Ovarian tissue cryopreservation: a narrative review on cryopreservation and transplantation techniques, and the clinical outcomes

Abstract

Fertility preservation (FP) includes all the methods to preserve germ cells, reproductive tissues, or embryos for the future reproduction of patients at risk of infertility. Cryopreservation is an essential step of FP, storing the specimens in subzero temperatures to suppress cellular metabolism and restore cryopreserved specimens for future use. Although oocyte cryopreservation (OC) and embryo cryopreservation (EC) are two accepted methods of FP in women, ovarian tissue cryopreservation (OTC) is a novel method that is favorable in patients who are not appropriate candidates for OC and EC, and those who suffer from irritating menopause symptoms caused by estradiol deficiency. OTC has shown promising results in restoring fertility and the endocrine function of ovaries. Slow freezing and vitrification are two well-established methods for cryopreservation of biological specimens. Despite recent developments in the vitrification of ovarian tissue and comparable results to slow freezing, we lack a standard protocol for ovarian tissue vitrification, and slow freezing is still the preferred method in most centers. Under an acceptable medical condition and desirability, transplantation of cryopreserved tissue is performed either in orthotopic sites (orthotopic transplantation, OT) such as the original site of the ovaries and uterus, or heterotopic sites (heterotopic transplantation, HT) like abdominal wall, forearm, and peritoneal lining. Although both sites of transplantation are associated with endocrine function recovery, OT better restores fertility. This review will focus on OTC and its types, ovarian tissue transplantation, and efficacy in clinical practice.

Introduction

Human ovaries contain a limited reserve of germ cells from the embryonal period. After puberty, germ cells develop into mature oocytes (MOs) and are released progressively until menopause. Menopause is defined as the permanent termination of menstrual cycles, which occurs physiologically in midlife because of the loss of ovarian follicular function. ¹ Almost 1% of women may experience premature menopause, which is the occurrence of ovarian failure before 40 years of age, and it is two standard deviations lower than the typical age. ² Primary premature menopause may spontaneously occur in women with similar family history or patients diagnosed with certain genetic conditions like Turner syndrome, autoimmune, and metabolic disorders. Also, it may be induced by interventions such as chemotherapy (ChT), radiotherapy (RT), and surgical bilateral oophorectomy. ² Regardless of the etiology, premature ovarian insufficiency (POI) is associated with loss of reproduction capacity, which is the primary concern of most of the women diagnosed with it. Besides, estrogen deficiency in these patients may lead to irritating climacteric symptoms.^3,4

Fertility preservation (FP) is an essential matter of concern, especially in patients who are at risk of experiencing POI because of receiving ChT and RT due to malignancies. Embryo cryopreservation and oocyte cryopreservation are the most common methods of FP.^5,6 However, they do not retain the hormonal status of the patients, and thus, they are not efficient in resolving other conditions associated with estrogen deficiency and climacteric syndrome. ⁷ Moreover, oocyte retrieval requires ovarian stimulation, which may be contraindicated in certain patients. Furthermore, the ChT should not be delayed in some cases, and the patient cannot undergo an ovarian stimulation cycle before initiating ChT.^8,9 Ovarian tissue cryopreservation (OTC) is the only suitable alternative in such patients. Surgical dissection of the ovarian cortical tissue fragments or a whole ovary is the suggested method for OTC. ¹⁰ In cases with a risk of malignancy reintroduction, isolation of primordial follicles is a safe method for OTC.^11,12 When the patient’s clinical conditions allow for the safe transplantation of the cryopreserved tissue, ovarian strips will be placed in orthotopic sites, such as the remaining ovaries or heterotopic sites, subcutaneously or retroperitoneal. ¹³

OTC does not require ovarian stimulation, which makes it a possible method for patients who need to start ChT immediately. ⁹ This method was suggested in the late 20th century when cryopreserved ovarian tissue was transplanted for oophorectomized animal models, and it was accompanied by promising results.^{14 –17} Regarding the promising results of OTC in animal studies, OTC was considered a practical method in humans. Several studies have begun to evaluate the prospect of developing functional follicles from human ovarian tissue. In recent years, OTC has progressed into a more established method, and up to this point, more than 200 live births have been reported in human studies. ¹⁸ Slow freezing was the first method described for OTC, resulting in the most reported live births. Vitrification has recently been described; it does not take too much time and expensive equipment. However, few studies have concentrated on this method, and more research is needed to confirm its efficacy.^19,20

The objective of the present manuscript is to provide a review of OTC, its indications, the current established methods for it, and an update on the most recent and robust research studies conducted so far. Herein, we describe the most recent reported clinical results related to the efficacy and safety of OTC in developing functional follicles, recovery of endocrine function, and occurrence of pregnancy and live births. Moreover, we discuss the critical ethical issues arising from OTC.

