Ovarian Tissue Cryopreservation Guidelines

Abstract

With the increase in the survival rate of cancer patients, there has been a growing interest in the field of fertility preservation. One of the main methods in this aim is ovarian tissue cryopreservation, especially for prepubertal girls. From the early time of introducing this opportunity as a chance to preserve future fertility in cancer patients, following gonadotoxic treatments, many guidelines have been published, to introduce the real indications. The need for these guidelines seemed very urgent, and attracted great interest, because this method was performed as an experimental and no standard clinical option, for many years. So patient selection should have been done with the most standard and highly accurate criteria, which could analyze the cost/benefit of this technique after multidisciplinary evaluation, for each patient, individually. For many years the specialist believed that all caution must be taken in referring patients for this technology. To ensure that cancer patients receive high-quality uniform treatment, evidence-based clinical practice guidelines (CPGs) are needed. CPGs are essential to enhance care quality and decrease heterogeneity in practice and costs. The guidelines can provide clear advice on the best practice in the field of female FP, based on the best available evidence.

1. Introduction

cancer survivors have increased dramatically with the improvements in cancer treatment [1]. However, cancer incidence, in the young age group, has increased, during the past 30 years, the mortality rates have declined and the 5-year survival rate has increased from less than 50% in the 1970s to 80% these days, and the 10-year survival rate is estimated 75% [2].

Cancer in adolescents and young adults (AYAs) occurs between 15 and 39 years old. Cancer-diagnosed AYAs are different from other age groups in many components, such as tumor etiology, biology, prognosis, intrinsic and extrinsic risk factors, cancer types and survivorship as well as the effectiveness of treatment. Besides, AYAs suffer more from long-term effects of treatment compared with those older ages, including sexual dysfunction, infertility, and future cancers [3].

Chemotherapy and radiotherapy may have gonadotoxic effects, and compromise ovarian function [4]. A positive correlation has been found between the risk of gonadal harmful effects and the patient’s age, which may be due to the previous reduction of the follicle numbers in older females. Additionally, the type of chemotherapy regimens and the dose are the other determinants of the degree of cytotoxicity [5, 6].

With the increase in the long-term survival rates of cancer patients who have experienced chemo/radiotherapy, there’s been greater attention to the long-term effects of treatments, such as premature ovarian failure (POF) [6]. A mixture of health and quality-of-life problems have been created for the young survivors, many of which were not predicted at the time of cancer diagnosis [4].

It needed to be discussed with the patients the risks of cancer treatments on reproductive health, and the options for fertility preservation, before treatment. Consultation with a reproductive endocrinologist is of great value in providing adequate information regarding the side effect of cancer therapies on reproductive consequences and the chance of success rates for the various fertility preservation strategies. It was shown that almost 30% of cancer patients under 50 years old request more information about premature ovarian insufficiency or the probable risks to their children’s health, and a third of them would have tended to have a fertility consultation before treatment started [7].

Gametes, embryo, and gonadal tissue cryopreserving may help to preserve fertility, and avoid damage to reproductive organs. Sperm and embryo freezing are well established (clinical stage), but oocytes and ovarian tissue freezing are still experimental [1].

However, in some parts, we referred to the other ways of fertility preservation (FP) options, but the main aim of this chapter is a review of the existing guidelines regarding ovarian cryopreservation, as a fertility preservation option. A comprehensive study of the available literature was conducted, to find updated and evidence-based guidelines and recommendations.

2. The emergence of oncofertility

Recently, with the increased survival rate of cancer patients, there have been much attention and interest in the long-term effects of treatment on future fertility [2].

Oncofertility is a domain that connects oncology with fertility and having a comprehensive view of these fields got it capable to introduce a standard of care in many institutions. This field has been developed simultaneously with the other life-preserving advancements in the oncology care unit, such as earlier diagnostics, targeted cancer therapies, new methods with less radiation dose, and local surgical procedures [8].

It passed more than half a century since the first time the concept of “ovarian tissue cryopreservation” was proposed, but it took 12 years, until achieving (reaching) the first successful human ovarian tissue cryopreservation, and the first live birth, following this technique has been reported in 2004. For many years, ovarian tissue cryopreservation and transplantation were done as experimental, in many fertility centers, until 2019, when this approach was accepted as a clinical technique, for fertility preservation [5].

This success has been achieved with the improvement in the technique, protocols, cryoprotectants agents, devices, as well the exact timing for equilibration, throughout many scientific studies, as it got qualified enough, to be considered as “the clinical approach” [9].

3. Female fertility preservation strategies

increasing the knowledge of the possibility of fertility loss following cancer treatment has led to huge growth in the fertility preservation field, throughout the last two decades. ESHRE 2020, described fully these options, based on the individual case [10].

There are some options for female fertility preservation before cancer treatment. For female fertility preservation, the most successful standard method is emergency IVF and embryo cryopreservation, as an established part of assisted reproduction, before cancer therapy [7, 10].

However, this method is not suitable for pre-pubertal girls, and young patients when there is no partner or when there is not enough time to delay cancer treatment. Oocyte and ovarian tissue cryopreservation are the other less effective options that are still experimental [10, 11].

Ovarian transposition or fertility-sparing cancer surgery is the other option that helps minimize the destructive effects of cancer treatments [10, 11]. Besides restoration of fertility, ovarian tissue transplantation in the POI can restore endocrine and hormonal function [12], also it can be done at any time during the menstrual cycle [13].

In addition, GnRH agonist co-administration also may provide some fertility protection against gonadotoxicity of the chemotherapy, but still prospective controlled trials are needed to approve this method as an established clinical method [7, 10]. In vitro oocyte maturation (IVM), and oocyte cryopreservation are the other choice methods, especially for women with age-related fertility loss, besides women who seek FP for medical indications [10].

Despite the introduction of these strategies, FP stays a particular challenge in the most common candidates such as hematological cancers (leukemia, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma) and breast cancer [14]. The most challenge is the patient’s selection criteria, under 35 years old, when the ovarian reserve is still high, a realistic chance of 5 years survival rate, and when there the risk of premature ovarian insufficiency is at least 50% [15].

