Sperm parameters in Israeli transgender women before and after cryopreservation

The application of fertility preservation, initially intended for oncological patients prior to gonadotoxic treatment, has extended in recent years to transgender and gender‐non‐conforming individuals undergoing therapy for gender compatibility.

Specimens collected after discontinuation of gender-affirming hormone treatments were further impaired. Moreover, post-thawing sperm total motile count, motility, and overall sperm survival were reduced in transgender women.

K E Y W O R D S fertility preservation, gamete cryopreservation, transgender INTRODUCTION
The application of fertility preservation (FP), originally intended for oncological patients prior to gonadotoxic treatment, has extended in recent years to transgender and gender-non-conforming individuals undergoing therapy for gender compatibility. 1 Gender incongruency is a term that describes the distress that a person may have experienced from a mismatch between their biological sex and their gender identity. 2 Individuals assigned male at birth who identify as female are defined as trans women.
There are no accurate universal epidemiological data regarding the prevalence of gender incongruency. Studies are limited as a result of cultural barriers and under-reporting. Nevertheless, a recent study based on a survey conducted in the United States estimated the prevalence of gender incongruency to be 0.6% of the population. 3 In recent years, there has been a significant increase in the number of young trans individuals presenting to specialist services and seeking gender-affirming treatments. 4 To achieve better alignment between their identity and their physical characteristics, optional treatments are available for transgender women, including gender-affirming hormone treatment (GAHT) and gender-affirming surgery. 5 Most of these interventions may limit the transgender's fertility options in the future. This may range from transient effects of short-term use of hormonal treatment with anti-androgens and estrogens to achieve feminization, to long-term use that may potentially have a detrimental, understudied effect on spermatogenesis. 6,7 Furthermore, gender-affirming surgery involving penectomy and bilateral orchiectomy will result in irreversible reproductive loss.
Most trans women desire to parent genetically related offspring or want to preserve the freedom to choose later in life whether to start a family, and as many as 76.6% will consider FP before starting a medical treatment. 8 Sperm cryopreservation before medical or surgical intervention may be a stressful and emotionally challenging process for some patients. If already initiated, temporary cessation of hormonal therapy to optimize sperm collection discourages some to initiate the procedure because of the associated physical changes. In recent years, scientific medical associations have highlighted the need to counsel transgender people about the effect of the medical transition on fertility and their available options for FP. [9][10][11] In Israel, each physician that intends to start gender-affirming treatment for a transgender patient must recommend comprehensive FP consultation before initiating the process. However, pursuing FP is not mandatory but rather up to the patient.
Spermatogenesis is a complex process involving mitotic cell division, meiosis, and spermiogenesis. The regulation of spermatogenesis involves both endocrine and paracrine mechanisms. 12 Measure of semen quality is an essential part of the infertility evaluation process. The basic requirements for semen analysis are standardized by the World Health Organization (WHO) guidelines. 13 Results of semen analysis are of greatest clinical significance when multiple abnormalities are present. The most severe clinical finding is azoospermia, defined as the absence of spermatozoa in the ejaculate after examination of a centrifuged semen pellet. Patients with azoospermia undergoing sperm retrieval should also undergo microdissection testicular sperm extraction (TESE). 14 Recent studies have shown that trans women who had undergone GAHT, presented with a significantly lower total sperm count (TC) than those who had not. Furthermore, semen abnormalities, such as lower TC and lower motility percentage, were also noted among trans women who had not undergone GAHT, compared to the WHO reference population. [15][16][17] This has been hypothesized to be a result of several risk factors associated with this population such as body mass index (BMI), recreational drug and alcohol consumption, non-reported gender-affirming hormone use, 18,19 as well as stress and effects of scrotal temperature with genital-tucking behavior. 20 Semen quality, and most importantly post-thaw sperm survival, determines the type of fertility intervention trans women and their partners may need. Good quality sperm sample enables less invasive and less expensive intra-uterine inseminations (IUI), whereas sperm sample of low quality requires in vitro fertilization (IVF), which is more invasive and expensive technique. 21 Estimating the post-thaw sperm parameters will enable andrology scientists to estimate the number of vials that can be divided from each sample. Furthermore, prediction of post-thaw sperm parameters will enable better consultation to patients undergoing FP prior to GAHT.
Although the storage costs of sperm samples for the trans population are not covered by the health public funding in Israel, the number of funded IVF treatments is not limited until the birth of the first two children. 22 For trans women from other countries attempting to conceive, the situation may be more challenging as it is for other infertile couples that often discontinue IVF treatments before a live birth is achieved because of high costs and sparse insurance coverage. 23 The aim of this study was to examine semen quality in our cohort of Israeli trans women, and to evaluate the post-thaw sperm parameters and suitability for the different types of assisted reproductive technology (ART), in order to optimize counseling before FP is carried out.

Study population
In this retrospective cohort study, we examined data on all consecutive transgender women who presented to the FP clinic at the Tel Aviv Sourasky Medical Center between January 2000 and July 2019.
Most of the patients were referred by the endocrinology transgender clinic in our center or by their primary physicians (endocrinologists or general practitioners). Their outcomes were compared with a control group of 100 consecutive candidates for sperm donation composed of young, unmarried males who were self-reported to be healthy.
The study was approved by the local institutional review board committee in accordance with the Helsinki Declaration of 1975 (#0472-17-TLV). Patient consent was waived because of the retrospective nature of our study and with the authorization of the ethical committee.

