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.

Impaired semen quality in trans women: prevalence and determinants.

STUDY QUESTION: What is the semen quality in trans women at time of fertility preservation, prior to the start of gender-affirming hormone treatment? SUMMARY ANSWER: Before the start of gender-affirming hormone treatment, semen quality in trans women was already strongly decreased compared to the general population. WHAT IS KNOWN ALREADY: Hormone treatment for -trans women (birth-assigned males, female gender identity) consists of anti-androgens combined with estrogens in order to achieve feminization and it is accompanied by a loss of reproductive capability. Trans women can opt for semen cryopreservation prior to their medical transition to retain the possibility to parent genetically related offspring. Post-thaw semen parameters determine which ART can be used. Knowledge of semen quality and the factors negatively influencing semen parameters in trans women are important to improve semen quality before fertility preservation. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study was performed between 1972 and 2017. In total, 260 trans women were included for this study. Due to the study design, there was no loss to follow-up or attrition. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied the quality of the preserved semen in trans women, prior to their medical transition, who visited our gender clinic. Semen parameters were collected, as well as data on age, alcohol consumption, smoking, cannabis use, BMI, previous use of estrogens or anti-androgens and endocrine laboratory results. Semen parameters were categorized using reference values for human semen of the World Health Organization (WHO) and compared with data from the general population. Logistic regression analyses were performed to analyze the extent to which factors known to have a negative impact on semen quality in the general population explained the impaired semen quality in the cohort. MAIN RESULTS AND THE ROLE OF CHANCE: The cohort consisted of 260 trans women between the age of 16 and 52 years. Semen quality in trans women was significantly decreased compared to WHO data from the general population. In total, 21 trans women had an azoospermia and median semen parameters for the remaining trans women and the general population, respectively, were as follows: volume 2.7 and 3.2 ml (P < 0.05), sperm concentration 40 and 64 million/ml (P < 0.05), total sperm number 103 and 196 million (P < 0.05) and progressive motility 41% and 57% (P < 0.05). Smoking (odds ratio (OR) 2.35 (95% CI 1.06-5.21)) and a higher age at time of fertility preservation (OR 1.04 (95% CI 1.00-1.08)) were found to correlate with an impaired progressive motility. Twelve trans women reported to have used anti-androgens and estrogens, and all had discontinued for at least 3 months prior to the first attempt for semen cryopreservation. No correlation was found between previous gender-affirming hormone use and decreased semen parameters. The median post-thaw total motile sperm count was 1.0 million per vial (interquartile range 0.1-3.1) and in only 26.4% of thawed semen samples was the quality adequate for a minimally invasive IUI. LIMITATIONS, REASONS FOR CAUTION: Limitations include the retrospective design and insufficient data on transgender-specific factors, such as bringing the testes into the inguinal position (tucking), wearing tight underwear and low masturbation frequency. WIDER IMPLICATIONS OF THE FINDINGS: Semen quality in trans women was decreased compared to the general population, which could not be explained by known risk factors, such as BMI, alcohol consumption, cannabis use, gender-affirming hormone use or abnormal endocrine laboratory results. Although a negative impact of smoking was observed, it was insufficient to explain the overall decreased semen quality in this cohort. Since low pre-freeze semen quality results in an even lower post-thaw semen quality, the majority of trans women and their female partner or surrogate may need an invasive and burdensome treatment to establish a pregnancy. STUDY FUNDING/COMPETING INTEREST(S): For this study, no external funding was obtained and there were no competing interests. TRIAL REGISTRATION NUMBER: NA.

Relation between acute-phase proteins and enhanced bleomycin-induced pulmonary fibrosis in the rat.

Intratracheal application of Bleomycin (Bleo) in rats induces interstitial pneumonitis followed by progressive fibrosis. As the presence of high levels of acute-phase proteins (= reactants = APR), especially alpha 2-macroglobulin of the rat (alpha 2M), enhances liver fibrosis, we investigated whether this phenomenon also occurs in rats with Bleo-induced lung fibrosis. The experiments showed that this is the case; lung fibrosis assessed by measuring hydroxyproline, hexosamine, and prolyl-4-hydroxylase was enhanced when just before Bleo application an acute-phase reaction was induced. This effect can be explained by the inhibitory effect of alpha 2M on collagenase. The experiments showed a significant positive correlation between alpha 2M and parameters of fibrosis. This is especially the case in the third week after Bleo application. Bleo itself does not induce a strong acute-phase reaction, notwithstanding the pneumonitis during the first weeks. The increased fibrosis is accompanied by progressive ventilatory disturbances demonstrated by high arterial pCO2 and low pO2. In patients undergoing Bleo treatment, varying levels of APR can be expected, and this could explain the rapid development of fibrosis in individual cases.

