Serum and intratesticular inhibin B, AMH, and spermatogonial numbers in trans women at gender-confirming surgery: An observational study.

BACKGROUND: Anti-Müllerian hormone and inhibin B are produced by Sertoli cells. Anti-Müllerian hormone secretion indicates an immature Sertoli cell state. Inhibin B serves as a marker of male fertility. Identification of markers reflecting the presence of germ cells is of particular relevance in trans persons undergoing gender-affirming hormone therapy in order to offer individualized fertility preservation methods. OBJECTIVES: Serum and intratesticular inhibin B and anti-Müllerian hormone values were assessed and related to clinical features, laboratory values, and germ cell numbers. MATERIALS AND METHODS: Twenty-two trans women from three clinics were included. As gender-affirming hormone therapy, 10-12.5 mg of cyproterone acetate plus estrogens were administered. Height, weight, age, medication, and treatment duration were inquired by questionnaires. Serum luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol were measured by immuno-assays. Serum and intratesticular inhibin B and anti-Müllerian hormone were measured by commercially available ELISAs. Spermatogonia were quantified as spermatogonia per cubic millimeter testicular tissue applying a morphometric analysis of two independent testicular cross-sections per individual after MAGEA4 immunostaining. RESULTS: Patients with high inhibin B levels presented with a higher number of spermatogonia (*p < 0.05). Furthermore, mean serum inhibin B was associated with low age (*p < 0.05), low follicle-stimulating hormone (*p < 0.05), and low testosterone (*p < 0.05). Serum anti-Müllerian hormone, however, was not related to spermatogonial numbers. It correlated with high testosterone (*p < 0.05) and high follicle-stimulating hormone (*p < 0.05) only. High intratesticular inhibin B was accompanied by high luteinizing hormone (*p < 0.05), high follicle-stimulating hormone (**p < 0.01), and high testosterone levels (**p < 0.01). Higher the intratesticular anti-Müllerian hormone levels, the longer gender-affirming hormone therapy was administered (*p < 0.05). DISCUSSION AND CONCLUSION: Serum inhibin B levels indicate the presence of spermatogonia, whereas anti-Müllerian hormone seems not to be a reliable marker concerning germ cell abundance.

Testicular blood supply is altered in the 41,XXY* Klinefelter syndrome mouse model.

Hypergonadotropic hypogonadism is a major feature of Klinefelter syndrome (KS), assumed to be caused by testicular hormone resistance. It was previously shown that intratesticular testosterone levels in vivo and Leydig cell function in vitro seem to be normal indicating other functional constraints. We hypothesized that impaired testicular vascularization/blood flow could be a co-factor to the observed hypergonadotropic hypogonadism. We evaluated the testicular vascular system by measuring blood vessel sizes during postnatal development and testis blood flow in adult 41,XXY* mice. Proportional distribution and size of blood vessels were analyzed during testicular development (1, 3, 5, 7, 10, 21 dpp, 15 wpp). While ratios of the vessel/testis area were different at 15 wpp only, a lower number of smaller and mid-sized blood vessels were detected in adult KS mice. For testicular blood flow determination we applied contrast enhanced ultrasound. Floating and reperfusion time for testicular blood flow was increased in 41,XXY* mice (floating: XY* 28.8 ± 1.69 s vs XXY* 44.6 ± 5.6 s, p = 0.0192; reperfusion XY* 19.7 ± 2.8 s vs XXY*: 29.9 ± 6.2 s, p = 0.0134), indicating a diminished blood supply. Our data strengthen the concept that an impaired vascularization either in conjunction or as a result of altered KS testicular architecture contributes to hormone resistance.

Differentiation of Testis Xenografts in the Prepubertal Marmoset Depends on the Sex and Status of the Mouse Host.

This study investigates the effects of the endocrine milieu of immunodeficient mouse host (intact vs. castrated male, intact male vs. intact female) on prepubertal marmoset (Callithrix jacchus) testicular xenografts. Previous marmoset xenografting studies used castrated nude mouse hosts which did not support efficient graft survival and maturation. Due to the distinct endocrine milieu in marmosets with a deletion of exon 10 in the LH receptor, we wanted to explore whether the most efficient xenograft development occurs in intact male mouse hosts compared to intact females or castrated males. We xenografted freshly isolated tissue from prepubertal marmosets (age range 4-6 months) into the back skin of three groups of nude mice (intact male, castrated male, and intact female). We collected serum for endocrine determinations and grafts after 20 weeks and determined hormonal/reproductive status, graft survival, somatic cell development and initiation of germ cell differentiation. Graft development, tubular integrity, and germ cell differentiation status in the grafts retrieved from different hosts was scored by morphometric analysis. The influence of the different endocrine status was compared between groups of hosts. Endocrine readouts and histological endpoints in xenografts substantiate that grafts were exposed to different microenvironments and responded with host specific developmental patterns. The intact male hosts supported the most significant progression of germ cell development. Our data provide evidence for the important role of the host milieu on survival and differentiation of marmoset xenografts. The xenografting model offers innovative avenues to exploit development and endocrine effects in the primate marmoset testis using limited numbers of non-human primates for the experimental settings.