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Indications of FP in women

POI is characterized by oligomenorrhea or amenorrhea before the age of 40 years, which lasts for more than 4 months, and high (>25 IU/L) levels of follicle-stimulating hormone (FSH) on two occasions at intervals of 4 weeks. ²¹ Moreover, about 5% of the women may be diagnosed with early menopause, which is defined as the loss of ovarian function occurring between 40 and 45 years of age. ²² As a consequence of estrogen deficiency, affected women may also experience irritating climacteric symptoms, increased risk of coronary heart disease, osteoporosis, and other chronic diseases, including arthritis, malignancies, cognitive disorders, cataracts, and skin and vaginal atrophy.^22,23 POI and early menopause are associated with infertility. Thus, these two conditions are essential indications of FP. Idiopathic POI, genetic syndromes (e.g., Turner syndrome), autoimmune disorders, and iatrogenic causes are the leading conditions associated with POI.^3,22,24,25

Based on the European Society of Human Reproduction and Embryology (ESHRE) guidelines (2020), FP could be considered for women with the aforementioned underlying conditions that predispose them to POI. ²⁶

OTC is a novel method of FP, especially in women ChT should initiate immediately or have already started, or patients with hormonal-sensitive tumors, who cannot undergo ovarian stimulation cycles.^26,27 OTC contributes to the recovery of the ovaries’ endocrine function as well as FP. Studies have reported the rate of ovarian function recovery to be more than 90% after OTC and transplantation.^{28 –31}

Current cryopreservation techniques in OTC

Slow freezing and vitrification are two essential methods of cryopreservation widely used for FP.^27,32 Cryoprotectants are agents that are used to preserve specimens from injuries during the freezing process. Different cryoprotectants used in cryopreservation could be categorized as below^19,33:

• - Permeating cryoprotectants: Due to their small size, these agents can diffuse through the plasma membrane and reduce the freezing temperature by forming hydrogen bonds with intracellular water, thus inhibiting ice crystal formation. ¹⁹ Ethylene glycol (EG) and dimethyl sulfoxide (DMSO) are the most common permeating cryoprotectants used for OTC. EG is frequently used in vitrification and has lower cytotoxicity. ³⁴ DMSO is commonly used in both vitrification and slow-freezing methods. Glycerol, propylene glycol, acetamide, and formamide are other agents in this category. ³⁵

• - Non-permeating cryoprotectants: These agents cannot diffuse through the membrane and prevent glass formation in the extracellular matrix. ¹⁹ Sugars are common cryoprotectants used in both vitrification and slow-freezing methods. Proteins and polymers are other non-permeating agents that increase the solution’s viscosity and adjust water’s osmotic diffusion. ³³

Below, we will briefly discuss slow-freezing and vitrification methods.

Slow freezing

In OTC using slow freezing, the temperature of the cells is reduced at a controlled rate by using special programmable freezers. As the cells are suspended in a solution containing cryoprotectant, they become dehydrated and reach equilibrium with the solution. With the progression of slow freezing and extracellular ice formation, the solution becomes concentrated, which leads to osmotic adjustment and dehydration of the cells.^19,36

Despite the variations between different slow-freezing protocols, all are based on the protocol described by Gosden et al. in 1994. Figure 1 illustrates this method. ¹⁵

Figure 1.

The schematic overview of steps of the slow-freezing protocol described by Gosden et al. ¹⁵

Currently, it is common to use a permeating cryoprotectant (usually 1.5 M) in combination with a non-permeating cryoprotectant (around 0.1 M), and the most established combination consists of DMSO and sucrose. ²⁰

Slow freezing is the standard method for OTC in most centers, and most of the reported live births and recovery of ovarian function in literature resulted from this method. ³⁶ In this method, the lower concentration of cryoprotectants minimizes their cytotoxic effects. ³⁷ The most critical challenge of slow freezing is the formation of ice crystals that cause cryoinjuries. ¹⁹ Moreover, consecutive changes in cell volume during slow freezing are injurious. One of the other challenges of slow freezing is the need for expensive equipment and a lot of time.^19,20,38

Vitrification

Vitrification is a well-established method for cryopreservation of oocytes, sperms, and embryos. However, this method has only recently been described for OTC, and a standard protocol has yet to be defined for it.^39,40 Vitrification is an ultra-rapid cooling method in which the super-cooled solution turns into a glassy solid without forming ice crystals and chemical or structural changes. In this method, the viscosity of the intracellular and the extracellular fluids increases significantly, and the solutions become dehydrated rapidly to form an amorphous vitrified solid. This amorphous state results from high concentrations of cryoprotectants and is not affected by cryoinjuries caused by ice crystals. Furthermore, unlike slow freezing, vitrification does not require expensive equipment and can be performed rapidly. ⁴¹

Current vitrification protocols are usually based on the protocol described by Kagawa et al., initially to cryopreserve mice ovaries and, after that, was used for human OTC in other studies. ⁴² Figure 2 illustrates the vitrification protocol described by Kagawa et al. ⁴²

Figure 2.

