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Testosterone administration increases leukocyte-endothelium interactions and inflammation in transgender men

  • Francesca Iannantuoni
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Juan Diego Salazar
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Aranzazu Martinez de Marañon
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Celia Bañuls
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Sandra López-Domènech
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Milagros Rocha
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain

    CIBERehd, Valencia, Spain
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  • Felipe Hurtado-Murillo
    Affiliations
    Gender Identity Unit, University Hospital Dr. Peset, Centro de Salud Sexual y Reproductiva Fuente de San Luis, Valencia, Spain
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  • Carlos Morillas
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Marcelino Gómez-Balaguer
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain
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  • Víctor Manuel Víctor
    Correspondence
    Reprint requests: Víctor Manuel Víctor, Ph.D., Department of Endocrinology and Nutrition, Hospital Dr. Peset, FISABIO, C/ Juan de Garay 21, 46017, Valencia, Spain.
    Affiliations
    Department of Endocrinology and Nutrition, Gender Identity Unit, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region (FISABIO), Valencia, Spain

    CIBERehd, Valencia, Spain

    Department of Physiology, University of Valencia, Valencia, Spain
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Open AccessPublished:October 06, 2020DOI:https://doi.org/10.1016/j.fertnstert.2020.08.002

      Objective

      To evaluate the effect of testosterone treatment on metabolic and inflammation parameters and leukocyte-endothelium interactions in transgender men (TGM).

      Design

      Prospective observational study.

      Setting

      University hospital.

      Patient(s)

      One hundred fifty-seven TGM.

      Intervention(s)

      Administration of testosterone undecanoate (1,000 mg, intramuscular) every 12 weeks.

      Main Outcome Measure(s)

      Endocrine parameters, adhesion molecules (vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and E-selectin), proinflammatory cytokines interleukin-6, and tumor necrosis factor alpha were evaluated in serum before and after treatment using Luminex’s xMAP technology. In addition, interactions between human umbilical vein endothelial cells and polymorphonuclear leukocytes were assessed by flow chamber microscopy.

      Result(s)

      Testosterone treatment led to an increase in leukocyte-endothelium interactions due to an increase in polymorphonuclear leukocytes rolling and adhesion and decreased rolling velocity. It also boosted levels of vascular cell adhesion molecule-1, E-selectin, interleukin-6, and tumor necrosis factor alpha. As expected, testosterone also produced a significant increase in free androgenic index, androstenedione, total testosterone, and atherogenic index of plasma and a decrease in sex hormone–binding globulin and high-density lipoprotein cholesterol.

      Conclusion(s)

      Treatment of TGM with testosterone induces an increase in leukocyte-endothelium interactions and adhesion molecules and proinflammatory cytokines. These effects are a reason to monitor cardiovascular risk in these patients.
      La administración de testosterona incrementa las interacciones endotelio leucocitarias e inflamación en hombres transgénero.

      Objetivo

      Evaluar el efecto del tratamiento con testosterona sobre los parámetros metabólicos y de inflamación y las interacciones endotelio leucocitarias en hombres transgénero (TGM).

      Diseño

      Estudio observacional prospectivo.

      Entorno

      Hospital universitario.

      Pacientes

      157 TGM.

      Intervención(es)

      Administración de undecanoato de testosterona (1000 mg, intramuscular) cada 12 semanas.

      Principales medidas de resultado(s)

      se evaluaron mediante la tecnología xMAP de Luminex los parámetros endocrinos séricos; moléculas de adhesión (molécula1 de adhesión celular vascular, molécula 1 de adhesión intercelular y selectina E), interleuquina 6 proinflamatoria y factor alfa de necrosis tumoral antes y después del tratamiento. Además, se evaluaron las interacciones entre células endoteliales de vena umbilical humana y leucocitos polimorfonucleares mediante microscopia de cámara de flujo.

      Resultados

      el tratamiento con testosterona llevó a un incremento en las interacciones endotelio leucocitarias debido a un aumento de la circulación y adhesión de leucocitos polimorfonucleares y disminución de la velocidad de circulación. También se elevaron los niveles de la molécula 1 de adhesión celular vascular, selectina E, interleuquina 6 y factor alfa de necrosis tumoral. Como se esperaba, la testosterona también produjo un incremento significativo en índice de andrógenos libres, androstenediona, testosterona total e índice aterogénico plasmático y una disminución de la globulina fijadora de esteroides sexuales y colesterol de lipoproteínas de alta densidad.

