Extended sperm search and microfreeze for fertility preservation after long-term hormone therapy in transgender women: a report of three cases

Introduction

When seeking gender affirming treatments, transgender individuals often need to navigate complex decisions surrounding medical interventions and personal goals. Many transgender women (TW) seek gender affirming therapy (GAT) including hormonal treatments and surgical procedures to minimize feelings of gender dysphoria. These interventions negatively impact fertility and reproductive potential to differing extents (1). Consequently, discussions surrounding fertility preservation (FP) are an important aspect of transgender healthcare.

While the guidelines of World Professional Association for Transgender Health (WPATH) version 8 state that it is preferable to undergo established FP techniques prior to the initiation of hormonal GAT, many TW seek FP after starting hormone therapy (2). A study on sperm cryopreservation trends in transgender individuals found that between 2006 and 2016, the incidence of TW sperm banking has increased, and that 6 patients of 84 total started hormonal therapy prior to sperm banking (3). However, gender-affirming hormone therapy for TW, particularly anti-androgens and estrogens, have been shown to impair sperm production and semen quality (4,5). Thus, research is needed to provide data on sperm retrieval and preservation after long-term GAT in TW.

Current traditional FP methods for TW include ejaculated sperm cryopreservation and testicular sperm extraction (TESE). For patients on gender affirming hormonal therapy, FP is often unsuccessful (6). A new method of sperm retrieval and preservation, extended sperm search and microfreeze (ESSM), is a novel and efficient technique that involves performing a thorough search on an ejaculated testicular sample. The entire sample is divided into droplets that are scanned using a high-powered microscope. Any sperm located with ESSM are placed on a specialized device, called SpermVD (MFC Global, Rishon LeZion, Israel), and frozen for use in in-vitro fertilization and intracytoplasmic sperm injection (IVF-ICSI). ESSM may provide hope to TW with previous unsuccessful FP attempts.

This case report highlights three TW patients that underwent ESSM after sustained hormone therapy. While ESSM and associated techniques have been described before, Miller et al. have focused on male infertility in cisgender individuals (7). This report demonstrates the utility of ESSM in TW who present with azoospermia with the additional challenge of reversing prolonged suppression of spermatogenesis. We present this article in accordance with the CARE reporting checklist (available at https://acr.amegroups.com/article/view/10.21037/acr-24-139/rc).

Case presentation

Human subject characteristics

The three TW women included in the analysis all presented for FP prior to gender affirming vaginoplasty with simultaneous orchiectomy. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Publication of this case report was waived from patient consent according to the New York University (NYU) Institutional Review Board. After conventional semen analyses demonstrated complete azoospermia after several attempts, all three patients were referred for ESSM, utilizing individual sperm vitrification of a semen sample 24 hours before surgery. If no or few sperm were recovered, there was a back-up plan to perform an ESSM on testicular tissue to evaluate for the presence of sperm during orchiectomy.

The characteristics of the three TW who underwent ESSM are shown in Table 1.

Case 1

A 40-year-old TW presented for FP after 20 years of estrogen therapy without anti-androgen therapy. She discontinued estrogen use with the intention of attempting FP, but after 10 months conventional semen analysis still demonstrated complete azoospermia. Her pre-operative testosterone level was 395 ng/dL, her follicle-stimulating hormone (FSH) level was elevated at 28.9 mIU/mL, and her estradiol level was low at 38 pg/mL.

Case 2

A 27-year-old TW had been on estrogen, progesterone, and spironolactone therapy for over 6 years before stopping spironolactone and lowering hormone replacement therapy 4 months prior to surgery with the intention of attempting FP. After decreased hormone therapy, she experienced changes to mood and skin with undesired effected. She began taking finasteride 1 month after lowering hormone therapy to alleviate these symptoms. After 3 months of decreasing hormones, conventional semen analysis demonstrated complete azoospermia. Along with ESSM, testicular tissue was also evaluated for the presence of sperm following orchiectomy. Her pre-operative testosterone level was 520 ng/dL, her FSH level was 7.9 mIU/mL, and her estradiol level was 95 pg/mL.

