Histological study on the influence of puberty suppression and hormonal treatment on developing germ cells in transgender women.

STUDY QUESTION: Can transgender women cryopreserve germ cells obtained from their orchiectomy specimen for fertility preservation, after having used puberty suppression and/or hormonal treatment? SUMMARY ANSWER: In the vast majority of transgender women, there were still immature germ cells present in the orchiectomy specimen, and in 4.7% of transgender women-who all initiated medical treatment in Tanner stage 4 or higher-mature spermatozoa were found, which would enable cryopreservation of spermatozoa or testicular tissue after having used puberty suppression and/or hormonal treatment. WHAT IS KNOWN ALREADY: Gender affirming treatment (i.e. puberty suppression, hormonal treatment, and subsequent orchiectomy) impairs reproductive function in transgender women. Although semen cryopreservation is generally offered during the transition process, this option is not feasible for all transgender women (e.g. due to incomplete spermatogenesis when initiating treatment in early puberty, in case of inability to masturbate, or when temporary cessation of hormonal treatment is too disruptive). Harvesting mature spermatozoa, or testicular tissue harboring immature germ cells, from orchiectomy specimens obtained during genital gender-affirming surgery (gGAS) might give this group a chance of having biological children later in life. Previous studies on spermatogenesis in orchiectomy specimens showed conflicting results, ranging from complete absence of germ cells to full spermatogenesis, and did not involve transgender women who initiated medical treatment in early- or late puberty. STUDY DESIGN, SIZE, DURATION: Histological and immunohistochemical analyses were performed on orchiectomy specimens from 214 transgender women who underwent gGAS between 2006 and 2018. Six subgroups were identified, depending on pubertal stage at initiation of medical treatment (Tanner stage 2-3, Tanner stage 4-5, adult), and whether hormonal treatment was continued or temporarily stopped prior to gGAS in each of these groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: All transgender women used a combination of estrogens and testosterone suppressing therapy. Orchiectomy specimen sections were stained with Mayer's hematoxylin and eosin and histologically analyzed to assess the Johnsen score and the ratio of most advanced germ cell types in at least 50 seminiferous tubular cross-sections. Subsequently, immunohistochemistry was used to validate these findings using spermatogonia, spermatocytes or spermatids markers (MAGE-A3/A4, γH2AX, Acrosin, respectively). Possibilities for fertility preservation were defined as: preservation of spermatozoa, preservation of spermatogonial stem cells or no possibilities (in case no germ cells were found). Outcomes were compared between subgroups and logistic regression analyses were used to assess the association between the duration of hormonal treatment and the possibilities for fertility preservation. MAIN RESULTS AND THE ROLE OF CHANCE: Mature spermatozoa were encountered in 4.7% of orchiectomy specimens, all from transgender women who had initiated medical treatment in Tanner stage 4 or higher. In 88.3% of the study sample orchiectomy specimens only contained immature germ cells (round spermatids, spermatocytes or spermatogonia, as most advanced germ cell type). In 7.0%, a complete absence of germ cells was observed, all these samples were from transgender women who had initiated medical treatment in adulthood. Cessation of hormonal treatment prior to gGAS did not affect the presence of germ cells or their maturation stage, nor was there an effect of the duration of hormonal treatment prior to gGAS. LIMITATIONS, REASONS FOR CAUTION: Since data on serum hormone levels on the day of gGAS were not available, we were unable to verify if the transgender women who were asked to temporarily stop hormonal treatment 4 weeks prior to surgery actually did so, and if people with full spermatogenesis were compliant to treatment. WIDER IMPLICATIONS OF THE FINDINGS: There may still be options for fertility preservation in orchiectomy specimens obtained during gGAS since a small percentage of transgender women had full spermatogenesis, which could enable cryopreservation of mature spermatozoa via a testicular sperm extraction procedure. Furthermore, the vast majority still had immature germ cells, which could enable cryopreservation of testicular tissue harboring spermatogonial stem cells. If maturation techniques like in vitro spermatogenesis become available in the future, harvesting germ cells from orchiectomy specimens might be a promising option for those who are otherwise unable to have biological children. STUDY FUNDING/COMPETING INTEREST: None. TRIAL REGISTRATION NUMBER: N/A.

Functional Outcomes and Urological Complications after Genital Gender Affirming Surgery with Urethral Lengthening in Transgender Men.