The schematic overview of the vitrification protocol described by Kagwa et al. ⁴²

Generally, the higher concentration of cryoprotectants required for vitrification than slow freezing (over 40% vs 10%–15%) is associated with more cytotoxicity.^19,20 To minimize the damage, it is essential to carefully manage the temperature, concentration, and exposure time to cryoprotectants. Also, it is necessary to use small sample sizes (under 1 mm thick or around 1 mm³ in volume) to increase the penetration of the vitrification solution into the sample and improve revascularization after transplantation.^19,20

Clinical outcomes of slow freezing and vitrification

Clinical evidence supports the efficacy of slow freezing as the standard OTC method.^{31,37,41,43,44} However, vitrification is a more novel method for OTC, and although it has resulted in few live births so far, histopathologic examinations are comparable to slow freezing.^37,41,44,45

A prospective cohort study between 2005 and 2015 included 800 patients undergoing OTC. In this cohort, transplantation was performed for 50 patients, of whom 6 wished for recovery of endocrine function, and 44 patients sought pregnancy. They observed improved endocrine function in 93.2% of the patients after a mean time of 94.3 days. In this cohort, three women experienced pregnancy, which resulted in four healthy deliveries. The results were not significantly inferior to the oocyte vitrification method. ³¹

In a cohort study between 1999 and 2010, 397 women with ChT-induced POI underwent OTC by slow freezing. Up to 2011, 12 patients underwent transplantation of the ovarian strips. All 12 patients showed recovery of ovarian endocrine function. Follicular growth was confirmed after a mean time of 19 weeks (8–26 weeks). Two patients experienced successful pregnancies and had live births by assisted reproductive technology (ART). Moreover, one of these patients experienced a second spontaneous pregnancy after giving birth to the first child, which resulted in a second live birth. ⁴⁶

OTC was performed using slow freezing in six patients with leukemia. After a median time of 74.5 months, completion of ChT, and complete remission, patients underwent transplantation. Ovarian endocrine function resumed in all patients after a median time of 3 months. Natural pregnancy occurred in two women, which resulted in one live birth in each. ⁴⁷

In a retrospective study, 30 women underwent OTC by slow freezing and transplantation between 2012 and 2023 due to malignancies. Pre-transplantation assessment revealed no malignant cell contamination in any of the ovarian strips. Of 20 women with POI before transplantation, 18 women achieved ovarian endocrine recovery after about 3 months. Regular menstrual cycles resumed in 10 women who underwent transplantation for reproductive purposes. Ten pregnancies were reported in this study, which resulted in four abortions, two ongoing pregnancies, and four healthy live births. ⁴⁸

Slow freezing was used for OTC in 75 patients with sickle cell disease before hematopoietic stem cell transplantation (HSCT). Five patients asked for transplantation of ovarian tissue. All of these patients achieved recovery of ovarian function. One patient conceived naturally, which resulted in a healthy live birth. ⁴⁹

A retrospective study between 2004 and 2020 in two centers revealed that 72 pediatric patients with a mean age of 9.3 years (0.2–17) had undergone OTC before receiving gonadotoxic treatments due to malignant or non-malignant diseases. For all patients, OTC was performed using slow freezing. One patient who was previously diagnosed with Hodgkin’s disease and received ChT and RT requested transplantation after 14 years; however, she did not experience any pregnancies. Another patient who did not accept transplantation had a live birth using ART. ⁴³

In a cohort of 17 women who were candidates for OTC due to malignancies and autoimmune and gynecological diseases, slow freezing was performed for all of them. The laparoscopic transplantation of the frozen-thawed strips was performed in the sub-peritoneal abdomen (15 patients), pelvic wall (12 patients), and ovarian site (10 patients). The mean follicular diameter after a cycle of stimulation was 14 mm. The proportion of MOs retrieved from three sites was 67%, 59%, and 68% for abdominal, pelvic, and ovarian sites. This cohort resulted in eight live births, one of which was the consequence of natural conception. ⁵⁰

A multicenter study of OTC in 14 Nordic centers revealed that slow freezing was the preferred method in most centers up to 2014. One center started vitrification besides slow freezing in 2009, and since then, almost half of the OTCs have been performed by vitrification. In one center, vitrification was tested between 2009 and 2011; however, slow freezing was still the method of choice in this center after this period. In total, 17 live births and 2 ongoing pregnancies have been reported in 14 centers up to 2014, all resulting from slow freezing. ⁵¹

Wang et al. conducted a retrospective study (2024) on patients with hematological disease who requested for OTC. OTC was performed by slow freezing for all cases. Out of 24 cases, 23 underwent HSCT, and all of them experienced amenorrhea or oligomenorrhea. Ovarian tissue transplantation (OTT) was performed for one patient with aplastic anemia. Six months after transplantation, the menstrual cycle was resumed. ⁵²

Abtahi et al. ⁵³ have reported two cases of POI who underwent OTC by vitrification and transplantation. Transplantation for the first case was performed 5 years after receiving ChT and RT due to invasive colon adenocarcinoma. Monitoring the patient’s hormonal status after transplantation demonstrated that although a transient decrease of FSH from 75.6 to 57.3 mIU/L was observed after 6 months, it suddenly increased after 7 months. The second patient received transplantation 4 years after hysterectomy and bilateral salpingo-oophorectomy due to adenocarcinoma. This patient did not exhibit any decrease in FSH level post-transplantation, which indicates that the ovarian grafts were non-functional. No pregnancies were reported in these two patients. ⁵³

