POLSKI
Introduction
The topic of pre- and pubertal female fertility preservation remains poorly described, despite the growing number of cases requiring intervention. Loss of fertility may be the result of cancer treatment, autoimmune diseases, infections, genetic diseases, or medical transition related to gender incongruence. The most frequently described issue is oncofertility, i.e., the preservation of fertility in children treated with oncological treatment [1–3]. There is also a growing interest in preserving fertility in transgender people in the context of puberty blockers and sex hormone therapy [4, 5]. The currently available techniques for ovarian tissue cryopreservation are not yet routinely used and are fraught with many medical, ethical, and social challenges [6]. Despite existing guidelines, their practical application is limited, and additional barriers include the lack of established standards, difficulties in assessing the child’s readiness, and issues related to the role of parents in the decision-making process [6, 7]. It is worth noting that not all children will have access to such procedures due to organizational, financial, or cultural barriers [8].
Causes of infertility
Among girls and women, non-iatrogen premature ovarian insufficiency (POI) is frequently diagnosed, unrelated to gonadotoxic therapy or procedures. Early POI is rare, with an incidence of < 1/10,000 cases before the age of 20 years. The POI is defined as amenorrhea for ≥ 4 months and follicle-stimulating hormone (FSH) > 25 IU/l before the age of 40 [9]. The most common condition associated with POI in young women is Turner syndrome [10]. Diseases such as cystic fibrosis, hemochromatosis, galactosemia, autoimmune ovarian failure, fragile X syndrome, or Down syndrome can disrupt the hypothalamic-pituitary-gonadal axis and also lead to POI [11]. If the diagnosis is made before the onset of POI, fertility preservation counseling is recommended [12].
Fertility preservation strategies in children should be adapted to the risk of infertility, planned treatment, and available methods. In patients with cancer and genetic diseases, multidisciplinary consultation is indicated. In practice, cryopreservation of immature gonadal tissue is mainly used in prepubertal children, while the use of gonadotropin-releasing hormone (GnRH) agonists remains controversial [11, 13, 14].
Methods of assessing gonadal reserve in pediatric patients
Diagnostics of infertility in children are mainly based on hormonal and imaging tests. Due to the immaturity of the hypothalamic-pituitary-gonadal axis, interpretation of results requires extensive experience and consideration of developmental age.
In girls, anti-Müllerian hormone (AMH) is crucial for assessing ovarian reserve. A low AMH level indicates a higher risk of POI. Currently, there is no clearly defined, universally accepted AMH threshold exists that could serve as a reliable marker of developing POI. Consequently, clinical management decisions should be based on an integrated evaluation comprising AMH measurement, ovarian ultrasound assessment (antral follicle count – AFC), and a thorough patient history and physical examination.
In patients with Turner syndrome, serum AMH concentration is serves as a sensitive and specific biomarker for premature ovarian insufficiency (POI). An AMH level < 8 pmol/l is associated with a 96% positive predictive value for POI (sensitivity 96%, specificity 86%). In prepubertal patients, AMH < 4 pmol/l predicts the absence of spontaneous pubertal onset, whereas in adolescents, AMH < 5 pmol/l indicates imminent POI [15, 16].
Additionally, FSH and luteinizing hormone (LH) are assessed – their high concentrations with low estradiol suggest primary hypogonadism [14, 17]. It should be mentioned that in girls aged ≥ 12 years at risk of POI (after gonadotoxic treatment), determining FSH concentration is the recommended and more sensitive screening test [18]. In the case of girls at pubertal age, in the context of planning and assessing the effectiveness of fertility preservation methods, there are no clear studies indicating which marker – AMH or FSH – is definitely better.
Image diagnostics using ultrasonography (USG) complements hormonal tests and provides information on the morphology of the gonads, their internal structure, volume, the presence of focal changes, hydroceles, cysts, or signs of atrophy. A decrease in the volume of the testicles may indicate a loss of spermatogenic function and requires further diagnostics [3].
In the case of girls, transabdominal or – in older girls – transvaginal imaging allows for the assessment of the number of antral follicles (AFC), i.e., those that are potentially capable of growth and maturation. The number of antral follicles correlates strongly with the level of AMH and is one of the best predictors of ovarian reserve. Additionally, ultrasound facilitates the identification of developmental defects in the uterus and ovaries, which can impact reproductive potential [19].
Magnetic resonance imaging is used in complex cases (e.g., suspected changes in the pituitary gland or gonads) and also allows for the differentiation of tumors and cysts.
The combination of hormonal and imaging tests enables the assessment of fertility potential and the implementation of protective procedures (e.g., cryopreservation), which is confirmed by the studies of Yang et al. [20].