      Conclusión (es)

      el tratamiento de TGM con testosterona induce un incremento en las interacciones endotelio leucocitarias y moléculas de adhesión y citoquinas proinflamatorias. Estos efectos son una razón para monitorizar el riesgo cardiovascular de estos pacientes.

      Palabras clave

      riesgo cardiovascular, citoquinas, leucocitos, testosterona, transgénero.

      Key Words

      Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/30437
      Transgender men (TGM) are people who were assigned a female gender at birth but who identify as male. Gender-affirming hormone therapy with testosterone is a cornerstone of medical treatment for TGM. These treatments are considered necessary to induce a masculine physical state and reductions in gender dysphoria, perceived stress, depression, and anxiety (
      • Irwig M.S.
      Testosterone therapy for transgender men.
      ). However, the use of testosterone can have metabolic side effects. In particular, androgen treatment can induce increased levels of low-density lipoprotein cholesterol, decreased high-density lipoprotein cholesterol (HDL-c), obesity, hypertension, or alterations in liver enzymes and emotional imbalances (
      • Dizon D.S.
      • Tejada-Berges T.
      • Koelliker S.
      • Steinhoff M.
      • Granai C.O.
      Ovarian cancer associated with testosterone supplementation in a female-to-male transsexual patient.
      ). Furthermore, androgens can reduce the release of nitric oxide (NO) in women (
      • Krishna M.B.
      • Joseph A.
      • Thomas P.L.
      • Dsilva B.
      • Pillai S.M.
      • Laloraya M.
      Impaired arginine metabolism coupled to a defective redox conduit contributes to low plasma nitric oxide in polycystic ovary syndrome.
      ), which leads testosterone to be related with endothelial dysfunction (
      • McCrohon J.A.
      • Jessup W.
      • Handelsman D.J.
      • Celermajer D.S.
      Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1.
      ). Testosterone has been associated with an increase in cardiovascular risk in women (
      • Stanhewicz A.E.
      • Wenner M.M.
      • Stachenfeld N.S.
      Sex differences in endothelial function important to vascular health and overall cardiovascular disease risk across the lifespan.
      ); however, the impact of chronic testosterone exposure on acute cardiovascular events, such as venous thromboembolism and ischemic stroke, among TGM has not been explored (
      • Getahun D.
      • Nash R.
      • Flanders W.D.
      • Baird T.C.
      • Becerra-Culqui T.A.
      • Cromwell L.
      • et al.
      ). High levels of testosterone can have deleterious effects by enhancing oxidative stress in different types of cells, including β-cells and neurons (
      • Paik S.G.
      • Michelis M.A.
      • Kim Y.T.
      • Shin S.
      Induction of insulin-dependent diabetes by streptozotocin: inhibition by estrogens and potentiation by androgens.
      ,
      • Holmes S.
      • Singh M.
      • Su C.
      • Cunningham R.L.
      Effects of oxidative stress and testosterone on pro-inflammatory signaling in a female rat dopaminergic neuronal cell line.
      ). Oxidative stress negatively affects male reproductive function and can induce infertility either directly or indirectly by disrupting crosstalk with other hormonal axes or by affecting the hypothalamus-pituitary-gonadal axis (
      • Darbandi M.
      • Darbandi S.
      • Agarwal A.
      • Sengupta P.
      • Durairajanayagam D.
      • Henkel R.
      • et al.
      Reactive oxygen species and male reproductive hormones.
      ). Moreover, high levels of testosterone in patients with polycystic ovary syndrome (PCOS) have also been related to oxidative stress (
      • Gonzalez F.
      • Rote N.S.
      • Minium J.
      • Kirwan J.P.
      Reactive oxygen species-induced oxidative stress in the development of insulin resistance and hyperandrogenism in polycystic ovary syndrome.
      ,
      • Zuo T.
      • Zhu M.
      • Xu W.
      Roles of oxidative stress in polycystic ovary syndrome and cancers.
      ).
      In this context, different studies have demonstrated that polymorphonuclear leukocytes (PMNs) produce high levels of reactive oxygen species (ROS) (
      • Gonzalez F.
      • Rote N.S.
      • Minium J.
      • Kirwan J.P.
      Reactive oxygen species-induced oxidative stress in the development of insulin resistance and hyperandrogenism in polycystic ovary syndrome.
      ,
      • Victor V.M.
      • Rocha M.
      • Banuls C.
      • Rovira-Llopis S.
      • Gomez M.
      • Hernandez-Mijares A.
      Mitochondrial impairment and oxidative stress in leukocytes after testosterone administration to female-to-male transsexuals.
      ) and proinflammatory mediators (
      • Victor V.M.
      • Rocha M.
      • Banuls C.
      • Alvarez A.
      • de Pablo C.
      • Sanchez-Serrano M.
      • et al.
      Induction of oxidative stress and human leukocyte/endothelial cell interactions in polycystic ovary syndrome patients with insulin resistance.
      ). Endothelial cells can be activated by ROS and proinflammatory cytokines, thereby leading to endothelial dysfunction and blood vessels alterations (
      • Kim F.
      • Tysseling K.A.
      • Rice J.
      • Gallis B.
      • Haji L.
      • Giachelli C.M.
      • et al.
      Activation of IKKbeta by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells.
      ,
      • Ley K.
      • Laudanna C.
      • Cybulsky M.I.
      • Nourshargh S.
      Getting to the site of inflammation: the leukocyte adhesion cascade updated.
      ). This process triggers a cascade of PMN-endothelium interactions, a process by which immune cells migrate to the inflammatory focus (
      • Ley K.
      • Laudanna C.
      • Cybulsky M.I.
      • Nourshargh S.
      Getting to the site of inflammation: the leukocyte adhesion cascade updated.
      ). This proinflammatory state and increased ROS content is characteristic of the high levels of testosterone in TGM, which favor leukocyte-endothelial interactions throughout the vasculature and not only at the site of inflammation. Therefore, the purpose of this study was to assess the effect of testosterone treatment on metabolic parameters, leukocyte-endothelium interactions, and inflammatory markers in TGM subjects.