In a study conducted by Zhao et al., ⁵⁴ the proportion of normal primordial follicles after thawing of cryopreserved strips by vitrification or slow freezing did not have a statistically significant difference (p > 0.05). The rate of stromal cell apoptosis was not statistically different (p > 0.05), but in both groups, this rate was higher than in the fresh tissue (p < 0.05). ⁵⁴ In addition, based on a meta-analysis comparing the results of slow freezing and vitrification in OTC, the proportion of primordial follicles with normal morphology was not significantly different in both groups (p = 0.390). ⁴¹

In a trial by Suzuki et al., ⁴⁵ out of 37 patients who underwent ovarian tissue vitrification, follicular growth was observed in 20 patients in histological analysis before transplantation. After transplantation, nine patients had follicular growth. In vitro fertilization (IVF) and embryo transfers were performed for four patients, which resulted in three pregnancies, followed by two live births. This study used EG, poly-vinyl-pyrrolidone, and sucrose as cryoprotectants. ⁴⁵ In a single-center study, 92 patients underwent OTC. For patients referred before and after September 2007, OTC was performed by slow freezing and vitrification, respectively. Thirteen patients returned for transplantation, of which vitrification was performed for 4. Recovery of ovarian endocrine function was observed in all of the patients 4–5 months after transplantation, which was demonstrated by regular menstruation and normal serum levels of FSH and anti-Müllerian hormone (AMH). Seven and two patients underwent slow freezing and vitrification, respectively, and experienced natural conceiving, giving birth to at least one child. Slow freezing and vitrification generally resulted in 11 and 2 live births. ⁴⁴

In a case report by Zhang et al., ⁵⁵ vitrification was performed for OTC in a 22-year-old woman diagnosed with aplastic anemia before HSCT. Histologic assessment after vitrification exhibited good follicular viability with a 20/2 × 2 mm² density. Four metaphase II oocytes were developed using in vitro maturation techniques and cryopreserved. ⁵⁵

Transplantation of vitrified, warmed ovarian tissue after ChT in a case of breast cancer resulted in restoring menstrual cycles after 2 months. After 3 months, a clinical pregnancy occurred, which resulted in spontaneous abortion. A second natural pregnancy occurred 6 months after transplantation, which resulted in a live birth. ⁵⁶

Anbari et al. ⁵⁷ report a patient diagnosed with leukemia who underwent OTC by vitrification before initiation of ChT. After achieving a complete response, the frozen-thawed tissue was transplanted orthotopically. The patient’s menstrual cycle and hormonal status were recovered successfully. No pregnancy was reported. ⁵⁷

Table 1 provides an overview of the clinical outcomes of OTC and transplantation, which is reviewed here.

Table 1.

An overview of the characteristics and clinical outcomes of OTC and OTT.

Author/year	Country	OTC method	Number of patients	Number of OTT	Outcomes
Diaz-Garcia/2018 31	Spain	Slow freezing	44	44	- Pregnancy in 3 women (spontaneous or using ART) - 4 live births
Sönmezer/2024 47	Turkey	Slow freezing	6	6	- Recovery of ovarian endocrine function in all after median time of 3 m - Natural pregnancy in 2 women  ✓ 2 live births from 2 women
Fabbri/2024 48	Italy	Slow freezing	30	30	- Recovery of ovarian endocrine function in 18/20 patients with POI after median time of 3 m - 10 pregnancies  ✓ 4 abortions  ✓ 4 live births  ✓ 2 ongoing
Missontsa/2024 49	France	Slow freezing	75	5	- Recovery of ovarian endocrine function in all - Natural pregnancy in 1 woman  ✓ 1 live birth
Grellet-Grün/2023 43	France	Slow freezing	72	1	- No pregnancies after transplantation - Pregnancy by ART in one patient without transplantation  ✓ 1 live birth
Gook/2021 50	Australia	Slow freezing	17	15	- Mean follicular diameter after stimulation = 14 mm - Mature oocytes retrieved from abdominal, pelvic, ovarian sites = 67%, 59%, 68% - 1 natural pregnancy  ✓ 1 live birth - 7 pregnancies by ART  ✓ 7 live births
Rodriguez-Wallberg/2016 51	Nordic countries	Slow freezing in 13 centers (vitrification was tested for 2 years beside slow freezing in one center), slow freezing +	1588	46	- 17 live births - 2 ongoing pregnancies
		Vitrification in one center		0
Wang/2024 52	China	Slow freezing	24	1	Recovery of ovarian endocrine function
Abtahi/2023 53	Iran	Vitrification	2	2	- No recovery of endocrine functions - No pregnancies
Suzuki/2015 45	Japan	Vitrification	37	20	- Follicular growth in 9/20 - 3 pregnancies by ART  ✓ 2 live births  ✓ 1 abortion
Silber/2018 44	United States	Before 2007: slow freezing	92	9	- Recovery of ovarian endocrine function - 11 natural pregnancies in 7 women  ✓ 11 live births
		After 2007: vitrification		4	- Recovery of ovarian endocrine function - 2 natural pregnancies in 2 women  ✓ 2 live births
Sänger/2024 56	Germany	Vitrification	1	1	- Recovery of ovarian endocrine function - 2 natural pregnancies  ✓ 1 live birth  ✓ 1 abortion
Anbari/2024 57	Iran	Vitrification	1	1	- Recovery of ovarian endocrine function - No pregnancies

ART, assisted reproductive technology; OTC, ovarian tissue cryopreservation; OTT, ovarian tissue transplantation; POI, premature ovarian insufficiency.