4. Ovarian tissue cryopreservation as an experimental method for FP

Nearly 20 years passed since the first human pregnancy report following ovarian tissue cryopreservation (OTC) [16], but until recently this method was being considered an experimental method. Embryo and gametes (sperm, and oocyte) cryopreservation are done as the standard practice worldwide, but OTC is a newly established method, which harbors many challenges. One of the reasons is that OT is a complex of various cell types with different physical structures and water permeability, that need a different process to survive during freezing [17].

The other challenge is the methods of cryopreservation, vitrification vs. slow freezing. There are two main methods for OTC; slow freezing (SF) and vitrification (VF). The conventional method is slow freezing, which is the base protocol for OTC worldwide, and nearly all live births have been reported to follow this method. The other is vitrification which compared to slow freezing is a new approach, with small numbers of live births. Some advantages and disadvantages have been reported regarding these methods [18].

The base for slow freezing is tissue exposure to the cryoprotectant agents (CPA), cooling of the tissue to a special temperature using programmable freezers, and final liquid nitrogen (LN₂) immersing (−196°C). DMSO (1.5 M) has been used as CPA in this method [19].

Vitrification protocol is done commonly using equilibration and vitrification solution exposure (increasing concentration of CPAs), and final LN₂ immersion. Ethylene glycol (EG) and dimethyl sulphoxide (DMSO) can be used as the CPAs in this method (7.5% for ES, and 20% for VS) [20].

However, SF has been used as the standard method, in many centers, but some properties of this method created an interest in vitrification. Besides this method is time-consuming, because of the need for a controlled-rate cooling device which is expensive. Also, the probability of cryoinjury is high in the formation of the intracellular ice crystal. Then vitrification was introduced for this purpose, instead of slow-freezing, because of time-saving, and rapid cooling with no need for expensive equipment [17]. Much research has been done to introduce the best method, but the results are controversial [21], but among numerous studies, rigorous documents have been earned that support VF as an alternative way to the SF method [22, 23, 24, 25, 26, 27, 28].

The other issue is the techniques of ovarian cryopreservation; ovarian cortical fragments or whole ovary.

Since most primordial follicles are located within the ovarian cortex, so cryopreservation of the small part of cortical tissue enables the storage of large numbers of oocytes. It mainly is recommended to obtain ovarian tissue before cancer treatment. For this purpose, ovarian tissue is commonly obtained by the laparoscopic approach, also it can be done by mini-laparotomy or during ovarian transposition [29].

For the fragment cryopreservation, the cortical tissue will be transferred to the lab, then thin cortical strips will be prepared, using a scalpel, the optimal size will be ∼5 × 10 × 1 mm³ thick, before freezing [18, 20, 30].

The whole ovarian cryopreservation consisted of ovarian removal with the vascular pedicle, through laparotomy or a laparoscopic approach. One of the big advantages is vascular anastomosis of the thawed ovary, which probably provides a larger follicular reserve and also a longer ovarian life span. But there are no currently rigorous documents to support this hypothesis, mainly because of the created damages by applying freezing procedures for a big sample as a human ovary [31].

In one study on sheep ovary, it was shown that the directional freezing method enhances the ovarian tissue cryopreserved viability, for both whole ovary, and ovarian fragments [31]. The physical concept for directional freezing is a sequence of four heat-conductive units positioned in a line. The blocks are set at different temperatures, which creates a temperature gradient. Freezing tubes will be passed, at 0.01 mm/s speed, along the thermal gradient, starting at +4°, and decreasing to −70°C. This results in a cooling rate of 0.3°C/min, and at the end, samples will be plunged into liquid nitrogen [32, 33].

The other problem is how to use cryopreserved ovarian tissue. However the OTC is now a relatively well-established procedure, but the restoration of fertility using the cryopreserved tissue remains a challenge. The only approach for the restoration of fertility is the re-implantation of ovarian tissue [34]. But the re-implantation process is accompanied by attrition of the non-growing follicles population, something about three-quarters [35]. Now the thawed ovarian tissue transplantation can be done in two ways; orthotopic or heterotopic. The other alternative approaches, such as in vitro follicular culture need additional research before accepting as an established practice for humans [34].

until 2017, worldwide over 130 babies have been born after ovarian tissue cryopreservation (OTC), and ovarian tissue transplantation (OTT). It was estimated that OTC resulted in 93% restoration of endocrine function [36], 29% successful pregnancy rates and 23% live birth rates [37].

One another important issue regarding OTC is how long the transplanted tissue will remain functional. However, there are some variations in the ovarian grafts’ lifespan after ovarian transplantation, it seems that the endocrine function duration is longer than what is expected. On average, the follicular density of nearly 4–5 years will be preserved well, after transplantation, but up to 7–10 years of ovarian function duration also has been reported [36, 38].

For the OTS two surgical processes are needed; one for the extraction and the other for re-implantation [39]. To date, no standard operative technique has been established for obtaining ovarian cortical tissue. Few publications have dealt with the operative technique and outcomes, and the mentioned methods were as follows; ovarian cortical biopsy, unilateral hemi-oophorectomy, as well bilateral hemi-oophorectomy, and unilateral salpingo-oophorectomy [40].

All of the mentioned challenges, together, cause us to consider the OTC as an experimental method for female fertility preservation. Besides, there are some information gaps, which need to be addressed.

5. Whom, what, when, which, who information gaps

After the creation of the new approach (fertility preservation), some important questions arose, but there were no unique answers for them. One of them was; for Whom must it be proposed? What were the indications? When must it be done? Which strategy is useful, for each case? Who must provide fertility preservation consultation?

There are some studies, which tried to answer these questions, and some indications have been introduced, a range from very commonly known cases such as cancer patients [41], the latter as the social reason for pregnancy delay [42, 43], and age-related fertility loss [10], to the new indications such as transgender (fertility preservation for trans men) [43, 44], or Endometriosis [45]. However many guidelines suggested that fertility preservation consultation should be done before treatment is a great information lack in this area [46].