Data collection
The following data were collected from the computerized database: Semen samples were frozen as previously described. 25 Each freezing procedure included freezing of the control sample in a small volume (0.1 ml) to evaluate the sperm quality of the frozen specimens. Frozen sperm samples were thawed on a hotplate at 37 • C for 5 min and then transferred to 1.5-ml tubes, which were mixed thoroughly to ensure a homogeneous mixture. Sperm parameters were analyzed as described above. Post-thaw sample was considered suitable for IUI if the TMC was more than 10 million; suitable for IVF if TMC was between 1 and 10 million; and suitable for IVF-ICSI if TMC was less than 1 million.

Statistical analysis
Statistical analysis was performed using IBM SPSS

RESULTS
Overall, 87 transgender women were referred for FP counseling at In transgender women who received hormonal treatment before sperm cryopreservation, the TC, motility rate, and TMC were further decreased (Table 1)   (p < 0.001) among transgender women with previous GAHT (Table 1 and Figure 1).
Analysis of the post-thaw sperm parameters showed that the motility rate remained lower in the transgender group compared to the control group (20.0% vs. 45.0%, respectively; p < 0.001). TMC remained lower as well (2.7 vs. 9.0, respectively; p < 0.001), and the post-thaw decreases in total motile sperm count were higher in the transgender group compared with the control group (91.5% vs. 90.0%).
Further subdivision of the transgender group showed that the decrease in TMC was lower for transgender women who did not use GAHT compared to those who did (−89.7% vs. −92.6%, respectively, p < 0.01).
(  17 This mounting evidence suggests the presence of one or more transgender-specific factors that negatively affect semen quality. Some suggest that high prevalence of factors associated with a lower semen quality in the transgender population, such as psychological stress, depression, and anxiety, may play a role in the negative effect on sperm parameters. 17,19,28 We have shown that not only were reduced fresh sperm parameters related to transgender women but that the TMC was also significant lower in their post-thaw spermatozoa, further suggesting an inherent factor affecting sperm quality and durability.
The use of GAHT prior to specimen collection is negatively associated with semen quality parameters. 7 Our data showed that transgender women who have frozen their spermatozoa before the initiation of GAHT had better results compared to transgender women that froze their spermatozoa after medication cessation. Furthermore, transgender sperm parameters after thawing showed to be inferior compared to sperm donor candidates and to an even greater extent after GAHT. This is supported by a previous publication that demonstrated semen parameters were inferior for transgender women with concurrent history of GAHT. 18 Except for a small part of transgender women in whom normal sperm quality was reported upon estrogen cessation, 18 most evidence supports that prolonged exposure to estrogen therapy causes testicular atrophy, absent or severely impaired spermatogenesis, and loss of Leydig cells. 29,30 Our data support that previous exposure to GAHT is associated not only with reduced sperm parameters but also reduced survival of spermatozoa after thawing. A possible explanation for this latter finding might be that poor-quality semen may be more prone to DNA damage and cell death after cryopreservation than good-quality semen samples. 31 Although the 12 transgender women who had GAHT had a minimum of 2 months of discontinuation before sperm cryopreservation, they had reduced semen parameters and thaw survival. The duration of use, although varied in our study, is not associated with any differences in testicular pathology parameters as observed in patients undergoing gender-affirming surgery. 32 Given the small sample size of our cohort, no conclusions can be drawn to link the type or the duration of GAHT with its effect on semen parameters.
The current study cannot establish the mechanisms involved in the altered semen parameters; however, the clinical association with reduced TMC can be explained by altered hormonal axis in the group that was previously exposed to hormones. Such an explanation cannot be offered in the general transgender population, and the mechanism is still unclear. The retrospective design of this study limited the ability to collect data on endocrine profiles for all patients, and there were incomplete clinical data on certain lifestyle factors known to influence semen quality, such as obesity, alcohol and drug consumption, and cigarette smoking, 17,19 as well as non-medical gender-affirming behaviors, such as "tucking." Furthermore, genetic or epigenetic factors remain a source of concern after use of GAHT, 33  Finally, eight transgender women who presented to our clinic were unable or unwilling to ejaculate. This is likely not representative of all transgender women who might be reluctant to turn to FP in the first place because of emotional stressors involving masturbation and are unaware of the option for surgical TESE that is offered at our center, which has shown to be effective. 36,37 Moreover, if GAHT has already been initiated, a therapeutic window could be difficult, as cessation of the treatment can lead to significant physical and psychological distress.
Offering FP prior to treatment initiation is essential. Therefore, information about availability, utility, and outcomes for patients and healthcare providers is of utmost importance in the provision of FP.
Although the fertility of transgender women was not initially a priority, most transgender men and women report that they want to become parents. However, only few benefit from FP. 37-39

CONCLUSIONS
The current results demonstrate that information about fertility preservation options should be provided before or early during the transition to enable ideal care and to avoid the need to resort to a therapeutic window, which will further decrease sperm quality and durability. The etiology of the differences found in the sperm param-