Over the last two decades, advances in reproductive technology (ART), such as in vitro fertilization, and cryopreservation technology have given rise to newer areas of reproductive tissue banking such as the freezing of sperm, eggs, embryos, testicular tissue, strips of intact ovarian tissue, or even entire ovary [1]. Cryopreservation of sperm, oocytes, and embryos is now routine in infertility management, while the other above-mentioned techniques are still in their early stages of development.

Though cryopreservation is routine in managing chemo or radiation-therapy-induced infertility and specific non-cancer indications (e.g., advanced female age), a new need has more recently emerged: patients who plan medical gender transition. This transition process commonly includes surgical removal of reproductive organs as well as hormone therapy, with resulting infertility. Recent studies have shown that many transgender patients desire to have children, and the success of cryopreservation in other circumstances suggests that it is also a viable option for preserving fertility in transgender patients.

Although fertility preservation presents an exciting opportunity for transgender patients, there are potential problems. Lack of awareness is one major issue. Recommendations exist from organizations such as the Endocrine Society to counsel transgender people about the possibility of preserving fertility prior to transition, but this counseling is often inconsistently performed ^[2][3][2,3]. This mirrors past experience with cancer patients, where counseling about fertility preservation has historically been underutilized [4]. In addition, sociological and psychological factors play a role, with social determinants of health having a profound impact on perceptions of parenthood ^[5][6][7][5,6,7]. Should the above be overcome, economic barriers still remain, such as the expense of the preservation procedure often deemed “elective” and storage fees for preserved material, which are often not covered by insurance in the United States.

2. Fertility in Transgender Patients

Patients undergoing medical transition have a high rate of fertility loss due to their treatments during the transition. The most common gender transition therapies among male-to-female (‘male at birth’) individuals include feminizing estrogen hormone therapy, breast enhancement, chondrolaryngoplasty reduction, penectomy, orchiectomy, and vaginoplasty. For transgender men (‘female at birth’), treatments include masculinizing testosterone hormone therapy, bilateral mastectomy, and hysterectomy/oophorectomy. While surgical procedures such as hysterectomy/oophorectomy or orchiectomy obviously preclude conception due to loss of gamete production, pharmacological treatments such as cross-sex hormone therapy also have an impact. For patients on estrogen therapy who do not undergo orchiectomy, estrogen may have permanent deleterious effects on sperm quality [8]. For patients on testosterone therapy without hysterectomy/oophorectomy, there does not appear to be a permanent impact on fertility; indeed, many patients who are actively on testosterone can still become pregnant. However, testosterone is a known teratogen and cannot be continued during pregnancy [9]. Many transgender individuals are of reproductive age at the time of gender transition. While parenthood is generally not an immediate priority at this time, studies have indicated a strong desire among transgender persons to be parents at some point in their lives. A 2012 Dutch study found more than 54% of transgender women who had undergone surgical transition expressed a current or previous desire to procreate [10]. This would entail possibly delaying hormone therapy to allow sperm cryopreservation before transition, or utilizing donor sperm banks. However, another study in 2012 found that 37.5% of transgender men reported that they would have considered freezing reproductive tissue had the option been available, and 54% of them expressed a desire for children. In the US, a 2016 study of transgender men in San Francisco found that 15% reported a desire to become pregnant [11].

3. Cryobiology of Gonadal Tissue Freezing

Thanks to advances in ART, the loss of fertility consequent to gender reassignment therapies need not be inevitable. Rigorous pre-procedure counseling followed by preservation of reproductive cells or tissue is a clinically viable alternative. The cryopreservation of cells and biological tissue is a process that entails brief exposure to high molar concentrations of a mixture of a variety of cryoprotectant agents (glycerol, propanediol, dimethyl sulfoxide, or sucrose), which leads to complete dehydration of the cell contents to prevent ice crystal formation that may damage the cells, followed by vitrification of specimens at extremely low temperatures (−196 °C) in liquid nitrogen. In vitrification, a solution transitions from a liquid state to a vitreous state at low temperatures, circumventing the formation of ice crystals to prevent tissue damage—a significant advantage over previously-common slow freezing. Cryopreservation is an effective way to preserve fertility and the use of preserved tissue frequently results in a successful pregnancy ^[12][13][12,13]. However, the success of the preservation process depends greatly on the quality of the material to be preserved. There is a narrow window of only a few years to undergo preservation of reproductive tissue, especially for eggs, embryos, or ovarian tissue. The older the patient, the harder it becomes to achieve current or future parenthood even through highly successful ART [14].

