, in an area where retroperitoneal capillaries are visible .
Ovarian tissue could also be preserved as an entire ovary with its vascular pedicle, preventing ischaemic damage occurring between transplantation and revascularization [bib_ref] The role of cryopreservation for women prior to treatment of malignancy, Donnez [/bib_ref]. Fertility restoration after whole ovary preservation requires retransplantation of the whole organ accompanied by vascular anastomoses of the blood vessels. However, although recent evidence from large animals, such as sheep, suggests that natural fertility can be fully restored following autotransplantation of whole ovaries and their supporting vascular pedicle after slow freezing and thawing [bib_ref] The effects of whole ovarian perfusion and cryopreservation on endothelial cellrelated gene..., Onions [/bib_ref] [bib_ref] Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation..., Campbell [/bib_ref] , cryopreservation of the whole ovary is likely to be more problematic in adult women owing to the increased size of their ovaries, the difficulty of achieving adequate perfusion and penetration of the cryoprotectants agents through the whole organ, and the inherently different freezing and thawing optima for the different cell types in both the ovary and blood vessels. Although several methods have been developed to help overcome these barriers [bib_ref] Cryopreservation of intact human ovary with its vascular pedicle, Bedaiwy [/bib_ref] [bib_ref] The effects of whole ovarian perfusion and cryopreservation on endothelial cellrelated gene..., Onions [/bib_ref] [bib_ref] Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation..., Campbell [/bib_ref] [bib_ref] Obstetric outcome after oocyte vitrification and warming for fertility preservation in women..., Martinez [/bib_ref] , more research is needed before this technique can be translated into clinical practice.

## Fertoprotective agents
GnRH analogues/agonists (GnRHa) may protect follicles from destruction during chemotherapy, probably by suppression of gonadotrophin levels and reduction of utero-ovarian perfusion [bib_ref] Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential..., Meirow [/bib_ref]. These agents have long been used for the prevention of ovarian damage, despite their efficacy being a subject of debate owing to inconsistent results from randomized trials using GnRHa [bib_ref] Gonadotropin-releasing hormone agonists for prevention of chemotherapy-induced ovarian damage: prospective randomized study, Badawy [/bib_ref] [bib_ref] Adjuvant goserelin and ovarian preservation in chemotherapy treated patients with early breast..., Sverrisdottir [/bib_ref] [bib_ref] Effect of the gonadotropin-releasing hormone analogue triptorelin on the occurrence of chemotherapy-induced..., Mastro [/bib_ref] [bib_ref] Effect of luteinizing hormone-releasing hormone agonist on ovarian function after modern adjuvant..., Gerber [/bib_ref] [bib_ref] Randomized trial using gonadotropinreleasing hormone agonist triptorelin for the preservation of ovarian..., Munster [/bib_ref]. Two meta-analyses of randomized trials have found an overall significant reduced risk of POI in young breast cancer patients [bib_ref] Gonadotropinreleasing hormone analogues for the prevention of chemotherapy-induced premature ovarian failure in..., Mastro [/bib_ref] [bib_ref] Ovarian suppression using luteinizing hormone-releasing hormone agonists during chemotherapy to preserve ovarian..., Lambertini [/bib_ref]. GnRHa increased the pregnancy rate and had no negative impact on prognosis [bib_ref] Ovarian suppression using luteinizing hormone-releasing hormone agonists during chemotherapy to preserve ovarian..., Lambertini [/bib_ref]. This protective effect was not as clear in other cancer patients (ovarian and lymphoma) (Del [bib_ref] Gonadotropinreleasing hormone analogues for the prevention of chemotherapy-induced premature ovarian failure in..., Mastro [/bib_ref]. Recently, no protective effect at all was found in young patients with lymphoma [bib_ref] No evidence for the benefit of gonadotropin-releasing hormone agonist in preserving ovarian..., Demeestere [/bib_ref]. Still, the quality of evidence is relatively low given the number of women included, relatively short-term follow-up hitherto and significant heterogeneity. Further high quality studies are needed to study the (long-term) effects of GnRHa use on POI.