5.1 The challenge of “for whom?”

Ovaries or testes will not be affected in many cases, by the cancer treatment and physicians can reassure the patients. But there are also other cases, in which, the impact of treatment on fertility is unknown due to a lack of valid studies. Also sometimes the problem is the medical oncologists when there is a belief that there is no good choice/or chance, so discussing it will not help.

The related risk for premature ovarian insufficiency (POI), after chemotherapy, is influenced by some factors such as age, body mass index (BMI), type of treatment, and duration. For example, in breast cancer, the risk of amenorrhoea, after six cycles of CMF (cyclophosphamide, methotrexate, fluorouracil) was estimated at 33% for patients below 40 years and, 81% for patients ≥40 years old, also the risk for amenorrhoea was estimated 51.4% in women under 30 years old and 95.0% for the women above 30 years, after 8 sessions for dose-escalated of BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone) [47].

5.2 The challenge of “which strategy?”

Which option is appropriate for each patient is the other gap, which must be exactly described in the guidelines. Some important criteria must be fulfilled in the aim to classify a new technology and treatment as an established technique. They are efficacy, safety, procedure, and effectiveness to assess [48]. The ovarian tissue cryopreservation, as a new field of oncofertility, at first must be set up on nonhuman primate models and then would become appropriate for humans, the necessary step to move from the bench to the bedside [4].

Ovarian tissue cryopreservation is done by obtaining ovarian cortical tissue, the part that is rich in primordial follicles, before gonadotoxic treatment, by laparoscopy or laparotomy. Small fragments are prepared and then cryopreserved by the slow freezing or vitrification technique. After cancer treatment, the tissue will be transplanted in two ways of orthoptic (the pelvis) or heterotropic (outside the pelvis-abdominal wall, and forearm) transplantation. Orthotopic transplantation the most successful method has been accompanied by spontaneous pregnancies, but in heterotopic transplantation, IVF is necessary [46].

Because of the nature of being experimental, it is needed to identify the accurate indication for this technique [15].

In this regard, there are some guidelines by the American Society for Reproductive Medicine (ASRM) and American Society of Clinical Oncologists (ASCO) which recommend cancer patients must be referred for fertility preservation counseling, related to the risk of infertility after cancer treatment and must be aware of the appropriate plans [49, 50, 51]. But, there is not, always, appropriate consultation regarding the potential side effects of cancer treatment on future fertility, and also fewer patients are aware of the fertility preservation program [52].

The other big gap is the information delivery, while the medical oncologists are not exactly aware of the effect of their treatments on reproductive health and outcomes, clinical cancer patients are not routinely treated by reproductive endocrinologists. Some physicians also believe that it involves ethical issues to enable a patient to bear a child while the parent has a lowered life span or cannot take care of a child [1].

5.3 The challenge of “when”?

when must ovarian tissue cryopreservation be done? It was mentioned that over 90% of patients undergoing high-dose chemotherapy may suffer from ovarian failure [53] and cryopreservation should always be offered when the risk of POF is high (>30–50%) and the risk of ovarian metastasis is low [54]. It suggested that the best time is before the patient receives any chemotherapy [2, 15, 53].

Among many of the guidelines, ASCO mentioned individual cytotoxic treatment regimens and their effects on fertility. The guidelines mentioned that counsel the patient that this method is experimental [50, 55].

The patients’ age was one of the most challenges which have been discussed in the various version. Some believed to refer all the patients of reproductive age [50], but some others considered age limitation, and recommended this to special ages, E.g. below 35, 37, or < 42 [51, 56, 57]. The reason for the age limitation is confirmation of the appropriate ovarian reserve [57].

A framework of guidance is needed for healthcare professionals to provide evidence-based care to the women and girls who are a candidate for fertility preservation [43].

5.4 The challenge of “who”?

Who must provide the fertility preservation consultation? The oncologists and hematologists may be the first ones, in the health care provider team, who find the need for fertility preservation. After referral, the other healthcare professionals which have an important role as the oncologists, in the management of women with cancer, are obstetrics-gynecologists, endocrinologists, andrologists, nurses, embryologists, reproductive biologists, reproductive medicine specialists, psychologists, counselors, and general practitioners [8, 43, 49].

6. Fertility preservation guidelines

Fertility preservation, nowadays, is a necessary professional domain. It needs close coordination between teamwork which includes oncologists, reproductive biologists, and reproductive medicine specialists in various fields [8]. Many guidelines had been provided to describe the situations, in which fertility preservation is needed to be considered with the patients. However, there is still no general agreement between different guidelines in introducing criteria for ovarian tissue cryopreservation, such as patients’ age limits or having a history of chemotherapy.

since the first time scientists succeeded in mammalian ovarian tissue cryopreservation, in 1994 [58], new indications have been introduced and the circumstances have been determined. It had been tried to introduce true indication, in each version. There are many heterogeneities in the criteria for introducing real candidates, such as patients’ age limits, excluding patients with a history of chemotherapy, or specific diagnoses which preclude re-transplantation. Tissue preparation and freezing/thawed procedure are the other diverse worldwide [59].

To ensure that cancer patients receive high-quality uniform treatment, evidence-based clinical practice guidelines (CPGs) are needed. CPGs are essential to enhance care quality and decrease heterogeneity in practice and costs [52]. The guidelines can provide clear advice on the best practice in the field of female FP, based on the best available evidence [60]. Several guidelines have been published worldwide. Some of the guidelines focused on efficacy, and safety, while others have paid attention to the aspects of feasibility, and acceptability, and few had ethical considerations. A comprehensive guideline that encloses all aspects of FP, from patients’ consultation to the outcome of techniques, would additionally help clinicians in this field.

These guidelines could introduce the potential candidates for OTC, when a woman is at risk for iatrogenic infertility, due to medical or surgical cancer treatment, for a benign or malignant condition [51].

Guidelines are set to address oncologists, gynecologists hematologists, endocrinologists, as well other healthcare providers, such as nurses, counselors, psychologists, and general practitioners who have a role in giving fertility preservation to cancer patients.