PURPOSE: We determined the urological complications and lower urinary tract function after genital gender affirming surgery with urethral lengthening in transgender men. MATERIALS AND METHODS: A single center, retrospective cohort study was performed from January 2013 to January 2018. Patient demographics, medical history, perioperative data, surgical and urological complications, and preoperative and postoperative urological outcomes were obtained. RESULTS: Of the 63 patients included in the study 8 (13%) underwent metoidioplasty and 55 (87%) phalloplasty, comprised of 27 (43%) free radial forearm flap, 19 (30%) anterolateral thigh flap and 9 (14%) superficial circumflex iliac artery perforator flap surgeries. In phalloplasty the types of urethral lengthening were tube-in-tube free radial forearm flap in 27 (49%), free radial forearm flap (second fasciocutaneous flap) in 18 (33%), superficial circumflex iliac artery perforator flap in 5 (9%) or labial in 5 (9%). Mean followup was 23 months (range 12 to 71). Stricture formation occurred in 35 (63%) phalloplasty and 5 (63%) metoidioplasty cases. Urethral fistula formation occurred in 15 (27%) phalloplasty and 4 (50%) metoidioplasty cases. Mean time to strictures and fistulas was approximately 3 months. Overall 46 (73%) patients needed revision surgery because of fistulas/strictures. After treatment 44 (70%) patients were able to void from the tip of the phallus. No clinically relevant differences in International Prostate Symptom Scores, frequency volume charts and uroflowmetry were found preoperatively vs postoperatively. CONCLUSIONS: Genital gender affirming surgery with urethral lengthening is a complex procedure with a high complication rate. After treating complications no clinically relevant differences in urological functioning were recorded. The majority of transgender men could void from the tip of the penis and showed favorable urological outcomes.

Refractory diversion neovaginitis in a sigmoid-colon-derived neovagina: clinical and histopathological considerations.

Colonic segments are being used as pedicled grafts in neovaginoplasty, a surgical procedure to (re)construct a (neo)vagina. A disadvantage of using colonic grafts is the potential occurrence of neovaginal complications due to diversion from the faecal stream. Here, we report a case of severe, refractory diversion colitis of the sigmoid neovagina, so-called 'diversion neovaginitis', in a 42-year-old woman with complete androgen insensitivity syndrome. Neovaginal biopsy specimens showed colonic-type mucosa with strong increase of lymphoplasmacellular infiltrate in the lamina propria, ulceration with fibrinoid deposition and some crypt irregularity. Endoscopy showed erythematous mucosa, superficial ulceration, mucus discharge and multiple pseudopolyp-like lesions. Local application of mesalazine foam enemas and sodium butyrate enemas initially gave symptom relief. However, this was a temporary effect, ultimately necessitating removal of the neovaginal construct. It is important that all patients are informed about neovaginal bowel complications, for example, diversion neovaginitis. Regular medical and endoscopic follow-up appears recommendable.

Promoter methylation analysis of WNT/ß-catenin signaling pathway regulators to detect adenocarcinoma or its precursor lesion of the cervix.

OBJECTIVE: Cervical adenocarcinoma (AdCA) and adenocarcinoma in situ (ACIS) are frequently missed in cytology-based screening programs. Testing for high-risk human papillomavirus (hrHPV) improves their detection, but novel ACIS/AdCA specific biomarkers are needed to increase specificity for these lesions. Novel markers may be deduced from the WNT/ß-catenin signaling pathway, which is aberrantly activated during cervical carcinogenesis. METHODS: Promoter methylation of nine WNT-antagonists (APC, AXIN2, DKK3, SFRP2, SFRP4, SFRP5, SOX17, WIF1 and WNT5A) was evaluated by methylation-specific PCR (MSP) on a small series of cervical tissue specimens, including AdCA and SCC. To estimate the diagnostic potential of the genes most frequently methylated in AdCA an extended series of ACIS, AdCA, CIN3, SCC, and normal cervical tissue specimens (n=131) as well as 49 hrHPV-positive scrapings were analyzed by quantitative MSP (qMSP). RESULTS: The frequency of DKK3 and SFRP2 methylation was significantly higher in AdCA compared to SCC, i.e. 82% vs. 18% (p<0.01) and 84% vs. 39% (p<0.01), respectively, while SOX17 methylation frequency was significantly higher in SCC than AdCA, i.e. 89% vs. 62% (p<0.05). Methylation of WIF1 was common in both AdCA (71%) and SCC (54%). Methylation frequencies ranged from 4% to 55% in precursor lesions and from 0% to 5% in normal biopsies. When tested on HPV-positive cervical scrapings, qMSP of the best ACIS/AdCA discriminator genes, i.e. DKK3 and SFRP2, detected all women with underlying ACIS/AdCA, compared to 3% of controls. CONCLUSIONS: DKK3 and SFRP2 promoter methylation is highly indicative for the presence of ACIS/AdCA, thereby providing promising triage markers for HPV-positive women at risk of ACIS/AdCA.