Case 3

A 39-year-old TW had been on estrogen and progesterone therapy for over 21 years without anti-androgen therapy. She discontinued hormone replacement therapy for one year prior to surgery with the intention of attempting FP. After 4 months of hormone cessation, conventional semen analysis demonstrated complete azoospermia. After the patient waited an additional 4 months without hormones, conventional semen analysis continued to demonstrate azoospermia. Along with ESSM, the patient also requested evaluation of testicular tissue for the presence of sperm following orchiectomy on the day of her surgery. Her pre-operative hormones levels included a testosterone level of 359 ng/dL, an FSH level of 10.6 mIU/mL, and an estradiol level of 24 pg/mL.

Materials and methods

All patients underwent at least two standard semen analyses that demonstrated azoospermia, with at least one analysis conducted within the month prior to surgery. The initial semen analyses were performed at various laboratories, and while specific protocols were not provided, it is presumed that these analyses followed the World Health Organization (WHO) guidelines, including centrifugation to evaluate the pellet for sperm. The subsequent ESSM analyses were conducted at Maze Laboratories, which are outlined in Figure 1 (8).

Figure 1 Flowchart illustrating the step-by-step process for fertility preservation in transgender women, including pre-surgical preparations, semen analysis, and advanced sperm retrieval techniques performed at Maze Laboratories. ESSM, extended sperm search and microfreeze; NYU, New York University; rcf, relative centrifugal force; PVP, polyvinylpyrrolidone; SpermVD, Sperm Vitrification Device; HTF, human tubal fluid.

In preparation for semen sample collection, the patient was advised to abstain from ejaculation for 7–10 days prior to extended search (7). After approximately 20 minutes liquefaction time, a 5-µL droplet of the sample was placed in a 60-mm petri dish, flattened gently by tapping on the laboratory benchtop, and evaluated under inverted microscope with phase contrast at 100× magnification.

The semen sample was divided into 0.5 mL aliquots and loaded on PureCeption Sperm Separation Media (Sage IVF 135 Inc., Trumbull, CT, USA) gradient of 0.5 mL 40% upper phase and 0.5 mL 80% lower phase and centrifuged for 20 minutes at 300 relative centrifugal force at room temperature. The supernatant was removed, and the pellet was resuspended in Sperm Washing Modified Human Tubal Fluid (HTF) Medium with 5 mg/mL Human Serum Albumin (Vitrolife, 141 Goteborg, Sweden) and centrifuged again for 10 minutes at 600 revolutions per minute at room temperature.

After this procedure was performed twice, the pellet was resuspended in 100-µL sperm washing medium. Approximately 60-µL of this sample was plated in 5-µL droplets on a 100-mm petri dish lid. The droplets were flattened by gently tapping the plate on the work surface to minimize droplet depth and debris clustering. A 1-µL droplet of polyvinylpyrrolidone (PVP) 7% solution (Sage IVF Inc.) for filling the microcapillary, and several collection droplets of 0.6-µL Sperm Washing Medium (VitroLife) were placed on the dish and covered with light mineral oil (Sage IVF Inc.).

The droplets were thoroughly searched for sperm under 100× or 200× total magnification, and any spermatozoa found were transferred to a collection droplet. This procedure was performed with the use of a beveled non-spiked glass microcapillary with a 10-µm inner diameter tip.

For cryopreservation, a 10-µL droplet of a 50/50 v/v mixture of Quinn’s Advantage Sperm Freezing Medium (Sage IVF Inc.) and Sperm Washing Medium (VitroLife) was prepared, and 0.8–1 µL droplets were placed onto each well of a SpermVD (MFC Global). The SpermVD was submerged into oil on the search plate and motile spermatozoa were transferred from the collection droplets to the droplets on the SpermVD wells. The SpermVD was then placed inside a labeled 1.8 mL cryovial (VWR, Lueven, Belgium) and immediately submerged into liquid nitrogen. Therefore, sperm samples are stored in liquid nitrogen tanks with other sperm samples. Other facilities were encouraged to store these samples with their oocytes and embryos, as they have a rapid thaw time closer to these specimens than the standard sperm vial. Vial caps were color coded in the lab so we can quickly differentiate which vials contain rapid-thaw samples.

Testicular tissue was processed and evaluated for ESSM in cases two and three. Testicular samples from both testes were suspended in HTF and thoroughly minced, resulting in 5.0 mL suspension of right and left testicular tissue. The resulting suspensions were processed using collagenase and gradient washed. ESSM was performed on the testicular suspension.