6.1 Edinburgh guideline (2005)

Edinburgh Criteria is one of them that was firstly published in 1996, and then slightly revised in 2000 [15]. Edinburg criteria once again and after multidisciplinary discussion and the report from the Royal College of Obstetricians and Gynecologists working group, described some criteria especially age limit) for patient selection, this guidance is applicable in a patient-specific manner and must be updated by the emerging new evidence and experience [61]. Besides introducing the patient selection criteria, two issues have been addressed; the probability of the congenitofabnormalities in children following chemotherapy, and the legal and ethical issues. Regarding the concern for child abnormalities, the guideline reassures that a large study did not show any link between these. The guideline mentioned that this technique (FP) raised some critical ethical and legal issues, which must be considered before use. The costs/benefits of any intervention or decision must be evaluated, and all the advantages and disadvantages, in the short, and long term, must be considered. These proposed opportunities should not create unrealistic expectations, and should not bring adverse effects for the subsequent offspring.

Valid informed consent from the patients must be earned voluntarily, and from a competent person. Legal competence was discussed in this guideline, and also described that obtaining valid consent becomes more complicated according to the patient’s age and their level of understanding of the discussed issues [61].

Edinburgh criteria validation for ovarian tissue cryopreservation had been done, in 2014, for young women and girls (younger than 18 years). The results validate the guidelines criteria for patient selection for ovarian tissue cryopreservation and show it can identify accurately the girls and young women at risk for premature ovarian failure [15].

Summarized recommendations of the Edinburgh Criteria for Ovarian Tissue Cryopreservation (2005, 2014):

1. Age < 30 (in 2005), Age <35 (in 2014)

2. Having no history of previous chemotherapy or radiotherapy (if the patients bellow15 years old, previous mild and low-risk chemotherapy can be considered)

3. A chance of long-term survival for 5 year

4. High-risk estimation for premature ovarian failure (>50%)

5. Informed consent from the patients or the parents (in children)

6. Negative syphilis, HIV, and hepatitis serology

7. No pregnant or existing children

But, the later study, again challenged one of Edinburgh’s criteria, having no history of previous chemotherapy or radiotherapy, while Vatanparast and her colleagues 2021, presented a young girl with acute lymphocytic leukemia, which had a history of chemotherapy (15 sessions, with 30 mg vincristine and 975 mg Adriamycin) before referring. An anti-Mullerian hormone (AMH) had been requested, to survey the patient’s fertility situation. It showed a premenopausal situation when reported within the normal range (3.66 ng/mL). after ovarian biopsy, the histology survey also showed a normal follicular density [62]. In the later guidelines, in 2022, it also was described that the OTC can be done also when the gonadotoxic risk is very high, but before the patients undergo chemotherapy is the ideal situation [39].

However, many studies have addressed fertility preservation before starting chemotherapy, fewer paid attention to the survey of pre-menopausal ovarian function [1, 50, 56, 57]. This gap in the available published guidelines is fully answered in the ESHRE guideline, 2020 [10]. There are some hormonal markers as well as ultrasound parameters that can estimate the ovarian reserve [63], behind the age or a history of chemotherapy! Unfortunately, ovarian biopsy only can be done through surgery or laparoscopy, to obtain ovarian reserve from the histological survey. But, one of the most common indirect signs of ovarian reserve is hormone assay; Anti-Mullerian hormone (AMH). In the studies, AMH is accepted as a marker of ovarian reserve [64]. As it has been mentioned that a history of cancer alone does not provide enough documents to decide on patient sterility [7], and does not exclude the patient from the fertility preservation program. It seems that a big gap in the guidelines is the confirmation of pre-menopausal ovarian function, both before or after starting treatment. Also for the aim of ovarian reserve prediction, the other well-validated biomarkers, such as; FSH, LH, estradiol, Inhibin-B levels, ovarian volume, and the antral follicle count (AFC, by transvaginal ultrasound) also may be helpful. Serum AMH is reliable for the assessment after the age of 5, in addition, in older children, ovarian volume and antral follicle assessment can be recommended [65].

It’s believed that small ovarian volumes, maybe a more acute marker in the aim of ovarian reserve estimation compared to abnormal hormonal concentrations,18 of which are indirect. Besides AFC, which shows the size of the existing primordial follicle pool may be a more sensitive marker of the ovarian reserve in comparison to ovarian volume. AMH which is produced by the growing follicles may be the most valuable representation of ovarian reserve [6].

6.2 American Society of Clinical Oncology (ASCO) guideline

The other guidelines also were prepared by the American Society of Clinical Oncology (ASCO) [49] and the American Society of Reproductive Medicine (ASRM) [1].

The ASCO guideline which was published in 2006, has been updated periodically. It was recommended that oncologists should consult with the patients regarding the possibility of infertility through their cancer treatment, before cancer therapy, and be aware of possible fertility preservation options to refer the patients to reproductive specialists.

Sperm and embryo cryopreservation which were introduced as the standard practices are widely available and the other options which were considered investigational should be performed in specialist centers [49]. This version was updated in 2013 [50]. In the new version, it was stressed that health care providers advise the patients regarding the potential effects of treatment on their fertility and inform them about a wide range of fertility preservation options, as soon as possible, during their treatment, although the patients initially focus on their cancer diagnosis. In this version, oocyte cryopreservation is mentioned as a standard practice. In this version, ovarian tissue cryopreservation is still considered experimental and not an established technique [50].

Summarized recommendations of the ASCO (2013):

1. Inform the patients of the individual risk of infertility and discuss the fertility preservation options with all patients of the reproductive age.

2. Refer both patients who intend to fertility preservation and are ambivalent to reproductive specialists.

3. Refer them for fertility preservation before treatment starts, as early as possible.

4. Fertility preservation consulting must be documented in the medical record.

5. Discuss common concerns

   • available fertility preservation (standard and experimental methods)

   • refer to consultation with an appropriate reproductive specialist, if the patients would like to learn more

   • discuss the possible impact of fertility preservation on the cancer treatment

6. Time:

   • before cancer treatment

   • sperm cryobanking is available for male cancer patients and can be repeated every 24 hours until collecting the necessary number of samples

   • for female patients, a period between 2 and 4 weeks may be needed for clinical techniques, or another experimental approach, so time is important for referring to a reproductive specialist.