Transplantation of cryopreserved ovarian tissue

When POI is confirmed and the patient has an appropriate medical condition, it is important to safely transplant the ovarian strips and ensure the survival and functionality of the transplanted strips. ⁵⁸ Two main surgical procedures of transplantation include orthotopic transplantation (OT) and heterotopic transplantation (HT).^18,20

It is suggested that less than a third to half of the cryopreserved strips be transplanted because, in some cases, further transplantations may be required due to failure or exhaustion of the transplanted strips. ⁵⁹

There is no standard protocol for tissue processing size of ovarian tissue for transplantation. ⁶⁰ Cutting the tissue into smaller pieces is associated with a faster restoration of endocrine function, ⁶¹ and a lower risk of ischemic injuries. ⁶² Diaz et al. ⁶⁰ have conducted a systematic review of the effect of graft size on transplantation outcomes. They categorized the tissue sizes as square, strips, and fragments, from more significant to smaller. It was observed that an earlier recovery of ovarian function accompanied fragment transplantation, although the difference was insignificant. Smaller sample size was associated with a higher pregnancy rate. Regarding the small sample size in this systematic review and the lack of essential information for analysis, these results do not provide robust evidence to be adopted in clinical practice. ⁶⁰

Orthotopic transplantation

OT refers to laparoscopic or mini-laparotomy transplantation of the ovarian tissue in the pelvic cavity close to the original site of the remaining ovaries, a peritoneal window, or in the uterus. The peritoneal window is created by an incision in the broad ligament beneath the ovary.^63,64 The obstetrical outcomes are not significantly different between these sites. ⁵⁹

OT provides a favorable environment for follicle development, including paracrine factors, blood supply, and temperature. This procedure allows the woman to recover endocrine function and experience natural conception or choose IVF. ⁶³

The first live birth following transplantation of cryopreserved ovarian tissue was reported in 2004. In this case, ovarian strips were cryopreserved for about 6 years. Almost 11 months after OT, intrauterine pregnancy was demonstrated, which resulted in a live birth. ⁶⁵ However, it is unclear whether this pregnancy resulted from ovulation from the remaining ovary or the graft. ⁶⁶

In a multicenter study between 2004 and 2018, 60 patients underwent OTs, which resulted in recovery of endocrine function in 90.4% of the patients, and the median FSH decreased from 68to 19 IU/L post-transplant. In total, 50 pregnancies and 44 live births have been reported in this cohort, and 50% of the patients have experienced at least one pregnancy. ²⁸ In a review of five European centers up to October 2020, in 97.5% of the cases, the ovarian tissue was transplanted in an orthotopic site. In this study, 40% of the women who wished for natural conceiving and 36% of the women who underwent IVF experienced conception. Finally, 30% of naturally conceiving pregnancies and 21% of IVF pregnancies ended up with live births. All of the live births in this study resulted from OT, and no pregnancies took place by HT. ⁵⁹

Heterotopic transplantation

HT includes the transplantation of tissue outside the peritoneal and pelvic cavity, in the subcutaneous area and peritoneal lining. ¹³ Despite the occurrence of follicular development in different heterotopic sites, the development of dominant follicles is uncommon in subcutaneous transplantation; the follicles undergo atresia before reaching the appropriate size for ovulation, making retrieval of MOs difficult. The peritoneal lining is the only favorable heterotopic site that supports follicular growth to achieve a standard diameter for ovulation.^67,68 The peritoneal pocket in HT is usually created in the parietal peritoneum beneath the abdominal wall.^67,69

Oktay et al. ⁷⁰ reported the first embryo development using oocytes retrieved from HT of ovarian tissue. In this case report of a patient diagnosed with breast cancer, frozen-thawed ovarian strips were transplanted subcutaneously beneath abdominal skin after completion of ChT. The ovarian endocrine function was recovered after 11 days. Following the ovarian stimulation cycles and oocyte retrieval, IVF was performed, and ultimately, one of the fertilized oocytes developed into a four-cell embryo, which was transferred to the patient. This article did not report the outcome of this pregnancy. ⁷⁰

