To evaluate Tribulus terrestris and Mucuna pruriens for inducing all-male tilapia, mixed-sex Nile tilapia, Oreochromis niloticus, (mean weight 0.025 ± 0.009 g; mean length 1.25 ± 0.012 cm), were given a meal supplemented with either T. terrestris powder (commercial fish feed, 40% crude protein) (TT group), M. pruriens seed extract (MP group), MP + TT (mixed group), 17α-methyl testosterone (MT, control positive), or without supplements (control negative). The MP extracts significantly increased (P < 0.05) the final weight, weight gain, weight gain rate, and specific growth rate while feed conversion ratio was significantly decreased (P < 0.05). Plant extracts markedly improved (P < 0.05) the survival rate, proportion of males, and total testosterone compared to control and MT. Estrogen levels were lower in groups with plant extract than other groups. Fifteen days post-feeding, the Amh gene was expressed in the brain of O. niloticus fries with higher levels in MP, TT, and MT groups. Additionally, the expression of the Sox9 and Dmrt1 genes as a male related genes in fish fry gonads revealed significantly (P < 0.05) higher levels in groups fed on MP, TT, and MT compared to control after 30-day post-feeding, whereas; Foxl2 gene expression as a female related gene was significantly (P < 0.05) lower in fish fed on MP, TT, and MT compared to other groups after 30 days post feeding. Histologically, MT, MP, TT, and the mixture all exhibited solely male reproductive traits without noticeable abnormalities. This study concluded that each of the TT or MP extracts can induce sex reversal in tilapia while having no negative health impact compared to MT as the growth and survival rate in the treated groups with TT and MP were higher than control and group treated with MT.

OBJECTIVE: The aim of this study was to compare the outcomes of in vitro fertilization (IVF) in patients with a poor ovarian response who used methyltestosterone, versus those using a placebo, in an infertility clinic setting.
METHODS: This clinical trial included 120 women who had undergone IVF with intracytoplasmic sperm injection due to poor ovarian reserve and infertility. The study took place at the Yas Infertility Center in Tehran, Iran, between January 1, 2018 and January 1, 2019. In the intervention group, 25 mg of methyltestosterone was administered daily for 2 months prior to the initiation of assisted reproductive treatment. The control group was given placebo tablets for the same duration before starting their cycle. Each group was randomly assigned 60 patients. All analyses were performed using SPSS ver. 23 (IBM Corp.).
RESULTS: The endometrial thickness in the intervention group was 7.57±1.22 mm, whereas in the control group, it was 7.11±1.02 (p=0.028). The gonadotropin number was significantly higher in the control group (64.7±13.48 vs. 57.9±9.25, p=0.001). However, there was no significant difference between the two groups in the antral follicular count. The chemical and clinical pregnancy rates in the intervention group were 18.33% and 15% respectively, compared to 8.33% and 6.67% in the control group. The rate of definitive pregnancy was marginally higher in the intervention group (13.3% vs. 3.3%, p=0.05).
CONCLUSIONS: The findings of this study suggest that pretreatment with methyltestosterone significantly increases endometrium thickness and is associated with an increase in the definitive pregnancy rate.

It is widely known that all-female fish production holds economic value for aquaculture. Sebastes schlegelii, a preeminent economic species, exhibits a sex dimorphism, with females surpassing males in growth. In this regard, achieving all-female black rockfish production could significantly enhance breeding profitability. In this study, we utilized the widely used male sex-regulating hormone, 17α-methyltestosterone (MT) at three different concentrations (20, 40, and 60 ppm), to produce pseudomales of S. schlegelii for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of S. schlegelii, while short term had no significant impact. Histological analysis confirmed sex reversal at all MT concentrations; however, both medium and higher MT concentrations impaired testis development. MT also influenced sex steroid hormone levels in pseudomales, suppressing E2 while increasing T and 11-KT levels. In addition, a transcriptome analysis revealed that MT down-regulated ovarian-related genes (cyp19a1a and foxl2) while up-regulating male-related genes (amh) in pseudomales. Furthermore, MT modulated the TGF-β signaling and steroid hormone biosynthesis pathways, indicating its crucial role in S. schlegelii sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in S. schlegelii and offers an initial insight into the underlying mechanism of sexual reversal in this species.

