The current options available for fertility preservation in transgender men are as follows:

• Oocyte/Embryo cryopreservation;
• Ovarian tissue cryopreservation (OTC) and in vitro maturation (IVM).

Oocyte or embryo cryopreservation is an established technique that can be performed before or after HT [8,9,10]. However, there is no unanimous opinion on the actual effects of androgenic therapy on fertility outcomes. The literature in this regard is not yet sufficient and clear. Recently, Israeli et al. retrospectively compared embryos from 7 testosterone-treated transgender men with a control group of 34 cisgender women, 10 of whom underwent embryo cryopreservation, and 24 of whom underwent fertility treatment. They found no significant differences between the transgender men and the cisgender women who preserved fertility in terms of the number of cryopreserved embryos or the distribution of embryo age at cryopreservation, while they found a significantly higher mean number of oocytes retrieved in the transgender group compared to the cisgender women fertility treatment group [11]. However, the follow should be noted: the heterogeneity of the two groups, the small sample size, the study design, and the younger age of the transgender men patient group. There is no clear consensus on the timing of controlled ovarian stimulation (COS) after the suspension of HT as well [8]. Steinle et al. suggest that HT should be discontinued for at least three months before starting COS, in order to return to more physiological hormonal levels, and to minimize the risk of ovarian hyperstimulation syndrome (OHSS) [12]. Other studies, however, report that the resumption of menses after the cessation of HT could be expected to occur after at least 6 months of discontinuation [13]. Interestingly, a recent case report involving two transgender men without suspension of GAT showed a total number of 39 metaphase II (MII) oocytes retrieved in the two patients [14]. Further studies with larger sample sizes and better designs are needed in order to upgrade the treatment protocols for AFAB patients. After COS, the oocytes obtained can be cryopreserved for future use, or fertilized with donor sperm or by a cisgender sperm partner, depending on the male or female partner of the patient. Similarly, the embryos eventually collected can be cryopreserved, or freshly transferred into a gestational carrier, in the uterus of the individual if not removed surgically, or in the uterus of the female partner, depending on the sex of the partner. Ovarian or embryo cryopreservation are standardized treatments widely used in the field of Assisted Reproductive Technology (ART) and, thus, currently represent the treatments of choice for FP in transgender patients [9,10,15]. Despite this, both techniques could cause discomfort to the subjects, mainly due to the following side effects: the need to suspend HT in order to start COS; the process of COS itself can bring both physical changes and psychological distress; and frequent vaginal ultrasound monitoring may not be easy to bear for these patients [16]. All these factors can negatively impact the FP journey, increasing the chances of FP rejection and/or drop out.

Ovarian tissue cryopreservation and in vitro maturation are two developing techniques for FP in transgender patients that are not as broadly spread among practitioners as oocyte or embryo cryopreservation. Nevertheless, these two methods of treatment could bring some benefits to transmen patients and, therefore, open new scenarios in the FP protocol.

OTC consists of surgical excision of part of the ovarian tissue or the whole organ and its cryopreservation for a future use. It is generally performed in laparoscopy under general anesthesia. The tissue picked up can be subsequently thawed, reimplanted into the patient’s body, and eventually stimulated to mature and produce viable oocytes at a later time. The possible advantages of this technique are represented by the possibility of not stopping the hormonal therapy for the patients; it could be performed at the same time as the GAS. Additionally, the eventual ovarian stimulation could be postponed until a later or further usage [15]. Furthermore, it constitutes the only viable option for FP in patients of prepuberal age. As a transplantation site, the orthotopic should be referred, rather than the heterotopic, for the higher success rate of the procedure [17]. Regarding the cryopreservation method, slow freezing is considered the standard method for human ovarian tissue, [10] but in recent times a number of studies have demonstrated positive outcomes through the utilization of the vitrification method [18]. More recently, Borrás et al. compared the two freezing methods in 18 AFAB individuals whom underwent OTC, demonstrating a similar accuracy in both procedures [19]. For subsequent handling and culturing, a diameter of the fragments of ovarian cortex of 4 mm appears to be the most suitable [20]. There is a lack of data in the literature about the technique and/or pregnancy outcomes in transgender patients, while it has been used with success in several cases of FP for cancer patients and Premature Ovarian Insufficiency (POI) patients. In particular, OTC represents the treatment of choice in prepubertal cancer survivors [21], and so far more than 130 babies are born from cisgender women across the globe thanks to this technology [22].