## Men
Sperm cryopreservation is the only established FP method in adult and adolescent males. Alternatives to the procurement of semen samples by masturbation include assisted ejaculation methods such as penile vibratory stimulation or electroejaculation. A recent paper on the European Algorithm for the cryopreservation of testicular tissue/sperm in pre-pubertal and adolescent patients at high risk of infertility. Clinical assessment for puberty should be carried out by a clinician with experience in pubertal assessment. It must be stressed that no clinical parameter can accurately predict the presence of sperm. The proven treatment option for pubertal and adolescent boys who are considered capable of producing a semen sample is semen collection and cryopreservation. If sufficient sperm are recovered the gametes can be banked using commercial glycerol-based sperm cryomedia. For those young patients who are clinically pre-pubertal and for whom semen cryopreservation is not possible the fertility preservation strategy should include collection of a testicular biopsy by an experienced surgeon. The tissue should be cryopreserved with a protocol optimized for preserving immature germ cells, (immature testis protocol). Patients who are pubertal but are unable to produce a suitable semen sample may proceed to testicular biopsy, with intra-operative analysis. Techniques for intra-operative analysis may vary between institutions but should be aimed at identifying tissue containing (or likely to contain) sperm. This should be carried out by an individual with experience in analysis of testicular tissue (e.g. surgeon, embryologist or andrologist). When sperm are not identified or deemed unlikely the tissue should be frozen with the immature testis protocol used for pre-pubertal patients. For patients in whom sperm are identified or considered likely to be present, tissue should be split into two portions for storage. One portion should be cryopreserved using the immature testis preservation protocol, whilst the second portion should be stored using a protocol aimed at preserving mature sperm cells with glycerol as the main cryoprotectant. As stated in the text at present there are several protocols for cryopreservation of immature testicular tissue and there is no clear evidence at the time of writing to demonstrate which is optimal. Reprinted with permission from [bib_ref] A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal..., Picton [/bib_ref].

## Patient semen analysis testicular biopsy

# Intra-operative analysis

# Method of cryopreservation
perspective on testicular tissue cryopreservation for FP in pre-pubertal and adolescent boys by the ESHRE Task Force on Fertility Preservation for severe diseases recommends an algorithm for sperm and testicular tissue cryopreservation in pre-pubertal boys and adolescent males at high risk of fertility loss [bib_ref] A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal..., Picton [/bib_ref]. This algorithm includes alternative experimental techniques, such as testicular sperm extraction (TESE), for patients presenting oligo-or azoospermia at the time of cryopreservation or those with necrozoospermia or ejaculation disorders, or immature testicular tissue cryopreservation or SSC cryopreservation when no sperm can be collected .