Results

A summary of ESSM results can be found in Table 2. Pathology outcomes can be found in Table 3.

Case 1

The evaluation of all sperm in a single 5-µL droplet revealed a sperm density of approximately 8 cells per micro-liter, and a motility percentage of 55%. Only motile sperm were collected for cryopreservation. A total of 278 motile sperm were recovered from the sample, 195 of which were graded as ‘progressive’, 83 of them graded ‘non-progressive’.

For cryopreservation, between 8–13 sperm were placed per droplet on the SpermVD, for a total of 19–26 sperm per device. A total of 13 SpermVDs were vitrified.

Testicular tissue demonstrated bilateral atrophic changes. Pathology revealed that less than 10% of the tubules exhibited active spermatogenesis with complete maturation, accompanied by a smaller number of mature spermatids. The sperm maturation process was interrupted in the remaining 90% of the tubules, and notably 0% of tubules showed a complete absence of maturation.

Case 2

The initial evaluation of a single 5-µL droplet did not reveal any sperm. After complete analysis of the 100 µL pellet, 8 sperm were identified in the ejaculate, with a motility percentage of 63%, and 7 were cryopreserved; 5 were motile, with 3 being graded as ‘progressively motile’ and 2 graded as ‘non-progressive’; 2 immotile sperm were also retrieved and vitrified; 1 amorphous sperm was observed and discarded. For cryopreservation, each immotile sperm was placed in an individual collection droplet, the 3 progressively motile sperm were placed in one droplet, and the 2 non-progressive sperm were placed in one droplet. The droplets were divided between two devices, for a total of 3–4 sperm per device. A total of 2 SpermVDs were vitrified.

Testicular tissue demonstrated mild to moderate and extensive atrophy, and mild thickening and hyalinization of the basement membrane; 90% of tubules contained mainly primary spermatocytes in maturation arrest and the remaining tubules had germ cells only. Leydig cells were present but relatively fewer in number. ESSM of testicular tissue resulted in 5.0 mL suspensions of right and left testicular tissue; 0 sperm were recovered from testicular tissue.

Case 3

The ESSM of the patient’s ejaculate revealed 0 sperm present. No sperm were recovered.

Surgical pathology of testicular tissue revealed mild to moderate and extensive testicular atrophy and the tubular basement membrane was mildly thickened; 5% of tubules were in maturation arrest with early round spermatids, 90% were in arrest at the spermatocyte stage, and the remaining 5% of tubules contained germ cells only. Leydig cells were present in increased numbers. ESSM of testicular tissue resulted in 10.0 mL suspensions of right and left testicular tissue; 2.5 mL of each were searched extensively; 0 sperm were recovered from testicular tissue.

Discussion

In this case report, all three patients had unsuccessful conventional sperm analysis after stopping or lowering hormone therapy before being referred for ESSM. With ESSM, two patients out of three were able to successfully retrieve motile spermatozoa despite several years of GAT. This suggests that ESSM can be useful in successful sperm retrieval when conventional semen analysis fails. ESSM is likely to be relevant to other TW interested in FP following long term GAT.

Case three was the only patient that had both an unsuccessful conventional semen analysis and an unsuccessful ESSM. Notably, this patient was on GAT for 21 years, the longest time of all three patients. Additionally, this patient’s testosterone level prior to surgery was the lowest despite stopping GAT for a year.

While patients in both case one and case two had successful ESSM, Patient 1 was able to recover and vitrify almost 40 times the number of sperm compared to Patient 2. Importantly, Patient 1 stopped hormone therapy completely while Patient 2 tapered hormones but never completely stopped. On one hand, this result demonstrates the increased success of FP via ESSM when hormones are discontinued. Cessation of hormones, and even decreasing without complete cessation caused patients in this case report mood lability and distress over de-transitioning. On the other hand, hormone discontinuation does not guarantee a successful ESSM, as seen with Patient 3. Moreover, when taking into account the psychological effects testosterone re-exposure can have on TW, ESSM may provide a way for patients to undergo FP without stopping hormones completely, thus potentially mitigating these damaging effects.