      Material and Methods

       Subjects

      Our study population was composed of 157 TGM receiving medical attention at the Department of Endocrinology and Nutrition at the University Hospital Dr. Peset, Valencia, Spain. The following criteria were applied: having been assigned 12-week treatment with testosterone undecanoate (intramuscular) at a dose of 1,000 mg; no treatment that could have interfered with the physiological function of the gonadal-pituitary-hypothalamic axis in the previous 6 months; no infectious, inflammatory, malignant, hematological, or organic disease; and absence of history of heart disease, stroke, thromboembolism, hyperlipidemia, hypertension, PCOS, or diabetes mellitus. Transgender men were evaluated and managed according to Diagnostic and Statistical Manual of Mental Disorders V criteria. Eligibility of the subjects for gender-affirming hormone treatment was determined following the guidelines of the World Professional Association for Transgender Health and those of the Endocrine Society. All the subjects were naive to hormonal treatment and eugonadal, which were confirmed by medical history, physical examination, and biochemical criteria before initiating treatment. Oophorectomy was not performed in any of these patients.All participants were informed about all the methods and procedures of the study and gave their written consent. The study was conducted in accordance with the Helsinki Declaration and approved by the Ethics Committee of University Hospital Dr. Peset (ID: 98/19).

       Main Outcome Measures: Anthropometric and Metabolic Parameters

      During the medical examination that was performed to assess subjects for inclusion in the study, anthropometric and metabolic parameters were measured, including body weight, waist circumference, body mass index, and blood pressure, as shown in Table 1. To evaluate hormone levels, blood samples were collected from the antecubital vein between 8:00 a.m. and 10:00 a.m. Total testosterone (TT), sex hormone–binding globulin (SHBG), and androstenedione levels were evaluated by specific chemiluminescent microparticle immunoassay (Architect 2nd Generation Testosterone Assay, Abbott Diagnostics). Free androgenic index was calculated with the formula ([TT]/[SHBG]) × 100.
      Table 1Anthropometric, clinical, and metabolic parameters in transgender men before and after 12 weeks of testosterone treatment.
      VariableTGM baselineTGM post-treatment
      n157157
      Age, y26.2 ± 7.5
      Body mass index, kg/m223.4 ± 3.124.1 ± 3.3
      Weight, kg62.8 ± 1066.4 ± 8.9
      Waist circumference, cm85.6 ± 7.587.8 ± 7
      Systolic BP, mm Hg109 ± 9118 ± 17
      Diastolic BP, mm Hg66 ± 972 ± 11
      TT, ng/mL0.60 ± 0.758.39 ± 5.43
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      SHBG, nmol/L41.6 ± 22.633.5 ± 41.6
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      FAI3.74 ± 2.5131.79 ± 88.1
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      Androstenedione, ng/mL3.54 ± 1.444.66 ± 1.85
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      hs-CRP, mg/dL3.38 ± 6.203.35 ± 4.44
      Total cholesterol, mg/dL172.2 ± 40.1167.7 ± 31.57
      HDL-c, mg/dL49.23 ± 11.943.35 ± 9.42