Wølner-Hanssen et al. ⁷¹ reported HT of cryopreserved ovarian tissue to the forearm of a woman 4.5 years after HSCT. The graft survived for 7 months, and during this period, two follicles with maximum sizes of 12.6 mm (after 18 weeks) and 6.7 mm (after 30 weeks) were developed. The estradiol level decreased from 6861 pmol/L 1 week after transplantation to <70 pmol/L after 7 months. ⁷¹ Although HT does not appropriately support follicular development, induction of ovulation in the remaining ovary due to the endocrine function of the transplanted ovarian tissue has been reported in a woman who underwent HT, which resulted in natural conceiving and three live births.^13,72 The first pregnancy resulted from the follicular development of HT ovarian tissue in a woman after bilateral oophorectomy was reported by Stern et al. ⁷³ in a 32-year-old woman. The ovarian strips were transplanted in the anterior abdominal and lateral pelvic walls. After 4.5 months, an FSH level of 11.6 IU/L and an E2 level of 219 pmol/L demonstrated the endocrine function of the ovarian tissue. A second transplantation was performed for the patient in the anterior abdominal wall 2 years later. After 7 months, two follicles with 16 and 18 mm diameters were developed in the right anterior abdominal wall following a stimulation cycle with FSH, Luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH) antagonists. Two oocytes were retrieved from the grafts and fertilized, and subsequently, two embryos were transferred, which resulted in a twin pregnancy and led to the delivery of two healthy babies.^73,74 A cohort of 17 patients who underwent OTC and transplantation was assessed to compare the outcomes of HT to the sub-peritoneal abdomen (15 patients) with OT to the ovary (10 patients) and peritoneal pelvic area (12 patients). Analysis of the follicle aspiration after a mild stimulation cycle demonstrated that the mean follicular diameter was similar in all three sites (~14 mm). Moreover, the proportion of MOs did not have significant differences. The fertilization rates of the retrieved oocytes from these three sites did not show significant differences (90%, 76%, and 96% in abdominal, ovarian, and pelvic sites, respectively). Eight live births have been reported in this cohort, of which three babies were from the abdominal site, three from the ovarian site, one from the pelvic site, and one from a mixed transfer of abdominal and ovarian sites. ⁵⁰

Generally, recovery of the ovaries’ endocrine function, required time for graft function, and graft life span do not differ between HT and OT. However, HT is associated with a lower fertilization rate in the retrieved oocytes and embryo generation, leading to a higher chance of embryonic arrest. ¹³

Table 2 summarizes the studies on clinical outcomes of OTT.

Table 2.

Summary of the studies on clinical outcomes of ovarian tissue transplantation.

Author/year	Technique of transplantation	Number of patients	Outcomes
Oktay/2004 67	OT	1	- Recovery of endocrine function - Spontaneous pregnancy after 11 months
Shapira/2020 28	OT	60	- Recovery of endocrine function in 90.4% - 50 pregnancies - 44 live births - 50% experienced at least one pregnancy
Dolmans/2021 65	OT	277	- Pregnancy in 106 women ✓ 67 natural pregnancies ✓ 95 live births
	HT	5	No pregnancies
	OT + HT	3	No pregnancies
Oktay/2004 71	HT	1	- Recovery of endocrine function after 11 days - 1 pregnancy using ART  ✓ Not reported the outcomes
Wølner-Hanssen/2005 72	HT	1	- Recovery of endocrine function after 1 week - Development of dominant follicles
Oktay/2024 13	HT	6	- Recovery of endocrine function in all patients - 4 spontaneous pregnancies in 1 patient  ✓ 3 live births
Stern/201375,76	HT	1	- Recovery of endocrine function after 4.5 months - Development of dominant follicles - 1 pregnancy using ART  ✓ 2 live birth
Gook/2021 50	HT	17	- Recovery of endocrine function in all patients - 10 pregnancies using ART  ✓ 8 live births

ART, assisted reproductive technology; HT, heterotopic transplantation; OT, orthotopic transplantation.

The life span of transplanted cryopreserved ovarian tissue

The life span of the transplanted strips in heterotopic sites varies between 9 months and 3 years in different studies. ⁶⁷ The median graft survival after OT was observed to be around 5 years. This survival can increase to 11 years after a second transplantation. ⁷⁵ In a long-term follow-up of five patients who underwent OTC and transplantation between 2001 and 2010, the function of transplanted ovarian tissue was evaluated by measuring endocrine function with hormonal status and ultrasonography. The endocrine function was recovered for 12–20 weeks after transplantation. The endocrine function in four patients who underwent a second transplantation lasted longer, between 9 and 84 months. In one patient, the ovaries remained functional for over 7 years. ⁷⁶

Different factors, such as patients’ age and stage of pubescence, ovarian function, and follicle density before OTC, freezing and thawing methods, the transplantation procedure, and injuries during these processes, are essential factors that influence the life span of the graft.^67,76