Studies demonstrating the effectiveness of stopping hormone therapy to restore spermatogenesis are limited. One study by Alford et al. demonstrated the restoration of successful ejaculated sperm after cessation of estrogen therapy and commencement of FSH and clomiphene (9). The patient was a 26-year-old TW who had been on 16 months of GAT consisting of estrogen injections and oral spironolactone. Spermatogenesis was restored by 6 weeks after GAT was stopped. While this patient spent substantially less time on GAT than the patients presented in this case report, this study provides some insight into the factors that may lead to a successful ESSM in future cases. Of note, Patient 3 in our study was prescribed clomiphene after stopping hormones for 2 months. However, she never started clomiphene and her hormone levels responded to cessation of hormone therapy. Her ESSM and TESE were ultimately unsuccessful. More research is needed to determine if stopping hormones alone is sufficient to restore spermatogenesis or if adding FSH and clomiphene is necessary in select cases.

Another study by Adeleye et al., 2019 compared sperm quality in three groups: TW who had never undergone hormonal GAT, TW who currently took hormonal therapy, or TW who discontinued use of hormonal medication (10). The study demonstrated that TW who had discontinued hormonal therapy had comparable semen parameters to TW who had never taken hormonal treatment. However, the TW in the “previous users” cohort were only on GAT for three to 4 years. The study further showed that, in alignment with the WPATH guidelines, patients that underwent FP prior to hormone therapy had better sperm parameters than those that were on hormonal therapy during specimen collection.

The use of anti-androgens further complicates the picture of FP for TW, as these medications can suppress testosterone and exacerbate azoospermia (11-13). In our case report, only Patient 2 used an anti-androgen before stopping 4 months prior to ESSM, which may have contributed to her lower sperm retrieval compared to Patient 1, who only used estrogen therapy. However, a previous study has described spermatogenesis restoration after cessation of anti-androgen. The case report describes nine trans women in The Netherlands and Australia describes nine women who all took anti-androgens, either spironolactone or cyproterone acetate (14). Six of the nine had sperm identified on conventional analysis after cessation of GAT, suggesting that restoration of spermatogenesis is possible. Furthermore, Patient 2 used spironolactone, which has an unclear timeline and extent of disruption of spermatogenesis in humans (11). Thus, it remains unclear whether the difference in sperm retrieval outcomes between Patient 1 and Patient 2 can be attributed more to the complete versus incomplete cessation of hormone therapy or to the prior use of spironolactone. The lack of data on the long-term effects of spironolactone, combined with the variable outcomes seen with different hormone cessation strategies, makes it difficult to determine which factor had the greater influence on the observed differences between the two patients.

The hormone levels observed in the three patients may offer valuable insight into the differing outcomes of ESSM. Patient 1, who had discontinued all hormone therapy 11 months prior to ESSM, exhibited a testosterone level of 395 ng/dL (reference range, 300–1,000 ng/dL), an elevated FSH level of 28.9 mIU/mL (reference range, 1.5–12.4 mIU/mL), and a low estradiol level of 38 pg/mL (reference range, <50 pg/mL). These levels suggest a recovery of the hypothalamic-pituitary-gonadal (HPG) axis with partial recovery of endogenous testosterone production, which may have contributed to her higher sperm retrieval compared to the other patients.

In contrast, Patient 2, who only partially reduced her hormone therapy and ceased spironolactone 4 months before ESSM, had a higher testosterone level of 520 ng/dL, suggesting that despite only a partial reduction, this patient had less testosterone suppression. Her FSH level was 7.9 mIU/mL, and her estradiol level was 95 pg/mL, both of which are indicative of ongoing hormone therapy. Patient 3, who had been off hormone therapy for 12 months, showed a testosterone level of 359 ng/dL, FSH of 10.6 mIU/mL, and estradiol of 24 pg/mL. Despite similar hormonal levels to Patient 1, Patient 3 had an unsuccessful sperm retrieval.

The varying testosterone levels among the patients highlight the inconsistency of using serum testosterone levels as a biomarker for spermatogenesis. While serum testosterone is used to assess the success of treatment for spermatogenesis, serum testosterone measurements are known to be imprecise, varying based on the time of day, sample, or laboratory used (15). Furthermore, intra-testicular testosterone rather than serum testosterone is essential for spermatogenesis. While higher serum testosterone levels, as seen in Patient 2 (520 ng/dL), might suggest a partial recovery of spermatogenesis, they do not necessarily predict the degree of success in sperm retrieval through ESSM. Patient 1, with a slightly lower testosterone level (395 ng/dL), achieved a much higher sperm retrieval rate compared to Patient 2, despite the latter’s less suppressed testosterone. This discrepancy suggests that testosterone levels do not reliably predict the extent to which ESSM will be successful.