Hormonal and imaging diagnostics not only enable the detection of disorders in the functioning of the reproductive system but also have a prognostic and educational function. It provides information that is the basis for discussions with the patient and family about future reproductive possibilities, as well as the need to take action to prevent irreversible loss of fertility.
Cryopreservation
Cryopreservation and autotransplantation of ovarian tissue as a method of fertility preservation are available for prepubertal girls and women at high risk of premature ovarian failure in more than 20 countries [21].
In 2019, the American Society for Reproductive Medicine (ASRM) stopped considering cryopreservation as an experimental method [22] because of more than 140 reported live births after ovarian tissue transplantation [23]. As of 2,022, 1,019 OTC procedures have been performed in patients aged 0–20.4 years, including 298 in girls below 13 years of age [24]. To date, more than 200 births have been reported, including after transplantation of prepubertal tissue [25, 26]. The first documented case involved a girl with thalassemia whose ovarian tissue was cryopreserved for 14 years, which allowed her to achieve a pregnancy by in vitro fertilization [27]. Importantly, this method does not require puberty or ovarian stimulation [13, 28]. The reason for the low number of these procedures is likely due to the limitations associated with the surgical removal of gonadal tissue and the uncertainty surrounding their reuse by autograft or in vitro maturation.
Despite in vitro maturation of infant germ cells is possible, it has not yet led to the birth of a live child [29].
For girls, cryopreservation of ovarian tissue remains the best option, as immature oocytes (from follicles < 6 mm) are rarely used. In some centers, immature oocytes can be used as part of maturation protocols such as in vitro follicular maturation (IVM) and in vitro follicular growth (IVFG) [6, 30–32]. Hormonal function after retransplantation has been achieved in > 95% of cases [33, 34], and the pregnancy rate is 50%, with a live birth rate of 41% [14, 35, 36]. Standardly, ovarian tissue is cryopreserved by slow freezing or vitrification. The tissue is frozen to –140°C and then stored at –196°C in liquid nitrogen [37]. Recent data suggest that the strategy of vitrification and warming of oocytes is superior to slow freezing and thawing in terms of clinical outcomes [38].
Graft survival depends on time, location (preferably orthotopic sites – such as the original site of the ovaries e.g. atrophic cortex of existing ovaries or in peritoneal pockets formed in the pelvis, broad ligament or ovarian fossa), and level of revascularization. Heterotopic grafts (outside the anatomical location of the ovaries e.g. lower abdominal wall) have been observed to cause the formation of a large number of empty follicles, inhibition of follicle development (< 15 mm), poor oocyte regeneration, and low fertilization rate [34]. A summary of available fertility preservation methods in pediatric patients is presented in Table I.
Table I
Review of methods for preserving fertility in children
| Sources, ref. | Method | Sex | Pre-puberty | Post-puberty | Clinical Status | Efficacy | Requires Surgery | Oncofertility | Clinical notes / limitations |
|---|---|---|---|---|---|---|---|---|---|
| Pampanini et al. [13] Donnez and Dolmans [32] Demeestere et al. [37] Emrich et al. [39] Rodriguez-Wallberg et al. [40] Imbert et al. [41] | Ovarian tissue cryopreservation | F | ✔ | ✔ | Clinical (established in children) | > 200 documented births; success post-transplantation | ✔ | ✔ | Only established option for prepubertal girls |
| Nikiforov et al. [30] | In vitro maturation of oocytes (IVM) | F | ✔ | ✘ | Experimental | No births (due to oocyte collection from children) | ✔ | ✔ | Low availability, lack of standardization |
| McClam and Xiao [2] Cobo et al. [38] | Cryopreservation of mature oocytes | F | ✘ | ✔ | Routine | High (20–40% per cycle) | ✔ | ✔ | Requires hormonal stimulation |
| McClam and Xiao [2] Alteri et al. [42] | Embryo cryopreservation | F | ✘ | ✔ | Routine | High; depends on embryo quality | ✔ | ✔ | Requires IVF and sperm source |
Efficacy of cryopreservation
Cryopreservation of ovarian tissue is currently the only available method of preserving fertility in girls before puberty and in patients who cannot undergo stimulation therapy due to clinical contraindications or the need to start oncological treatment quickly. In recent years, there has been an increasing number of reports confirming the efficacy of OTC also in the pediatric population. A summary of clinical results after cryopreservation of reproductive material in the pediatric population is presented in Table II. One of the first documented cases was a patient whose ovarian tissue was collected at the age of 13 years and 11 months, and after retransplantation 13 years later, a spontaneous pregnancy and delivery of a healthy child occurred [37]. Similarly, in a patient with acute lymphoblastic leukemia, in whom OTC was performed at the age of 14 and 15 years after cryopreservation, a pregnancy was achieved after IVF and another one spontaneously. A retrospective analysis by Imbert et al. [41] showed that in 3 of 8 patients who underwent retransplantation after previous OTC (including prepubertal patients), menstruation returned, and pregnancy was achieved.