7. Costs:

   • insurance coverage and list the benefits for the patients

   • some organizations may provide cost-saving programs

8. Risk estimated for future pregnancy and children after cancer treatment

9. Consultation with appropriate specialists:

   • reproductive specialists

   • mental health professionals

   • advocacy organizations.

6.3 American Society for Reproductive Medicine Clinical Practice Guideline

In ASRM (ethic committee) recommendations (2005), also as the other guidelines, consulting the patients regarding fertility preservation has been considered as the patient’s right. This guideline, in a new approach, pertained to ethical and legal issues, in detail, for physicians and fertility specialists, as well cancer patients and offspring welfare, and the other issues are experimental vs. established options, the minor children’s ability to give consent, the welfare of probably resulting children, and posthumous issues. This guideline described that concerns about the welfare of future offspring should not impede cancer patients from fertility preservation programs, also this guideline emphasizes the need for precise instructions in the case of a patient’s death, unavailability, or other contingency after doing fertility preservation (disposition of cryopreserved gametes, embryos, or tissues). The necessity for Preimplantation genetic diagnosis to prevent the birth of children with a high risk of hereditary cancer is ethically accepted [1].

The Practice Committee of ASRM, in 2014, after searching the existing literature, to evaluate the efficacy and safety of ovarian tissue cryopreservation, published new recommendations [29]. In this version, the indication for OTC has been described in detail, and the technique (cortical strips and whole ovary), methods of cryopreservation (slow freezing and vitrification), and transplantation have been discussed. In this guideline slow freezing has been reported as standard protocol. One of the concepts which were discussed in this version is safety concerns regarding the risk of re-implantation of cancer after transplantation. They suggest an alternative way for transplantation when there is a risk of reintroducing; if it is possible to isolate and recover immature oocytes from the ovarian cortex and use them for subsequent IVF, after doing maturation in vitro (either mature oocytes or embryos cryopreservation).

The guideline described the circumstances which must be realized until an experimental procedure is considered an established medical practice. The evidence published in the medical regarding their risks, overall safety, benefits, and efficacy must be documented from the only valid studies which were appropriately designed and performed by several independent researchers [29].

Summarized recommendations of ASRM (2005)

1. Cancer patients must be informed by their physicians about fertility preservation options before treatment.

2. Sperm cryopreservation and embryo cryopreservation are established methods.

3. Oocyte or ovarian tissue cryopreservation are experimental methods and should be offered only in a research setting.

4. The issue of the welfare of the offspring should not deny the patients receiving fertility preservation.

5. Parents may decide the preserve fertility for their minors.

6. Need for precise instructions about the disposition of the cryopreserved gametes, tissue, embryo, or oocytes of the posthumous.

7. The necessity of preimplantation genetic diagnosis to avoid the risk of inherited cancer.

Summarized recommendations of ASRM (2014)

1. Ovarian tissue cryopreservation and transplantation must be considered as an experimental option.

2. OTC is offered when immediate gonadotoxic treatment is required and for prepubertal girls.

3. OTC should not be done in benign conditions or for delaying childbearing.

4. The options for humans’ OTC are cortical strips or a whole ovary.

5. Ovarian tissue transplantation sites may be done in both orthotopic and heterotopic.

6. Pregnancies and live births have been reported only after orthotopic.

7. No pregnancies have been reported from the heterotopic site or whole ovary transplantation.

8. One of the concerns of ovarian tissue transplantation is the potential risk of reintroducing malignancy.

6.4 Backhus et al. guideline (2007)

A guideline has been written by Backhus et al. [51] that was retrieved from the ASCO and ASRM’s criteria. This version is wider than Edinburg’s.

These guidelines help to identify correct candidates for OTC, when a woman with a benign or malignant condition is at risk for fertility loss, following medical or surgical treatment. In this guideline, also three methods of ovarian cryopreservation and transplantation, and follicles in vitro maturation were considered experimental and should not be recommended currently for patients who are candidates for fertility preservation, whenever there is no immediate or iatrogenic threat [51].

Summarized recommendations of Backhus et al. criteria [51].

1. Age < 42 years

2. IVF cycle cannot be done, regardless of the presence of the partner

3. The pre-menopausal status is demonstrated or assumed

4. There is a significant risk for the acceleration of ovarian function loss

5. Informed consent must be earned from the adult patient

6. Informed assent is needed when the patient is below 18 years, besides informed consent from parental/guardian

7. patient can undergo an elective surgical operation

8. The patient plans to have a child in the future

9. when ovarian stimulation for oocyte retrieval is contraindicated, such as hormone-sensitive tumors.

6.5 Ferti-PROTEKT network¹ (2011–2019)

FertiPROTEKT network which first was established in 2006, encompasses the university-based, hospital-based, and private infertility and oncology centers. The main aim was to introduce local fertility preservation programs in Germany, Switzerland, and Austria, the techniques which enhanced the chance of achieving a pregnancy [57, 66].

The main theory of the formation of this network was that “a close coordination is needed between oncologists and reproductive medicine specialists, and reproductive biologists”, so fertility-preservation activities should be organized, in a network structure, both as the medical-logistic network and as a professional medical society [8]. The three major networks introduced were as follows: (1) the Danish Network,² (2) The German-Austrian-Swiss network FertiPROTEKT,³ and (3) The Oncofertility Consortium.⁴ As these networks’ structures are different in goals and logistics, so all the aspects of possible network structures are covered [8]. For example, the Danish network, gives practical implementation in the field of fertility-preserving techniques such as ovarian tissue cryopreservation and transplantation, in a small country. The second, the German-Austrian-Swiss network (FertiPROTEKT) organizes these (fertility-protective techniques) in a large country, and the Oncofertility Consortium facilities knowledge transfer among its members.

In an attempt to introduce indications, in 2011, the Ferti-PROTEKT network prepared a practically oriented recommendation [57], and it was updated in 2018 [47]. This new version has a process-oriented approach, which stressed three main topics; disease prognosis, disease-specific treatment and risks of infertility, and disease-specific cryopreservation measures. Besides the risk for ovarian metastasis is also assessed which with the other mentioned topics are critical in deciding if fertility preservation is needed, or not. It could provide a disease-specific recommendation for fertility preservation measures [47].