Despite WPATH’s recommendation and research demonstrating the advantage of undergoing FP before initiating hormone therapy, many transgender patients pursue FP after hormone initiation. A previous study found around 7% of a sample of 84 TW that banked sperm had already started hormone therapy (3). Given the increasing number of transgender individuals in the United States, it is likely the number of TW who desire FP after starting hormone therapy will continue to increase. This divergence from the WPATH guidelines can be attributed to several causes. Systemic barriers to care are substantial in FP due to high cost of both lab and medical procedures and preservation, insufficient insurance coverage, lack of provider knowledge and resources, and discrimination (1,16). Many TW are unable to delay hormone therapy for FP in order to alleviate gender dysphoria (16). Furthermore, a questionnaire-based study by Strang et al. demonstrated that while many transgender youths were uninterested in FP, they acknowledged that their desire may change overtime (17).

While ESSM can provide another option for TW with unsuccessful conventional semen analyses, it is significantly more expensive than conventional semen cryopreservation, with standard cryopreservation costing approximately 30% of the price of ESSM. For example, Maze Laboratories charges approximately $2,000 for the process of extended sperm search, not including the cryopreservation. The increase cost is largely due to the increased time, training, and technical difficulty involved, as well as the higher cost of consumable lab equipment. Compared to a standard semen analysis, where a single drop of the semen sample is examined under a microscope, ESSM involves an exhaustive examination where the lab technician evaluates the entire semen sample under a microscope, regardless of how many hours this process might take. Given the complexity and resources required, ESSM is more comparable in cost to oocyte cryopreservation than standard sperm freezing. While ESSM could be considered as a standard procedure, its higher cost may limit its routine use to cases where conventional methods are ineffective, such as in patients with azoospermia following long-term hormone therapy. Despite this, ESSM may be a valuable option in specialized scenarios, particularly for patients who have failed to achieve viable sperm through conventional methods.

A potential criticism of this study is the inter-laboratory variability in semen analysis results. This variability could raise concerns about the reliability of azoospermia diagnoses based on limited testing. To mitigate this concern, all patients in this study underwent at least two standard semen analyses, both of which demonstrated azoospermia, with one analysis conducted within a month of the ESSM procedure. Although the possibility of intermittent sperm production cannot be entirely excluded, the consistent findings across multiple analyses strengthen the reliability of the azoospermia diagnoses in these patients. Moreover, the ESSM procedure itself is designed to maximize the likelihood of detecting even the smallest quantities of sperm, further addressing the limitations of conventional semen analysis.

Due to the limited and irreplaceable nature of the samples, we cannot perform a ‘test’ post-thaw and discard patient samples. Our quality assessment is done by requesting outcomes from the IVF labs that thaw the samples for IVF. They are requested to provide post thaw spontaneous motility (not synonymous with post thaw survival), fertilization rate, blastocyst formation rate, and live birth. To date, all the TW patients have performed FP only and the samples have not been thawed for IVF yet.

Furthermore, our case report demonstrates a potentially successful avenue for FP for TW on longstanding GAT. Future research involving larger cohorts and follow up for the success of conception using the extracted sperm in IVF-ICSIs is necessary to evaluate the fertility viability of the sperm after thawing.

Conclusions

FP is a crucial component of healthcare for TW that can be provided before and during GAT. The success in two cases from this study underscore the potential use for ESSM as a tool for FP in TW who have undergone multiple years of hormonal therapy and have unsuccessful semen analyses and testicular extractions.

Acknowledgments

Contents of this article have been presented as an abstract at the American Urology Association Conference, 2024.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://acr.amegroups.com/article/view/10.21037/acr-24-139/rc

Peer Review File: Available at https://acr.amegroups.com/article/view/10.21037/acr-24-139/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://acr.amegroups.com/article/view/10.21037/acr-24-139/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Publication of this case report was waived from patient consent according to the NYU Institutional Review Board.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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