3.1. Sperm Freezing

Frozen sperm obtained via masturbation or through testicular or epididymal needle aspiration has been successfully utilized for procreative purposes for decades [15]. While the characteristics of transgender women’s semen specimens may not be optimal [16], there is evidence that such samples can, nevertheless, be used to initiate conception ^[17][18][17,18]. Transgender individuals face unique challenges with sperm quality: transgender women taking hormone therapy have been observed to have significantly altered sperm parameters, which may persist for months after treatment discontinuation [19]. Even individuals not taking hormones may experience difficulty: genital tucking and/or wearing tight undergarments is common among transgender women, and these practices negatively impact sperm quality due to increased heat stress and greater production of reactive oxygen species ^[20][21][20,21]. The sperm abnormalities in these individuals range from low count, low motility, and low normal forms, to azoospermia, in which case fine needle aspiration from the epididymis or the testis may be necessary [22]. Cryopreservation of sperm inevitably leads to a reduction in the usable quality and quantity of sperm at the time of thawing. While this scenario is not any different from that of non-transgender fertility patients, it may nevertheless constitute a major setback to many transgender patients; suboptimal samples inevitably require the use of expensive assisted reproductive techniques such as in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (ICSI). These techniques, collectively known as ART, tend to be quite expensive and insurance usually does not cover them. Only an insignificant minority is likely to conceive without ART, using less expensive methods such as intrauterine sperm insemination. Even cryopreserved sperm from optimal samples show significant increases in abnormal morphology and decreases in motility after thawing; current research focuses on altering the cryoprotectant medium to prevent damage [23]. Currently, limited information is available regarding pregnancy rates using cryopreserved sperm collected from transgender patients prior to transition. However, other patient populations have long had success with the use of cryopreserved sperm, with fertilization rates not significantly different from those of fresh sperm even for patients with suboptimal semen samples; e.g., patients with azoospermia ^[24][25][26][24,25,26]. As for prepubertal patients, the preservation and use of testis tissue from such patients is not yet feasible. The preservation of fertility in prepuberty is also of particular relevance to childhood cancer patients, and this is a topic of research [27]. Currently, no standardized protocols exist for the preservation of this tissue, and techniques vary with regard to cryoprotectant medium and freezing rate [28]. Protocols for the preservation of mature testicular tissue are not applicable; these focus on preserving spermatozoa, with no consideration for preserving spermatogonial stem cells, which are needed for spermatogenesis [28]. The best way to utilize such tissue, whether by grafting or in vitro spermatogenesis/maturation (IVM), is not known. Of these two techniques, tissue grafting has shown success in non-human animals, including primates, although ICSI was still required for fertilization [28]. IVM has been successful with rodent tissue, but in humans, it has only reached the spermatid stage [28]. As of 2022, some fertility centers have elected to cryopreserve testicular tissue from prepubertal patients in anticipation of technological advancements that would facilitate its use ^[29][30][29,30]. However, to our kno wledge, no live births have yet occurred using cryopreserved tissue harvested from prepubertal individuals [28].