Ischemic injuries

Ischemia is the leading cause of follicular damage in OTC. It has been demonstrated that tissue perfusion and re-oxygenation initiate 5 days post-transplantation. ⁷⁷ The age of the patient, ChT before ovarian tissue retrieval, OTC process, and transplantation procedure can affect this time. ⁷⁷ Thus, it is important to reduce ischemic injuries to prevent the loss of follicles. Using FSH as a gonadotropin factor during vitrification has improved vascularization and graft survival. ⁷⁸ Anti-apoptotic factors, such as Kit ligand, basic fibroblast growth factors (GFs), c-Kit, and insulin-like GF I and II, could also reduce follicular loss.^{79 –81} Preparation of a vascular bed before grafting, administration of melatonin for the patient, and a shorter interval between oophorectomy and grafting are possible ways to enhance revascularization.^77,82 Incubation of the graft with hyaluronic acid, VEGF, and vitamin E has been shown to improve follicular survival.^77,83 It has been recommended that a two-step transplantation is a suggested technique to optimize neovascularization in which a few ovarian strips are transplanted in the first step, and local inflammation is triggered to enhance neo-angiogenesis. In the second step, the remaining strips are transplanted. ⁸⁴ In another study, a scaffold made of a human extracellular matrix was used to improve revascularization. ⁸⁵ Using platelet-derived factors encapsulated fibrin hydrogel in animal studies has enhanced the neovascularization of ovarian tissue. ⁸⁶

Reperfusion injuries

Reperfusion is another primary mechanism of follicular loss following ischemia, which is caused by the production of high levels of reactive oxygen species (ROS) during the ischemic period that occurs after initiating tissue perfusion. ROS is responsible for deteriorating endothelial function, oxidative damage of the cells, and, finally, follicular loss. ⁸⁷ It is demonstrated that remote-ischemic preconditioning (R-IPC) has promising results in kidney and liver transplantations by modifying the expression of inflammatory genes and VEGF.^88,89 In addition, better function of ovarian tissue was observed using R-IPC compared to the control group, confirming the efficacy of R-IPC in reducing follicular loss after transplantation. ⁹⁰ Treating the ovarian tissue with antioxidant agents such as N-acetylcysteine, ⁹¹ Mitoquinone, ⁹² erythropoietin, ⁹³ and melatonin ⁹⁴ was accompanied by promising results in preclinical studies.

Cryoinjuries

Changes in the temperature during freezing and thawing procedures lead to various thermal, mechanical, and chemical injuries that mainly occur in temperatures between 0°C and 15°C. ⁹⁵ One of the manifestations of cryoinjuries during the freezing and thawing process is the detachment of follicular cells from the basement membrane leading to follicular loss; because the basement membrane provides a supporting structure and necessary GFs for the follicular cells. ⁹⁶ On the other hand, the integrity of the stroma deteriorates during the freezing process, which results in decreased follicular density. ⁹⁶ The formation of ice crystals, mainly during slow freezing, is related to physical damage to the cells. Vitrification results in a lower degree of cryoinjuries than slow freezing. However, follicular density in both methods is lower than in the non-cryopreserved tissue.^19,96 Antifreeze proteins, modified cryoprotectants, an open freezing system, and decreased transportation time are suggested to reduce cryoinjuries effectively. ⁹⁷

Complications of OTC and OTT

Generally, it is suggested that OTC and transplantation are safe both in prepubertal and postpubertal women, and most of the patients do not experience any adverse events. ⁹⁸ In any case, every surgical procedure to remove and transplant the ovarian tissue may result in surgical and anesthetic complications. The risk of damage to the vascular, urological, and gastrointestinal systems is about 0.1% during every laparoscopy. ⁹⁹ Surgical site infection, postoperative hemorrhage, and intraoperative blood loss are rare and have an incidence of <1%; however, this risk should be considered. ⁹⁸

In patients with malignant disease, the risk of reseeding harbored malignant cells in cryopreserved ovarian tissue should be considered.^9,47,59 Transplantation of any cryopreserved ovarian tissue with minimal residual disease is accompanied by the risk of relapse. Ovarian tissue should be evaluated for contamination with malignant cells before transplantation by pathologic and molecular techniques. ¹⁰⁰ If the tissue is positive for malignant cells, using ART to avoid reintroducing cancer cells or ex vivo eliminating the cancer cells by photodynamic therapy of cryopreserved ovarian tissue should be considered to decrease the risk of relapse.^101,102 Making an artificial ovary without malignant cell contamination is a safe alternative in such patients. ¹⁰³ In patients diagnosed with leukemias, harvesting the ovarian tissue after remission induction with non-gonadotoxic ChT may eliminate the risk of contamination.^47,104

Ethical concerns

OTC in healthy post-menopausal women

Using OTC to restore the endocrine function of ovaries in healthy post-menopausal women is accompanied by critical ethical issues. The safety of OTC in post-menopausal women is not appropriately evaluated. Exposure to high levels of endogenous estrogen after OTT results in a higher risk of breast cancer. Moreover, the superiority of OTC to hormone replacement therapy has not been studied yet. ¹⁰⁵ In addition, OTC and transplantation are associated with a prolonged reproductive life span of the patient, which may lead to spontaneous high-risk pregnancies. ¹⁰⁵ In any case, it is essential to perform patient selection carefully and based on the accepted Edinburgh selection criteria to identify those who benefit from OTC to prevent introducing unnecessary harm to the patient.^106,107