Direct but no transgenerational effects of decitabine and vorinostat on male fertility.

Establishment and maintenance of the correct epigenetic code is essential for a plethora of physiological pathways and disturbed epigenetic patterns can provoke severe consequences, e.g. tumour formation. In recent years, epigenetic drugs altering the epigenome of tumours actively have been developed for anti-cancer therapies. However, such drugs could potentially also affect other physiological pathways and systems in which intact epigenetic patterns are essential. Amongst those, male fertility is one of the most prominent. Consequently, we addressed possible direct effects of two epigenetic drugs, decitabine and vorinostat, on both, the male germ line and fertility. In addition, we checked for putative transgenerational epigenetic effects on the germ line of subsequent generations (F1-F3). Parental adult male C57Bl/6 mice were treated with either decitabine or vorinostat and analysed as well as three subsequent untreated generations derived from these males. Treatment directly affected several reproductive parameters as testis (decitabine & vorinostat) and epididymis weight, size of accessory sex glands (vorinostat), the height of the seminiferous epithelium and sperm concentration and morphology (decitabine). Furthermore, after decitabine administration, DNA methylation of a number of loci was altered in sperm. However, when analysing fertility of treated mice (fertilisation, litter size and sex ratio), no major effect of the selected epigenetic drugs on male fertility was detected. In subsequent generations (F1-F3 generations) only subtle changes on reproductive organs, sperm parameters and DNA methylation but no overall effect on fertility was observed. Consequently, in mice, decitabine and vorinostat neither affected male fertility per se nor caused marked transgenerational effects. We therefore suggest that both drugs do not induce major adverse effects-in terms of male fertility and transgenerational epigenetic inheritance-when used in anti-cancer-therapies.

Experimental endocrine manipulation by contraceptive regimen in the male marmoset (Callithrix jacchus).

Marmosets are used as preclinical model in reproductive research. In contrast to other primates, they display short gestation times rendering this species valid for exploration of effects on fertility. However, their peculiar endocrine regulation differs from a those of macaques and humans. We subjected male marmosets to previously clinically tested hormonal regimens that are known to effectively suppress spermatogenesis. Beside a control group, seven groups (each n=6) were investigated for different periods of up to 42 months: regimen I, (four groups) received testosterone undecanoate (TU) and norethisterone enanthate (NETE); regimen II, (two groups) received TU and NETE followed by NETE only; and regimen III, (one group) received NETE only. Testicular volume, cell ploidy and histology, endocrine changes and fertility were monitored weekly. TU and NETE and initial TU and NETE treatment followed by NETE failed to suppress spermatogenesis and fertility. Testicular volumes dropped, although spermatogenesis was only mildly affected; however, testicular cellular composition remained stable. Serum testosterone dropped when NETE was given alone but the animals remained fertile. Compared with controls, no significant changes were observed in sperm motility and fertility. Administration of TU and NETE affected testicular function only mildly, indicating that the regulatory role of chorionic gonadotrophin and testosterone on spermatogenesis is obviously limited and testicular function is maintained, although the endocrine axis is affected by the treatment. In conclusion, marmosets showed a different response to regimens of male contraception from macaques or men and have to be considered as a problematic model for preclinical trials of male hormonal contraception.

Testosterone-induced prostate growth is blocked by co- and preadministration of norethisterone enanthate in castrated cynomolgus monkeys.

INTRODUCTION: Prostate size and function are regulated by testosterone. However, the progesterone receptor is expressed in the primate prostate. Progestins affect the prostate by endocrine suppression, but can also act directly. Examining the role of progestins, we studied the effects of norethisterone (NET) on testosterone undecanoate (TU)-induced prostate growth in castrated macaques. MATERIALS AND METHODS: Two groups (n = 6 for each group) received TU every 9 weeks. Using a crossover setting, group I received norethisterone enanthate (NETE) 3 times at 3-week intervals, while group II received placebo. After 9 weeks, placebo was administered to group I, and group II received NETE. RESULTS: In group II, the prostate grew under TU and placebo over the first period. In group I, coadministered with NETE, the increase was lower. After the crossover, prostates of animals previously treated with NETE did not increase to normal values under placebo. Prostates of animals treated with TU and placebo in the first period shrank following NETE administration after the crossover. The long half-life of NET can explain the lack of a TU effect on animals coadministered with NETE after the crossover. CONCLUSIONS: Pre- and coadministration of NET reduces testosterone-induced prostate growth with possible implications for the treatment of benign prostate hyperplasia and hormonal male contraception.