3.2. Oocyte and Ovarian Tissue Freezing

Embryo-freezing technologies have existed for more than three decades, while technologies for cryopreservation of oocytes have advanced dramatically in the last decade after the switch from slow-freezing to vitrification as the preferred cryopreservation method. Oocyte cryopreservation has now become part of standard medical practice [31]. When desired, the oocytes can be thawed and fertilized with the sperm from the patient’s partner or a sperm donor. Current data show that the pregnancy rates achieved after uterine transfer of embryos derived from cryopreserved oocytes have paralleled the success rates achieved with the use of fresh non-cryopreserved oocytes and embryos [32] and the overall projected success rates based on extrapolations of current data are quite high [33]. However, some studies have shown that although pregnancy rates may be similar, the successful creation of an embryo is likely to require more thawed oocytes than fresh oocytes, and the number of oocytes required for successful outcomes increases with maternal age ^[34][35][34,35]. Among transgender patients, the use of cryopreserved oocytes as opposed to embryos is similarly novel. In 2013, in the first report of its kind among transgender people, a biological female, self-identified as male, underwent an oocyte cryopreservation procedure prior to undergoing gender transition [36]. Since then, this practice has become more common, but overall rates of oocyte preservation among transgender men remain low. ^[37][38][39][37,38,39]. Since testosterone generally induces an anovulatory state in biological females, it has been hypothesized that transgender men may experience issues with oocyte harvesting for cryopreservation. However, a 2023 study compared results of oocyte vitrification in testosterone-naïve patients vs. patients who had previously used testosterone, but had ceased treatment at least three months prior to oocyte harvesting, and found no difference in outcomes between the patient groups [37]. While the use of cryopreserved oocytes in transgender patients specifically remains low enough that overall rates of successful pregnancy cannot be extrapolated, the delivery of normal healthy infants has been reported in multiple cases ^[17][39][17,39]. This mirrors the experience of non-transgender patients, where the use of cryopreserved oocytes is also quite low, with the majority of patients not returning to use their oocytes; indeed, among patients who cryopreserve oocytes in anticipation of iatrogenic infertility (e.g., gonadotoxic chemotherapy), the utilization rate is under 10% [34]. Ovarian tissue cryobanking has only recently begun the move from an experimental procedure into clinical practice. Clinical and laboratory protocols for optimization of various steps involved in ovarian tissue cryobanking, i.e., obtaining ideal grafts, freeze-thaw protocols, transplantation, and successful regrafting, are the focus of vigorous research at fertility clinics worldwide [31]. Ovarian tissue freezing often entails the excision and freezing of several strips of ovarian cortical tissues [40]. The basis for this concept lies in the preservation of tissue in cancer patients undergoing treatment. The cortical strips can be re-transplanted on the same ovary when the patient is ready to resume reproductive processes [41]. Alternatively, the cortical strips can also be implanted heterotopically in the patient’s forearm [42]. Globally, the rates of successful live birth using preserved ovarian tissues are about 33% [43]. A comprehensive analysis of patients implanted with previously cryopreserved ovarian tissue at reproductive medicine centers throughout Germany, Austria, and Switzerland from 2007–2020 found ovarian tissue transplantation to yield pregnancy and live-birth rates of 32.7% and 26.5%, respectively, though these rates varied depending on the size and experience of the medical center [44]. Cryopreservation of ovarian cortical strips (or, potentially, the whole ovary) opens new opportunities. It is the only fertility preservation option suitable for prepubertal patients, as ovarian stimulation, oocyte harvesting, and cryopreservation are not feasible to perform in this age group [45]. It also has the advantage of being faster for the patient; there is no need for patients to potentially undergo multiple cycles of ovarian stimulation and egg retrieval [46]. This may be particularly useful for prepubertal transgender patients or patients concerned about delaying hormonal therapy, as it allows for fertility preservation without requiring the production of mature oocytes. However, pregnancy using these tissues requires discontinuation of testosterone therapy and may not be well-tolerated. This method is not without drawbacks; post-harvest graft ischemia, resumption of hormonal function and follicular development after transplantation, and in vitro maturation of oocytes continue to pose challenges [47]. Furthermore, there currently is some question as to whether vitrification (now the preferred cryopreservation method for sperm, mature oocytes, and embryos) [48] is truly superior to slow-freezing for ovarian tissue. Vitrification involves higher concentrations of cryoprotectants than slow-freezing, which can be toxic to cells and primordial follicles [49]. A 2021 meta-analysis found vitrification to preliminarily be superior, but noted that high variability in protocols and a lack of data on clinical outcomes suggest that further research is required before a definitive conclusion can be drawn [49]. As for immature oocytes, in a case report from 2020, physicians in Tel Aviv reported successfully harvesting and cryopreserving immature oocytes from a prepubertal girl with mosaic Turner syndrome [50], but cryopreserving oocytes from prepubertal patients is not yet common practice, and to date, there are no records of any pregnancies using oocytes harvested from such patients. These areas are the focus of intense investigation [51].