IVM of the oocytes from the ovarian tissue excised for cryopreservation could represent an option in order to avoid the negative features of the following tissue retransplantation after OTC, such as the need for a second intervention carried out under general anesthesia, the interruption of HT, and the multiple vaginal ultrasound checkups required for COS [23]. In addition, most patients decide to undergo OTC in the course of GAS, making reimplantation not feasible and, therefore, an alternative strategy necessary. Oocytes at different stages of maturation retrieved from the surgically excised tissue are cultured in vitro for ripening, and then thawed for fertilization through intracytoplasmic sperm injection (ICSI). In 2017, Lierman et al. collected data from a total number of 259 MII oocytes retrieved after IVM, showing a normal spindle structure analysis and chromosomal alignment after vitrification [24]. However, a more recent publication from the same group involving oocytes collected from 83 transmen patients under testosterone treatment showed high aberrant cleavage patterns and early embryo arrest after fertilization, suggesting a low development capacity of in vitro matured oocytes in this group of patients [25]. At the state of ART, IVM remains an experimental procedure and further studies are needed to upgrade the technique.

Oocyte cryopreservation has shown promising results among transgender men patients, as shown by mounting evidence. Leung et al. carried out a matched retrospective cohort study involving 26 AFAB patients under HT whom underwent FP between 2010 and 2018, compared with 130 cisgender controls [16]. The mean number of oocytes retrieved after COS was 19.9 ± 8.7, higher than the one in the control group (15.9 ± 9.6). Interestingly, the total dose of gonadotropins used in the transgender group was higher than the one in the cisgender group [16]. Amir et al. compared FP outcomes in nine adolescent transgender men patients before starting GAHT with thirty-nine cisgender patients whom underwent FP prior to cancer treatment [26]. The number of oocytes retrieved, of MII oocytes, and maturity rates did not show any significant differences between the two groups [26]. The same result was found by Yan et al. in a systematic review [27]. The amount of available evidence is still limited but may suggest that oocyte cryopreservation is an effective technique for these individuals which can lead to results similar to those of cisgender patients [26,27]. This observation is supported by the absence of statistically significant differences in the number of MII oocytes retrieved in the research cited, since the collection of high-quality oocytes is a crucial step in FP. Furthermore, evidence from the abovementioned studies showed that HT may not adversely affect the results of the technique, although more publications are needed.

The literature about FP outcomes after OTC/IVM is not as full of evidence. Lierman et al. performed a number of studies about IVM with different results, which have been discussed previously [24,25]. A very recent study by Christodoulaki et al. analyzed oocytes retrieved from the ovarian tissue of ovaries removed from 19 transgender patients whom underwent a bilateral oophorectomy from 2020 to 2022 [28]. The oocytes collected from transgender patients were subsequently in vitro matured, and compared with control in vitro and in vivo matured oocytes from cisgender patients whom underwent ART treatment for infertility. After IVM and ICSI, the rate of fertilized oocytes was higher in the control group than in transgender one. Following embryo development, the rate of day-5 blastocysts was significantly higher in the control group. Applying the experimental technique of Spindle Transfer (ST), the rate of day-5 blastocysts increased to similar levels of the cisgender group [28]. According to the authors, different cytoplasmic factors such as poor calcium release could be the explanation for poor embryo development of the oocytes retrieved from ovarian tissue in these patients; ST represents a promising technology in overcoming the issue [28].