17α-methyltestosterone (MT) hormone is a synthetic androgenic steroid hormone utilized to induce Nile tilapia transitioning for enhanced production yield. This study specifically focuses on the removal of MT through the utilization of photocatalytic membrane reactor (PMR), which employs an in-house polyvinylidene fluoride (PVDF) ultrafiltration membrane modified with 1% nanomaterials (either TiO2 or α-Fe2O3). The molecular weight cut-off (MWCO) of the in-house membrane falls within the ultrafiltration range. Under UV95W radiation, the PMR with PVDF/TiO2 and PVDF/α-Fe2O3 membranes achieved 100% MT removal at 140 and 160 min, respectively. The MT removal by the commercial NF03 membrane was only at 50%. In contrast, without light irradiation, the MT removal by all the membranes remained unchanged after 180 min, exhibiting lower performance. The incorporation of TiO2 and α-Fe2O3 enhanced water flux and MT removal of the membrane. Notably, the catalytic activity was limited by the distribution and concentration of the catalyst at the membrane surface. The water contact angle did not correlate with the water flux for the composited membrane. The degradation of MT aligned well with Pseudo-first-order kinetic models. Thus, the in-house ultrafiltration PMR demonstrated superior removal efficiency and lower operational costs than the commercial nanofiltration membrane, attributable to its photocatalytic activities.

Methyltestosterone (MT) is one of the most frequently misused anabolic androgenic steroids detected in doping control analysis. The metabolism of MT in humans leads to several phase І metabolites and their corresponding phase Ⅱ conjugates. Previous studies have postulated the 3α-sulfoconjugate of 17α-methyl-5β-androstane-3α,17β-diol (S2) as principal sulfate metabolite of MT, with a detection window exceeding 10 days. However, a final direct and unambiguous confirmation of the structure of this metabolite is missing until now. In this study, we established an approach to detect and identify S2, using intact analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) without complex sample pretreatment. An in vitro study yielded the LC-MS/MS reference retention times of all 3-sulfated 17-methylandrostane-3,17-diol diastereomers, allowing for accurate structure assignment of potentially detected metabolites. In an in vivo excretion study with a single healthy male volunteer, the presence of the metabolite S2 was confirmed after a single oral dose of 10 mg MT. The reference standard was chemically synthesized, characterized by accurate mass mass spectrometry (MS) and nuclear magnetic resonance (NMR), and quantified by quantitative NMR (qNMR). Thus, this study finally provides accurate structure information on the S2 metabolite and a direct analytical method for detection of MT misuse. The availability of the reference material is expected to facilitate further evaluation and subsequent analytical method validation in anti-doping research.

This study focuses on the comparative metabolic profiling and effects of two steroid types: natural and synthetic, specifically 17α-methyl testosterone (17α-MT) at varying concentrations (1.5, 2, and 3 mg/kg) in rainbow trout (Oncorhynchus mykiss). Over a 75-day feeding trial, growth metrics, such as feed efficiency, daily specific growth, live weight gain, total weight gain, and survival rate were systematically monitored every 15 days. At the end of the feeding trial, histopathology, immunohistochemistry, and metabolome analyses were performed in the high-concentration groups (3 mg/kg natural and 3 mg/kg synthetic), in which the lowest survival rate was determined. Key findings reveal that the type of hormone significantly influences growth parameters. While some natural steroids enhanced certain growth aspects, synthetic variants often yielded better results. The metabolomic analysis highlighted significant shifts in the metabolism of tryptophan, purine, folate, primary bile acids, phosphonates, phosphinates, and xenobiotics via cytochrome P450 pathways. Histopathologically, the natural hormone groups showed similar testicular, hepatic, muscular, gill, cerebral, renal, and intestinal tissue structures to the control, with minor DNA damage and apoptosis observed through immunohistochemistry. Conversely, the synthetic hormone groups exhibited moderate DNA damage and mild degenerative and necrotic changes in histopathology.