## Results of art after fp

## Women
As a well-established technology, embryo cryopreservation has high pregnancy success rates . However, outcomes in cancer patients are scarce. Recently, [bib_ref] Utilization rates and results of long-term embryo cryopreservation before gonadotoxic treatment, Dolmans [/bib_ref] reported a 44% live birth rate (LBR) per patient among 54 women with cancer undergoing IVF and embryo cryopreservation, with a cumulative live birth rate (CLBR) similar to that achieved with fresh embryos in non-cancer patients [fig_ref] Table I: Non-oncological conditions requiring fertility preservation [/fig_ref]. Similarly, Oktay et al. reported 18 pregnancies and 25 live births among 33 women with breast cancer, with a LBR of 45% per embryo transfer [bib_ref] Fertility preservation success subsequent to concurrent aromatase inhibitor treatment and ovarian stimulation..., Oktay [/bib_ref]. Success rates associated with oocyte cryopreservation have significantly improved in recent years, with vitrification success rates being superior to slow freezing [bib_ref] Age-specific probability of live birth with oocyte cryopreservation: an individual patient data..., Cil [/bib_ref]. A recent report of the outcomes achieved in oocyte donation showed a 6.5% oocyte-to-baby rate, with CLBR increasing with the number of oocytes used [bib_ref] Six years' experience in ovum donation using vitrified oocytes: report of cumulative..., Cobo [/bib_ref]. A similar report among women undergoing oocyte vitrification because of age or because of non-oncological medical conditions revealed a LBR per patient of 50% among women aged 35 years, and of 22.9% among those aged >36 years after the transfer of embryos obtained from vitrified oocytes. The CLBR was higher and increased faster among younger women [bib_ref] Oocytes vitrification as an efficient option for elective fertility preservation, Cobo [/bib_ref] [fig_ref] Table I: Non-oncological conditions requiring fertility preservation [/fig_ref]. It should be noted that these figures arise from patients with a good prognosis who are managed by a highly experienced team, and therefore may not be representative of other FP programmes [bib_ref] Oocyte vitrification for elective fertility preservation: lessons for patient counseling, Stoop [/bib_ref]. These success rates are comparable to those achieved with fresh oocytes [bib_ref] Embryo development of fresh 'versus' vitrified metaphase II oocytes after ICSI: a..., Rienzi [/bib_ref] [bib_ref] How does vitrification affect oocyte viability in oocyte donation cycles? A prospective..., Sole [/bib_ref]. Outcomes after oocyte vitrification among female cancer patients are scarce. reported fertilization rates up to 76.6% and a mean number of embryos transferred of 1.8  0.7 SD among 11 women with cancer, four of whom gave birth at term without negative perinatal outcomes. [bib_ref] Live birth using vitrified-warmed oocytes in invasive ovarian cancer: case report and..., Alvarez [/bib_ref] first reported a successful birth in a woman with invasive ovarian cancer. Despite being considered an experimental technique both restoration of ovarian function and spontaneous pregnancies after ART have been reported after orthotopic transplantation of cryopreserved ovarian tissue [bib_ref] Live birth after autograft of ovarian tissue cryopreserved during childhood, Demeestere [/bib_ref] [bib_ref] Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark, Jensen [/bib_ref] [bib_ref] First pregnancies, live birth, and in vitro fertilization outcomes after transplantation of..., Oktay [/bib_ref]. Only one case of a live birth after heterotopic transplantation has been reported up to 2013 [bib_ref] First reported clinical pregnancy following heterotopic grafting of cryopreserved ovarian tissue in..., Stern [/bib_ref]. Recently, Demestree et al. have reported the first live birth following re-grafting of ovarian tissue that had been cryopreserved during childhood in a 13-year old girl undergoing HSCT [bib_ref] Live birth after autograft of ovarian tissue cryopreserved during childhood, Demeestere [/bib_ref]. To date, a large series of 60 live births after transplantation of cryopreserved ovarian tissue has been reported, also showing that by repeating the procedure ovarian activity can be restored for more than 11 years . In a series of 111 cases, the conception rate was 29%. Two women delivered three babies each, proving the efficacy of the technique, as well as the possibility of conceiving naturally several times after only one transplantation procedure [bib_ref] Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark, Jensen [/bib_ref]. Although it is impossible to provide a fixed success rate for transplantation of ovarian tissue as long as it remains active [bib_ref] Success and challenges in fertility preservation after ovarian tissue grafting, Andersen [/bib_ref] , given these encouraging results, ovarian cortex transplantation is proposed as an open clinical application .

## Men
Success rates of semen cryopreservation have greatly increased with advances in ART, especially in ICSI, with pregnancy rates up to 57% [bib_ref] A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal..., Picton [/bib_ref]. With this technique, [bib_ref] Assisted reproductive outcomes of male cancer survivors, Garcia [/bib_ref] reported a LBR of 62.1% in a cohort of 272 men with cancer, which was significantly higher than that of the comparative normospermic non-cancer