Summarized some of the recommendations of the Ferti-PROTEKT network (2011):

1. In the case of breast cancers, fertility preservation must be considered especially in low-stage of cancer, in women below 35 years old, and in Hodgkin’s lymphoma when the women’s age is <40 years and the risk for POI is high.

2. In the case of Hodgkin’s lymphoma, the chemotherapy regimens have a big role in decision-making as they may have very low to very high gonadotoxicity.

3. In the case of borderline ovarian tumors, fertility preservation usually is done through fertility-sparing surgery. In some cases, ovarian stimulation may be useful.

4. In some other cancer patients, a set of several factors must be considered, since there is a need to make an individual, stage-dependent decision. Some clinical and key recommendations have been provided by the guideline in the case of rheumatic and autoimmune diseases, cervical cancer, colorectal carcinoma, and other malignancies such as Ewing sarcoma, endometrial cancer, Borderline ovarian tumors (BOT), non-Hodgkin lymphoma, leukemia, etc.

5. In acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) ovarian tissue cryopreservation was considered experimental because of the high-risk estimation for ovarian contamination with the malignant cells.

At last, they concluded that answering the challenge of fertility preservation necessity depends on several factors such as prognosis, the risks for fertility loss, and some individual factors such as future family planning.

6.6 Cancer Council Australia (2014)

This guidance which pertained to many aspects of FP in adolescents and young adults (AYA), in detail, has been published by the Clinical Oncology Society of Australia (COSA), assists health professionals by introducing evidence-based recommendations and ‘good practice points’, to provide an effective consultation for AYA patients and their families.

Three main objects are as follows:

• the potential risk of infertility following cancer treatments

• fertility preservation strategies for patients, diagnosed with cancer

• list of potential late effects and the need for subsequent reproductive, sexual and endocrine health follow-up after cancer treatment.

Summarized recommendations of the Cancer Council Australia (2014):

1. Discussing fertility with the cancer patients

2. Supervising the fertility preservation strategy

3. informed the patients regard to the potential effect of cancer treatment on their fertility and reproductive health

4. Discussing the fertility preservation options

5. The need for long-term follow-up of their reproductive health, endocrine, and sexual function.

Regarding OTC, the guidance considered this technique as an investigational technique, which is appropriate for young women who are at high risk for ovarian failure, or when the other options are not suitable. Consult the patient that this technique is not a routine clinical practice. The ovarian tissue must be monitored for the presence of malignant cells. The guidance recommended managing the pregnancy after cancer treatment as a high-risk pregnancy, preferably in a tertiary center.

The guide also has a recommendation for infertile AYA cancer survivors. They should bedsides providing infertility counseling, and be informed about the assisted reproduction technologies opportunities, such as sperm, egg, or embryo donation, surrogacy, and adoption.

The complete version of this guideline is available via: https://wiki.cancer.org.au/australia/COSA:AYA_cancer_fertility_preservation [67].

6.7 Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology Guideline for Ovarian Tissue Cryopreservation and Transplantation (CSGE-ISGE) (2018)

Besides some guidelines which have no special target, a guideline has been published by the CSGE-ISGE to introduce the standard application of OCT in China. Based on Chinese specific conditions and after assessing the international guideline, they formulated the selection criteria as follows.

1. Age preferred ≤35 years old, for the assurance of good ovarian reserve, but if the ovarian reserve is confirmed and the patient asks for it, age limitation can be changed.

2. excluded the malignant ovarian tumors or when there is a high risk of ovarian metastasis.

3. good prognosis.

4. when the risk of POI is high because of the primary disease or the treatment.

5. laparoscopic or open abdominal biopsy is not contraindicated.

6. Radiotherapy and chemotherapy can be delayed at least 3 days.

7. Informed consent (the patients or their parents or guardian).

In this guideline, the authors described the good indication as the patients with tumors and nonmalignant diseases, as the aim of OTC is to preserve fertility and ovarian endocrine function. This method is suitable for prepubertal girls, patients who do have not enough time to postpone chemo- or radiation therapy, and women with hormone-sensitive tumors [45].

This guideline described that there is no uniform standard protocol for the timing of transplantation. It depends on the primary disease and can be done when the disease is cured and clinical rehabilitation happens. After full situation consideration, the patient’s specific treatment is done.

commonly when the primary condition is cured, and the symptoms of menopause have been revealed due to ovarian function destruction; such as hot flashes and sweating, serum level of follicle-stimulating hormone (FSH) ≥ 25 IU/L, and anti-Mullerian hormone (AMH) <1.1 ng/ml ovarian retransplantation can be done at least 3–6 months after stopping chemotherapy [45].

Every month follow-up is needed to assurance of ovarian function recovery (reproductive and endocrine). After recovery, follow-up can be continued every 3–6 months.

For leukemia cases, there is no ideal fertility preservation program and OTC should be done before hematopoietic stem cell transplantation. Due to the high risk of reintroduction of malignant cells, ovarian transplantation should be performed with caution. This guideline also paid attention to ovarian endometrioma fertility preservation, while the inappropriate endometrioma removal can cause destruction effects on the ovarian reserve, and therefore assessment of ovarian function should be done before endometriosis surgery, since fertility preservation may be needed during the operation.

6.8 British fertility society policy and practice guideline (2018)

This guideline brought together the evidence literature for fertility preservation techniques and their outcome, as well as the associated risks, for medical reasons; both oncological and non-oncological cases. The guideline recommended consultation with women and girls about the risk of cancer treatment on their fertility and available preservation techniques. The four measures were: embryo, oocyte, and ovarian tissue cryopreservation, GnRH agonist administration, and ovarian transposition. In this version also ovarian tissue cryopreservation was still considered experimental. For benign and malignant conditions, current treatment modalities, which were accompanied by a better fertility-sparing profile, were described.

The guideline highlighted the role of psychological support in decision-making. The guideline also highlights that it is needed for the patients to meet the exact criteria to undergo invasive procedures, and FP must be done for curative intent [43].