It is well known that hormones influence and direct most facets of physiology; however, there is still contention regarding the directions of certain relationships, for example, between gonadal hormones and immunity. Among the many proposed relationships relating to gonadal-immune interactions, support for immunosuppressive effects of androgens remains prominent within physiological literature. Although ample study has been directed toward the immunosuppressive effects of androgens, considerable disagreement remains regarding their influence on immune function. In this study, we test the hypothesis that androgens inhibit immunocompetence in the American alligator (Alligator mississippiensis). Developing alligators were incubated at female-producing temperatures with a subset of individuals being exposed to 17-α-methyltestosterone (MT) before sexual determination. 17-α-methyltestosterone is a potent androgen, not aromatizable by crocodilians, that has been found to exert masculinizing effects in exposed crocodilian populations in vivo and in vitro. Additionally, a subset of animals was exposed to a novel antigen to quantify innate and acquired immune function. We recovered no significant differences in leukocyte ratios or proportions between groups and found no significant differences in innate immune function as measured by hemolysis-hemagglutination. However, we did find significant differences in acquired immune function, where masculinized individuals expressed greater antibody titers. Our findings reject the hypothesis that androgens suppress immune function; rather, androgens may be immunoenhancing to acquired humoral responses and neutral to innate humoral immunity in crocodilians.

The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia affinis) were continuously exposed to 17α-ethinylestradiol (EE2, 10 ng/L), methyltestosterone (MT, 10 ng/L (MTL); 50 ng/L (MTH)), and mixtures (EE2+MTL and EE2+MTH). Various endpoints, including sex ratio (phenotypic and genetic), secondary sex characteristics, gonadal histology, and transcriptional profile of genes, were examined. The results showed that G. affinis exposed to MTH and EE2+MTH had a > 89.7 % of phenotypic males in F1 generation, with 34.5 and 50.0 % of these males originated from genetic females, respectively. Moreover, females from F0 and F1 generations exposed to MTH and EE2+MTH exhibited masculinized anal fins and skeletons. The combined effect of MT and EE2 on most endpoints was dependent on MT. Furthermore, significant transcriptional alterations in certain target genes were observed in both the F0 and F1 generations by EE2 and MT alone and by mixtures, showing some degree of interactions. These findings that the effects of EE2+MTH were primarily on the phenotypic sex of G. affinis in offspring generation suggest that G. affinis under chronic exposure to the binary mixture contaminated water could have sex-biased populations.

In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures.

Methyltestosterone is one of the banned drugs in aquaculture, and it should be monitored in food-producing animals. 17α-Methyl-5β-androstane-3α,17β-diol, as the main metabolite of methyltestosterone in vertebrates, could be used as another marker for controlling the administration of methyltestosterone, due to its high residual concentration and slow elimination rate. In this study, an analytical method based on gas chromatography-mass spectrometry (GC-MS) was developed and validated for the simultaneous determination of methyltestosterone and its main metabolite in fish. After pretreatment by liquid-liquid extraction with n-hexane and solid phase extraction with C18 and NH2 columns, the target analytes in the muscle tissues were extracted and concentrated, and the influence of the sample matrix was eliminated. Then, the prepared samples were separated and detected with GC-MS in the selected ion monitoring (SIM) mode. Methyltestosterone-D3 was chosen as the internal standard for quantitation. After optimization, the limits of detection for methyltestosterone and 17α-methyl-5β-androstane-3α,17β-diol were 20 μg kg-1 and 15 μg kg-1, respectively. The limits of quantitation were both 50 μg kg-1. The calibration curves showed good linearity in the concentration range from 50.0 ng mL-1 to 500.0 ng mL-1. The correlation coefficients of methyltestosterone and 17α-methyl-5β-androstane-3α,17β-diol were more than 0.9990. The recoveries of the analytes in real samples were in the range of 99.7-116.6% with the relative standard deviation of 5.2-8.3%. The established method could meet the demand for simultaneous detection of methyltestosterone and its major metabolite, and it could be used to provide more information on the abuse of methyltestosterone in food-producing animals.