## Women

## Activation of ovarian follicles
Cryopreserved ovarian tissue from prepuberal patients and patients with primary POI contains immature primordial follicles that must be activated in order to start developing. It has been recently described that activation of primordial follicles can be induced in vivo by mechanically interrupting the Hippo signalling pathway (by ovarian fragmentation, drilling, laser) [bib_ref] Intraovarian control of early folliculogenesis, Hsueh [/bib_ref]. Follicle activation may also be achieved in vitro before autotransplantation by acting on the PI3K-PTEN-AKT-FOXO3 pathway [phosphatidylinositol 3-kinase(PI3K) activators and phosphatase and tensin homologue enzyme (PTEN) inhibitors, Protein Kinase B (AKT) stimulators, transcriptional factor forkhead box O3(FOXO3)] which has been shown to regulate primordial follicle dormancy at oocyte level [bib_ref] Intraovarian control of early folliculogenesis, Hsueh [/bib_ref]. This pathway has also a fundamental role in FSH stimulation of granulosa cell differentiation of antral follicles and in oocyte maturation of preovulatory follicles [bib_ref] Intraovarian control of early folliculogenesis, Hsueh [/bib_ref]. Using this double approach in women with POI, [bib_ref] Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment, Kawamura [/bib_ref] found rapid follicle growth after grafting ovarian tissue back to patients, obtaining mature eggs. A live birth was achieved after IVF and embryo transfer. In vitro activation protocols are under development with the aim of increasing the pool of viable activated follicles available for in vitro growth (IVG) procedures [bib_ref] Short-term PTEN inhibition improves in vitro activation of primordial follicles, preserves follicular..., Novella-Maestre [/bib_ref].

## In vitro follicle culture
Transplantation of cryopreserved tissue carries the risk of re-seeding original cancer cells into the patient, as recently highlighted in a series of recent reviews and reports [bib_ref] Legal termination of a pregnancy resulting from transplanted cryopreserved ovarian tissue due..., Ernst [/bib_ref] [bib_ref] The safety of transplanting cryopreserved ovarian tissue in cancer patients: a review..., Rosendahl [/bib_ref] [bib_ref] First reported clinical pregnancy following heterotopic grafting of cryopreserved ovarian tissue in..., Stern [/bib_ref] [bib_ref] A review of 15 years of ovarian tissue bank activities, Dolmans [/bib_ref] [bib_ref] Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue, Dolmans [/bib_ref] [bib_ref] Safety considerations for transplanting cryopreserved ovarian tissue to restore fertility in female..., Sorensen [/bib_ref] [bib_ref] No malignancy detected in surplus ovarian tissue from a former Ewing sarcoma..., Andersen [/bib_ref]. This risk can be minimized by using complete IVG and maturation of oocytes as the means of fertility restoration [bib_ref] The in vitro growth and maturation of follicles, Picton [/bib_ref] [bib_ref] Current achievements and future research directions in ovarian tissue culture, in vitro..., Smitz [/bib_ref]. The goal of complete IVG and maturation of oocytes is particularly challenging in human follicles because of the greatly extended developmental time-frame for follicles and oocytes, and increased size of ovulatory follicles and mature gametes [bib_ref] Ovarian follicle culture: advances and challenges for human and nonhuman primates, Telfer [/bib_ref]. Furthermore, recent evidence suggests that the dynamics of the in vivo and hence in vitro growth environments may differ between the pre-and post-pubertal human ovary [bib_ref] The immature human ovary shows loss of abnormal follicles and increasing follicle..., Anderson [/bib_ref]. To date, three-dimensional (3D) culture methods have proved most successful in supporting the demands of human follicle activation and IVG, as these approaches are best able to maintain the morphology of the follicles and preserve critical cell-cell interactions both between the different cellular compartments in the follicle and between the follicle and its surrounding stromal tissue thus better mimicking the in vivo ovarian growth environment. Several multi-step culture systems have succeeded in culturing human follicles [bib_ref] In vitro growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue, Newton [/bib_ref] [bib_ref] The in vitro growth and maturation of follicles, Picton [/bib_ref] [bib_ref] Oocyte development in bovine primordial follicles is promoted by activin and FSH..., Mclaughlin [/bib_ref] [bib_ref] Noninvasive index of cryorecovery and growth potential for human follicles in vitro, Barrett [/bib_ref] [bib_ref] Engineering the ovarian cycle using in vitro follicle culture, Skory [/bib_ref]. Critically, all IVG systems used for fertility restoration for FP patients must start with the in situ culture of primordial follicles from cryopreserved tissue. The recent production of meiotically competent metaphase II non-human primate and human oocytes following IVG of freshly isolated secondary follicles [bib_ref] In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon..., Xu [/bib_ref] [bib_ref] In vitro follicle growth supports human oocyte meiotic maturation, Xiao [/bib_ref] is encouraging. However, whether mature human oocytes can be obtained from primordial follicles grown from cryopreserved ovarian tissue using these culture strategies or whether the oocytes so derived are competent to complete cytoplasmic and nuclear maturation in a timely manner is yet to be confirmed. It also remains to be confirmed whether their genomic imprint establishment and maintenance is normal [bib_ref] Culture of oocytes and risk of imprinting defects, Anckaert [/bib_ref] and whether metaphase II gametes so produced are healthy and able to undergo fertilization and support normal early embryonic development until the embryonic genome is activated. Considerable further research effort is therefore needed to confirm the safety and efficacy of oocytes derived following extended IVG and the maturation of human oocytes from cryopreserved tissue before this technology can be used to restore fertility in FP patients.