The summary of its recommendations is as follows:

1. Reassure the patients that cancer treatment does not increase the risk of congenital anomalies or genetic disease

2. Radiotherapy to the uterus is probably with obstetric risks.

3. Inform cancer patients that the available data showed pregnancy does not increase the risk of cancer resuming, in many cancers.

4. The risk estimated for infertility, and premature ovarian failure should be done based on age, type of regimens, and dose of chemotherapy.

5. The risk of infertility, following pelvic, abdominopelvic, or craniospinal radiation depends on the field of exposure.

6. Inform the patients with breast cancer that they may suffer from delays in conception because of lengthy endocrine therapy, even if there is no need for gonadotoxic treatment.

Offer fertility preservation if:

1. There is a serious risk of infertility following the current cancer treatment.

2. There is a good chance for long-term survival.

3. The patient’s situation permits ovarian stimulation and oocyte collection and does not jeopardize prognosis.

4. Cryopreserved embryos and oocytes have the success rates as the fresh ones.

5. To shorten the treatment duration and prevent the risk of ovarian hyperstimulation syndrome (OHSS) antagonist protocols are usually recommended. An agonist trigger reduced the risk of OHSS.

6. An anti-estrogen [letrozole, clomifene, or tamoxifen] prescription during ovarian stimulation in the case of estrogen-sensitive tumors.

7. Patients should be aware of the time limitation for storing their oocytes/embryos.

8. Available evidence shows no influence of the duration of storage on the success rate of cryopreserved oocytes/embryos.

9. Child welfare assessment should be done at the time of using oocytes/embryos for conception [43].

6.9 ESHRE guideline (2020)

The last version of the guidelines has been published by the European Society of Human Reproduction and Embryology (ESHRE, 2020) [60]. In this guideline fertility preservation has been discussed in women and transgender men concluded assessment before FP, the interventions, and also the post-treatment recommendations. It includes 50 evidence-based recommendations which were approved by the ESHRE Executive Committee and the Guideline Group. They consider this version needs to be updated 4 years after this publication.

This guideline considers intrinsic and extrinsic factors for the patients’ assessment and selection. Intrinsic factors such as patient’s health status, age, consent obtaining, and ovarian reserve assessment, and extrinsic such as the risk for fertility, and risk for pregnancy, … are discussed. The needs and ways for “ovarian reserve testing” are argued. Some of the important recommendations regarding OTC have been brought below.

In preparing a guideline, it’s the first time that OTC methods have been reviewed, and the slow-freezing protocol is considered as standard protocol and offered the vitrification protocol only in the research program. One of the limitations of the present guidelines is the lack of discussion about the best cryopreservation method, although the choice of the method is selected by the embryologist and based on the conditions and facilities of the specialized centers, the freezing method is needed to be discussed in the guidelines.

Summarized recommendations of the ESHRE (2020):

1. OTC is recommended for the patients who must receive the high-risk gonadotoxic treatment and the oocyte or embryo cryopreservation is not feasible, or when the patient selects it.

2. OTC may not be useful for patients with low ovarian reserve⁵ or advanced age.

3. The efficiency of OTC in patients >36 years old is doubtful.

4. Patients who have a history of low gonadotoxic treatment or a previous history of chemotherapy, also can be considered for OTC.

5. There is no limitation for ovarian stimulation immediately after OTC

6. OTC can be done when oocyte pick-up is done, following ovarian stimulation only for research purposes.

7. Ovarian transposition also can be done when OTC is done in patients who need pelvic irradiation.

8. OTC cannot be considered the primary option in transgender men but can be performed as an experimental procedure during gender reassignment surgery.

9. Ovarian tissue cryopreservation and transplantation can be done in patients with genetic and chromosomal associated POI disorders, after genetic counseling and in a research approach.

7. Guidelines regarding the concerns of cancer reintroducing

One of the serious concerns regarding OTC and transplantation is the risk of re-implantation, by the possible existing malignant cells in the harvested ovarian tissue after remission and resulting in the exclusion of ovarian cancer or when there’s a high risk for ovarian metastasis [45, 68, 69]. It is of paramount importance in the cases of hematologic malignancies, which are at the most risk of ovarian metastasis, with the transferring of malignant cells via the bloodstream, and the highest risk was found with leukemia [69].

Recently, there is a new idea that ovariectomy after chemotherapy may be resulted in tissue cancer cell-free and increase transplantation safety in hematologic patients [12, 59, 70]. To evaluate the presence of leukemic cells in cryopreserved ovarian tissue histology and immunohistochemistry can be used to identify the ovarian infection by the malignant cell, but the molecular analysis is a better sensitive way. In leukemia patients, polymerase chain reaction (PCR) can reveal malignant cells in up to 75% of cases of thawed ovarian tissue [12]. RT-qPCR, and long-term (6 months) xenografting to immunodeficient mice, also have been used, in this aim [69, 71]. It must be mentioned that a disease-specific molecular marker may not be found in some cases of ALL, and AML [12]. In one study immunohistochemistry showed no evidence of the presence of residual leukemic cells, in the ovarian tissue after freeze and thaw. But the malignant cells were identified in 6 of 8 specimens, by the RT-PCR [72]. In the other study, it was shown that in the ovarian tissue harvested from ALL patients, there was no evidence of malignant cells in the ovarian grafts into immunodeficient mice, or in any of the other tissues, it’s despite 4 RT-PCR positive malignant cells of the 7 grafts [73].

However, in a report in 2013, the author strongly discouraged ovarian autotransplantation when there’s a risk of malignant cell reimplantation, especially in leukemia cases, and suggested patients consultation before reimplantation, in other cases in which the estimated risk is low [69], in the later publication in 2016, it was suggested that leukemia survivors also may benefit from OTC after providing maximal safety measures. These measures include harvesting after chemotherapy and an exact and intense search for leukemia cells within the graft. These cases must be informed that to date tissue involvement with malignant cells cannot be ruled out entirely with no measures [59]. Studies are needed to reassure the safety of this technique in different types of cancer.

In leukemia survivors, for ovarian tissue transplantation, careful consideration and evaluation must be taken to increase safety. Informed the patients thoroughly regarding the limitations of present investigation modalities, and the risk of cancer re-introducing should be weighed against the opportunity of being a biological parent [12].