Prepubertal patients

OTC in prepubertal girls is still experimental, and long-term efficacy and safety should be evaluated carefully. Prepubertal girls probably will need FP due to their reduced ovarian reserve. ¹⁰⁸ Ernst et al. ¹⁰⁹ reported the first case of puberty induction by OTC and transplantation. A 13-year-old girl diagnosed with Ewing’s sarcoma presented with RT and ChT-induced POI. After the auto-transplantation of cryopreserved ovarian tissue, she achieved Tanner stage 4, and her menstruation was initiated. ¹⁰⁹

The first human live birth following transplantation of prepubertal cryopreserved ovarian tissue was reported in 2015 in a case of sickle cell anemia presented with POI 10 years after HSCT and myeloablative conditioning regimen. She underwent OT at the age of 24, and after 4 months, the endocrine function was recovered. About 2 years later, natural conceiving occurred, leading to live birth. ¹¹⁰

Regarding the higher follicular density of prepubertal ovaries, transplantation of prepubertal ovarian tissue may result in a more prolonged graft survival. ¹¹¹ However, the life span of the tissue is limited, and hormone replacement therapy is inevitable. Moreover, the hypothalamic-pituitary-ovary axis is impaired, and the patient experiences a rapid increase in estrogen levels; thus, the patient is at risk of precocious puberty. ¹¹¹

Decision-making about OTC in prepubertal patients is a complex process that affects the child’s future reproduction system. ¹¹² Health professionals should provide information about FP options for each patient and assist the patients with decision-making. ¹¹³ The challenges of assent in prepubertal girls also influence the decision on posthumous use of the tissue in cases where the child does not survive. Regarding the challenges of obtaining consent in such cases, the parent’s decision on posthumous reproduction should be evaluated based on the deceased’s antemortem interests and their community’s values. ¹¹⁴

Research gaps in this field

Most of the clinical studies focus on slow freezing, and data about the efficacy of vitrification are limited. More robust clinical evidence and long-term follow-up are required for its applicability in clinical practice. ³⁶ On the other hand, there is no optimal combination of cryoprotectants for the vitrification of human ovarian tissue to achieve the best follicular integrity. ³⁴

There is no consensus on the protocol of tissue processing and graft size. ⁶⁰ An established protocol for the size of the retrieved and transplanted ovarian strips is required to ensure the success rate of transplantation and minimize the risk of ischemic injuries. ⁶⁰ In addition, evidence indicating the efficacy of rapamycin and AMH in retaining the follicular reserve in human ovarian tissue is restricted to preclinical studies.^115,116 Therefore, clinical studies are required to demonstrate the efficacy and safety of these agents. Furthermore, it is essential to explore how these interventions influence the long-term survival of follicles. Moreover, clinical studies are needed to provide more robust evidence demonstrating the efficacy and safety of experimental agents such as hyaluronic acid, VEGF, vitamin E, and Mitoquinone in enhancing follicular survival.^77,83,92

Ethical issues regarding the decision-making for OTC in prepubertal girls, the impact on their reproductive future, and their long-term safety are still a matter of concern. ¹¹³ A dedicated, ethical analysis of these challenges is required to warrant the reproductive autonomy of prepubertal girls.

Limitations of this review

Most published studies on clinical outcomes of OTC are case reports, observational studies, or cohort studies. Large randomized clinical trials are needed for more reliable results and increased clinical applicability.^117,118 Moreover, since most studies are case reports/series, reports with successful outcomes likely have a higher chance of publication, and limited data on unsuccessful grafts results in publication bias. ¹¹⁸ However, an inspection of the funnel plot and the Peters test in a systematic review and meta-analysis of obstetric outcomes following OTC by Finkelstein et al. indicated no publication bias. ¹¹⁸ In addition, there is high heterogeneity in patient selection, cryopreservation protocol and tissue treatments, transplantation techniques, and documentation of the outcomes. ⁶⁰

Conclusion

OCT is a valuable method that does not require ovarian stimulation or fertilization and can be used for prepubescent girls or those whose ChT/radiation has already begun. It restores endocrine function, reducing the need for hormone replacement. OTC is also effective for resuming puberty in individuals treated before puberty. This review highlights the progress in OTC methods and the clinical outcomes, including slow freezing and vitrification. Although most live births result from slow freezing, vitrification is a promising alternative that does not require much time and money.

Clinical outcomes of OTC, especially OT, demonstrate its efficacy in recovering the endocrine function of the ovaries and reproduction. Although few pregnancies and live births have been reported using HT, which occurred using ART, recent studies suggest that the sub-peritoneal abdomen is a more favorable heterotopic site for follicular development, which may increase the chance of oocyte retrieval for ART purposes.

Advances in cryopreservation and transplantation techniques, as well as strategies to decrease ischemic injuries, reperfusion damages, and cryoinjuries, will further improve the efficacy of OTC. Integrating OTC into clinical practice is a promising perspective for reproductive medicine. Future studies should be conducted to optimize the OTC protocols and reduce tissue injuries during processing and transplantation. Analyzing the ethical issues especially in prepubertal girls is also a matter of concern, which helps in a better clinical applicability.