## Artificial ovaries
An alternative to the in vitro culture of primordial follicles is their development into an engineered 'artificial ovary', consisting of isolated preantral follicles along with other ovarian cells assembled in a structure-3D matrix, or scaffold, which allow follicles to grow and develop in an ovarian-like environment [bib_ref] First transplantation of isolated murine follicles in alginate, Vanacker [/bib_ref]. Ovarian cells from fresh medullary tissue have been suggested as the best source of isolated stromal cells for the artificial ovary [bib_ref] The best source of isolated stromal cells for the artificial ovary: medulla..., Soares [/bib_ref]. Once transplanted to the patient, this artificial ovary would potentially restore fertility and endocrine function [bib_ref] Toward precision medicine for preserving fertility in cancer patients: existing and emerging..., Kim [/bib_ref]. Following this procedure, [bib_ref] Initiation of puberty in mice following decellularized ovary transplant, Laronda [/bib_ref] reported the production of estradiol in vitro by primary ovarian cells of mice seeds into a previously decellularized ovary. Moreover, when transplanted into ovariectomized mice, grafts from these cells were able to initiate puberty.

## New fertoprotective agents
The most recent theory of chemotherapy-induced follicle loss suggests that, simultaneously with large follicle apoptosis, chemotherapy also triggers activation of dormant follicle growth. Current research focuses thus on both agents with anti-apoptotic properties (imatinib, sphingosine-1-phosphate, thyroid hormone T3, granulocyte colonystimulating factor and tamoxifen) that have been shown to reduce follicle loss in animal models [bib_ref] Toward precision medicine for preserving fertility in cancer patients: existing and emerging..., Kim [/bib_ref] ,and on agents that also prevent follicle activation such as AS101, an immune modulator that acts on the PI3K/PTEN/AKT follicle activation pathway [bib_ref] Cyclophosphamide triggers follicle activation and 'burnout'; AS101 prevents follicle loss and preserves..., Kalich-Philosoph [/bib_ref] , and anti-Mullerian hormone [bib_ref] Prevention of chemotherapy-induced ovarian damage, Roness [/bib_ref]. Clinical applicability of these agents depends not only on their fertoprotective capacity, but also on their potential interaction with cancer treatments.