8. Is ovarian tissue cryopreservation and transplantation still experimental?

Until recently, and for many years, ovarian tissue cryopreservation has been considered an experimental method, in many guidelines [15, 49, 50, 74]. One study described that the studies on tissue cryopreservation are limited, and most of them were done with no control group. There’s a need for more experience, to accept this technique as a routine technique for fertility preservation. They said the published success of pregnancies may be because of the patient’s native ovarian tissue [75]. In one study, in 2018, authors discussed this subject with more caution, and said because a live birth has not been achieved in the woman who had ovarian cryopreservation in the prepubertal stage, the efficacy is not still approved in children, and also in women with leukemia, the safety of this technique is still poor, so they considered it as experimental in children and in adults’ patients which the risk for ovarian contamination with malignant cells is high.

For a technique to be defined as experimental or established some clear criteria are needed. European Society of Human Reproduction and Embryology proposed a platform to classify a technique as experimental, innovative, or established. The suggested applying criteria were efficacy, safety, procedure, and effectiveness, which all of them must be fulfilled, to accept a new treatment as established. These criteria also can be applied for the classification of the OTC and OTT in children, adult malignant cases with a high risk of ovarian contamination with malignant cells (e.g., leukemia), or low risk of ovarian involvement (e.g., breast cancer or Hodgkin lymphoma). In final, they classified this technique as established and no longer experimental when the risk of ovarian metastasis is low [48].

The first time, in 2016, in a study which was published by ASRM, it was concluded that there are enough national ethical and professional authorities for considering OTC as a standard modality, in fertility preservation programs [59]. Also, CSGE-ISGE (2018) mentioned that now there is large evidence of data that can confirm the effectiveness of the OTC, and it must be considered as a clinical and standardized procedure that would be promoted as soon as possible. The cryopreservation indications are extensive, and it is the only chance for prepuberty girls and patients who need emergency treatment (radiotherapy or chemotherapy) [45].

After many evidence-based studies and live birth reports, following transplantation, it’s time to consider this technique as a clinical approach for medical indications (by the American Society of Reproductive Medicine), which can restore both patients` fertility and ovarian endocrine function [76]. Subsequently to more and more achievements of OTC reports, in the literature, researchers concluded that we passed the experimental phase.

In one study, the pregnancy rate per re-implantation was calculated, in the presence of evidence. In this large series of 111 cases, which the results were collected from Denmark, Belgium, Spain, Germany, and also from Australia, 29% of the women conceived (n ¼ 32). Knowing the exact number of transplantations, the data was highly relevant and evidence-based. This study approved the efficacy of the OTC technique [9].

According to one of these studies on 20 cases, the success rate for restoration of hormone activity was calculated at 94% [77]. Other studies, also referred to the worldwide live birth rate of 30–70% and concluded that this is a rigorous document to consider OTC as a standard protocol, for female fertility preservation options [36, 77, 78]. Many pioneers believe that now enough evidence support OTC and we reach the time to stop considering it as an investigational or experimental procedure [79].

9. Conclusions and recommendations

While the aim is to gather more evidence of the efficacy of the OTC, it is essential to consult the children and their families about this chance. There is no doubt that OTCP will be accepted as a successful standard method, soon with the fast growth in the technologies. They may suffer by delaying the offer.

We must discuss with all the patients, who may be at risk for premature ovarian insufficiency, following chemo- or radiotherapy, about the FP techniques. Discuss FP using OTCP, to the patients who do have no enough time for ovarian stimulation, just the time the diagnosis is established.

Patients should be well-informed about the different choices to be able to make valuable decisions according to the available evidence, as it may be their only real chance to be genetic parents in their future lives. A multidisciplinary team is needed, that could support all the necessary aspects of the FP, such as clinical, psychological, as well as legal, and ethical issues. After a comprehensive review of the present literature, a flow chart of the guidelines regards to OTC for cancer patients has been provided (Figure 1).

Figure 1.

Flow chart of a comprehensive guideline for referring cancer patients for fertility preservation.

Regarding future FP programs, it must be stressed that however, the emerging options are promising, but their efficacy and safety of both established and newer techniques are needed to be confirmed by rigorous clinical trials, maybe with a focus on live birth, before introducing them as international clinical standards, and then can be offered as a medical intervention. Moreover, the exact criteria and relevant indication must be identified, in the aim to achieve the most efficacy of each FP method, also to reach more advancement in this field more research is needed on both established and newer techniques. Recent evidence has approved ovarian tissue cryopreservation as one of the promising options for female FP.

In the aim to give the patients appropriate and good clinical practice medical intervention about FP, widening access to the FP options and technologies is the key aspect (both appropriately patient selection and relevant indication).

However, negligence in referring the true cases to fertility preservation will result in depriving someone to have a family, applying FP to all cases, even with a low risk for gonadotoxicity, creating a large amount of unused stored reproductive cells and tissue, which burden costs for both patients and health services.

However, there are some new treatments in oncology with unknown gonadotoxicity effects, the risk of ovarian gonadotoxicity can be estimated in specific treatments which enough studies were done, and with sufficient detail, also ovarian reserve testing may be helpful, however, they have limited authority for predicting future fertility. So decision-making for many patients requires multidisciplinary discussion to evaluate the risk and benefits of FP interventions exactly. Providing comprehensive information is crucial to support the patients to have true decision-making.

Despite some general limitations in the current review of the published guidelines and the limited provided evidence, there is a hope that this document will help best practices in female FP.

From all guidelines, the following recommendations can be extracted:

1. It is needed to discuss with the patients the possibility of impaired fertility following cancer treatment.

2. The early referral to a reproductive specialist is a promising approach that gives a chance to improve fertility outcomes while minimizing the cancer treatment delay

3. It is necessary to identify malignant and benign conditions which approve the selected patient as a true candidate for ovarian cryopreservation.

4. Keep in mind all the current available established and experimental options to preserve female fertility

5. The difficulty in accurate estimation of the future impaired fertility in all cases

6. The difficulty in providing an accurate estimation of the risk for re-implantation after remission in blood-born hematologic cases.