The 20-year experience of the center in Bologna (over 1,000 cases, including 238 children and young women) showed the effectiveness in restoring ovarian function after OTT in 85% (orthotopic transplants) and 100% (heterotopic). Four deliveries and 2 miscarriages were recorded; the average time of tissue storage was 7.8 years [43]. Overall, the success rate of ovarian function restoration after OTC is 85–100%, with pregnancy rates ranging from 30–67%, depending on age, storage time, and underlying disease. Although pediatric data are limited, successfully transplanted tissue can lead to spontaneous and assisted pregnancies.
Table II
Clinical outcomes following cryopreservation of reproductive material performed in childhood
| Author, ref. | Gender of subjects | Fertility preservation method | Age at cryopreservation | Cryopreservation technique | Pregnancy | Time to retransplantation | Age | Transplantation method | Hormone levels before OTT | Hormone levels after OTT |
|---|---|---|---|---|---|---|---|---|---|---|
| Demeestere et al. [37] | F | OTC | 13 years and 11 months | Slow freezing (DMSO) | Yes (natural, 2 years after OTT) | 13 years | 27 years (at OTT) | Orthotopic | High FSH (POF), no menstruation | FSH 5 IU/l, E2 166 pg/ml, menses resumed |
| Rodriguez-Wallberg et al. [40] | F | OTC | 14 years | Slow freezing (propanediol) | Yes (IVF then spontaneous) | 15 years | 29 years | Orthotopic (laparoscopic) | Post-menopausal FSH, AMH undetectable | Normalized FSH, E2 rising, AMH 0.15 ng/ml |
| Imbert et al. [41] | F | OTC | Varied (mostly prepubertal) | Slow freezing (DMSO) | Yes (3 pregnancies in 8 patients) | Varied | Varied (> 20 years) | Orthotopic and heterotopic | High FSH in 10/13 women | FSH < 40 IU/l, resumption of menses |
| Emrich et al. [39] | F | OTC | Avg. 13–17 years | Slow freezing + vitrification | Yes (1 adolescent) | Avg. 10–14 years | 17–29 years | Orthotopic | High FSH, low AMH | Observed recovery |
| Gillipelli et al. [44] | F | OTC | Under 21 | Slow freezing (DMSO, propanediol) | Yes (6/9 patients) | 5–15 years | 18–30 years | Orthotopic and heterotopic | Elevated FSH | Reduced FSH, menses resumed |
| Fabbri et al. [43] | F | OTC | 2–38 years (avg. 12.9 for pediatrics) | Slow freezing | Yes (4 births, 2 miscarriages) | 2–17 years (avg. 7.8) | 36.2 years (avg. at OTT) | Orthotopic and heterotopic | Most in POI | Return of menses in 85% of women after orthotopic retransplantation and in 100% after heterotopic |
Discussion
Clinical practice and literature indicate that OTC is currently the only fertility preservation method available for prepubertal girls and for patients who are not eligible for hormonal stimulation. Over 200 births have been achieved with this technique, with the efficacy of retransplantation in restoring ovarian function being 85–100% and pregnancy rates being 30–67%, also in the pediatric population. The number of documented cases of children undergoing OTC is limited, and the results depend on many factors, such as the patient’s age, underlying disease, tissue storage time, and the cryopreservation protocol used.
There are numerous obstacles to the development of fertility preservation techniques in the pediatric population, such as moral conundrums, a lack of standardization in protocols, financial and technological constraints, and unequal access to procedures. It is also a challenge to take into account psychological and legal aspects in the care of pediatric patients and their families.
Conclusions
Pre-pubescent fertility preservation is a complicated and rapidly evolving area of medicine that calls for close interdisciplinary collaboration, early diagnosis, and risk assessment of each individual. The group of cancer patients may have a much higher chance of retaining reproductive autonomy as adults if gonadal tissue cryopreservation techniques are carried out safely and effectively.
Additional fundamental and clinical research is required to develop alternative approaches (e.g., in vitro gamete maturation), enhance the efficacy of retransplantation, and optimize cryopreservation techniques. Concurrently, it is imperative to establish a legal and ethical framework that is consistent and to establish an infrastructure that guarantees equitable access to care in the pediatric fertility preservation field.