## Men
In a similar way, the risk of reintroducing malignant cells via the graft might be overcome by in vitro spermatogenesis. As per follicle culture, SSCs (whole testicular biopsy or isolated SSCs) are cultured in 3D systems that resemble the in vivo situation [bib_ref] A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal..., Picton [/bib_ref]. Recently, Nickkholgh et al. have reported the genetic stability of a long-term culture of human SSCs from two prostate cancer patients and although changes in the methylation status were observed, the consequences of these epigenetic changes on the functionality of the sperm of the health of the offspring are unknown [bib_ref] Genetic and epigenetic stability of human spermatogonial stem cells during long-term culture, Nickkholgh [/bib_ref]. The fertilizing ability of in vitro-cultured sperm is to be established before assessing the clinical value of this technique. Moreover, fertility restoration strategies by autotransplantation of cryopreserved testicular tissue have not yet been tested for safe clinical use in humans [bib_ref] A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal..., Picton [/bib_ref].

## Both sexes

## Artificial gametes
To date, infertile patients lacking functional oocytes or sperm cannot benefit from currently available ART unless donor gametes are used. The use of primordial germ cells (PGC) present in the gonads, such as SSC, is a promising approach for treating infertility. However, the population of these cells is scarce and decreases with age. Pluripotent stem cells (PSC), such as embryonic stem cells or induced PSC, constitute other potential sources of gametes [bib_ref] Cells Esig S. Stem cells in reproductive medicine: ready for the patient?, Vassena [/bib_ref]. [bib_ref] Stepwise differentiation from naive state pluripotent stem cells to functional primordial germ..., Hayashi [/bib_ref] derived functional PGCs from PSC potentially able to generate functional oocytes and sperm [bib_ref] Cells Esig S. Stem cells in reproductive medicine: ready for the patient?, Vassena [/bib_ref]. Recently, [bib_ref] Complete meiosis from embryonic stem cell-derived germ cells in vitro, Zhou [/bib_ref] have reported the generation of haploid mouse spermatid-like cells able to produce viable and fertile offspring. Notwithstanding these advances, a recent critical review of available evidence in humans and animal models carried out by the ESHRE Special Interest Group in Stem Cells concluded that 'to date there are no proven stem cell-based means to improve reproductive function, either by producing functional gametes in vitro, or stimulating the resident stem cell population in the ovary to elicit de novo oocyte production' [bib_ref] Cells Esig S. Stem cells in reproductive medicine: ready for the patient?, Vassena [/bib_ref].

## Summary
The Expert Working Group made the following recommendations:
- Several oncological and non-oncological diseases may affect current or future fertility, either due to the disease itself or to gonadotoxic treatment, and need an adequate FP approach. These patients should be counselled regarding potential fertility loss and should be referred to fertility specialists to discuss options for FP and current results. - Women wishing to postpone maternity and transgender individuals before starting hormone therapy or undergoing surgery to remove/ alter their reproductive organs, should also be counselled accordingly.
- Embryo and oocyte cryopreservation are first-line FP methods in post-pubertal women. Metaphase II oocyte cryopreservation (vitrification) is the preferred option. - Cumulative evidence of restoration of ovarian function and spontaneous pregnancies after ART following orthotopic transplantation of cryopreserved ovarian tissue supports its future consideration as an open clinical application. - Semen cryopreservation is the only established FP technique in men. - Testicular tissue cryopreservation should be recommended in prepubertal boys even though fertility restoration strategies by autotransplantation of cryopreserved testicular tissue have not yet been tested for safe clinical use in humans. - The establishment of international registries on the short-and longterm outcomes of FP techniques is strongly recommended.
[fig] Figure 2: Fertility preservation techniques in women. Experimental procedures are indicated in discontinuous boxes, while established ones (i.e. those proven to restore fertility, with live births reported) are indicated in shaded boxes. Vitrified-thawed oocytes can be fertilized by IVF/ICSI for embryo transfer. Immature oocytes can be matured in vitro (IVM) for IVF/ICSI. Research is undergoing on the potential use of oogonial stem cells to repopulate follicle-depleted ovaries or differentiating follicle somatic cells and oocytes from embryonic stem cells or induced pluripotent stem cells to assemble de novo follicles for transplantation or IVM and IVF/ICSI. *Cryopreserved. OT, ovarian tissue. [/fig]
[table] Table I: Non-oncological conditions requiring fertility preservation. [/table]