Is the magnetic resonance imaging proton spin-lattice relaxation time a reliable noninvasive parameter of developing liver fibrosis?

During the development of liver fibrosis in rats by an individual dose-titrated CCl4 administration, hepatic proton spin-lattice relaxation time (T1) has been measured in vivo every 2 weeks for 8 weeks. Liver content of collagen, triglycerides and water has been measured biochemically in biopsy material. After 4 weeks of CCl4 treatment, T1 increased significantly and remained at the same level, whereas liver collagen reached its maximum at 8 weeks. It is concluded that, under our experimental conditions, increased hepatic T1 represents drug-induced edema and that hepatic T1 is not a reliable noninvasive parameter for developing liver fibrosis in vivo.

Mechanisms by which acute phase proteins enhance development of liver fibrosis: effects on collagenase and prolyl-4-hydroxylase activity in the rat liver.

Previous experiments showed that the presence of high levels of acute phase reactants (APR) enhance CCl4-induced liver fibrosis in the rat. A high correlation was found between the degree of fibrosis and alpha 2-macroglobulin of the rat (alpha 2-macrofetoprotein, alpha M-FP) used for monitoring the acute phase response. This acute phase reaction was provoked by epinephrine just before CCl4 treatment was started. In the present study we analyzed the effect of APR by repeating these experiments and estimating liver neutral collagenase with a synthetic substrate and endogenous collagen as a substrate, and liver prolyl-4-hydroxylase. A strong depression of liver collagenase activity was found in rats with a preceding acute phase reaction contrary to the rats that underwent CCl4 treatment only. A high level of alpha M-FP correlated negatively with collagenase activity. Also in vitro alpha M-FP proved to inhibit collagenase activity. Prolyl-4-hydroxylase was increased in the rats during acute phase reaction and correlated highly and positively with alpha M-FP, haptoglobin, and ceruloplasmin. Thus high levels of APR promote development of CCl4-induced fibrosis, partly by anticollagenase activity and partly because of enhancement of prolyl-4-hydroxylase activity. The latter phenomenon can also be explained by the presence of APR, but this has to be proved.

Acute phase reactants enhance CCl4 induced liver cirrhosis in the rat.

High levels of acute phase proteins (acute phase reactants, APR) suppress acute inflammatory reactions in the rat. As many APR have antiprotease properties, including an anticollagenase activity, the effect of APR on the development of CCl4-induced liver fibrosis was investigated in rats. APR were provoked by repeated injections of epinephrine, inducing a broad spectrum of APR. This reaction can be monitored measuring alpha 2-macroglobulin levels in the rat (alpha 2-macrofetoprotein, alpha M FP). This protein was found to inhibit both acute galactosamine hepatitis and acute CCl4-induced liver toxicity. The animals with high levels of APR at the start of CCl4 treatment developed a more severe degree of fibrosis and cirrhosis than the control group in which no acute phase reaction was induced. Epinephrine alone had no such effects. Additionally, the APR positive group showed an initially lower degree of hepatocellular damage when compared to control animals. This uncoupling of liver cell damage and subsequent fibrosis may demonstrate that higher levels of APR might be important as to the development of cirrhosis, possibly based on the anticollagenase activity of these proteins.

Inflammation inhibiting properties of rat alphaM foetoprotein (rat-alpha2 macroglobulin), an acute phase reactant.

alphaMFoetoprotein of the rat (rat alpha2macroglobulin) is present in serum during foetal development. After birth, alphaMFP declines rapidly, but the protein returns after injury. The injury we used consisted of skin and muscle incision, laparotomy, BaSO4 i.p. The protein occurs also during liverregeneration, livercardinogenesis en during infections. Therefore, alphaMFP can be considered as an acute phase reactant. We found that this protein strongly inhibits inflammatory exudation caused by carrageenin, histamin, prostaglandin E2, 5HT and bradykinin. Furthermore, we found that alphaMFP suppresses completely inflammatory reactions during Ga1.N-hepatitis. In this condition, the primary biochemical lesion, consisting of liver UDPG depletion, does occur in spite of the protective effect of alphaMFP. Thus, the inflammatory inhibiting effects of alphaMFP seem to be an important mechanism reducing inflammatory reaction patterns.