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      LDL-c, mg/dL110.1 ± 38.1109.4 ± 28
      Triglycerides, mg/dL62 (49, 83)70 (61, 112)
      AIP, log10 triglycerides/HDL-c0.15 ± 0.230.23 ± 0.21
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      Glucose, mg/dL85.88 ± 6.185.01 ± 7.1
      Insulin, μUl/mL8.63 ± 2.838.03 ± 3.96
      HOMA-IR1.65 ± 0.711.68 ± 1.01
      Note: Comparison between baseline and after treatment was done using a paired Student’s t test. Data are expressed as mean ± standard deviation, except for triglycerides, which are represented as media and interquartile range. Triglyceride serum concentrations were normalized using a log transformation. AIP = atherogenic index of plasma; BP = blood pressure; FAI = free androgenic index; HDL-c = high-density lipoprotein cholesterol; HOMA-IR = homeostasis model assessment-insulin resistance; hs-CRP = high-sensitive C-reactive protein; LDL-c = low-density lipoprotein cholesterol; SHBG = sex hormone–binding globulin; TGM = transgender men; TT = total testosterone.
      P < .05; ∗∗P < .01; ∗∗∗P < .001.
      A latex-enhanced immunonephelometric assay (Boehringer Nephelometer II, Dade Behring) was used to evaluate high-sensitive C-reactive protein. Enzymatic assays were also used to evaluate total cholesterol and triglycerides: a Beckman LX-20 autoanalyzer (Beckman Coulter) was employed to determine HDL-c concentration, and Friedewald’s method was employed to evaluate low-density lipoprotein cholesterol. To calculate the atherogenic index of plasma (AIP) a logarithm of the ratio of plasma concentration of triglycerides to HDL-c was used. Glucose levels were measured using an enzymatic method with a Dax-72 autoanalyzer (Bayer Diagnostic); insulin was evaluated by enzymatic luminescence techniques; insulin resistance was calculated by homeostasis model assessment using glucose and baseline insulin according to the formula homeostasis model assessment = (fasting insulin (U/mL) × fasting glucose (mmol/L)/22.5.

       Cells

      Between 8:00 a.m. and 10:00 a.m., a citrate blood sample was collected from the antecubital vein of participants to perform PMN extraction. Samples were incubated with dextran 3% for 45 minutes, and the supernatant was resuspended over Fycoll-Hypaque and centrifuged at 250 g for 25 minutes. Pellets were lysated with a lysis buffer, centrifuged at 100 g for 5 minutes, and washed with Hank’s balanced salt solution (Sigma Aldrich). Cells were counted using a Scepter device (Millipore). An aliquot of 1.2 million cells was resuspended in 1.2 mL of complete Roswell Park Memorial Institute medium (Biowest-bw) to perform an ex vivo adhesion assay. Endothelial cells from human umbilical cords (HUVEC) were extracted by means of collagenase digestion, seeded in monolayers on fibronectin-coated plastic coverslips (Sigma Aldrich), and maintained in culture in EBM2 medium (PromoCell) supplemented with Supplement Pack Endothelial Cell GM2 (PromoCell) until leukocyte-endothelium microscope interaction analysis.

       Leukocyte-Endothelium Cell Interaction Assay

      Adhesion assays were evaluated using an inverted microscope (Nikon Eclipse TE 2000-S) with a flow chamber in continuous and stable flow conditions (flow rate = 0.36 mL/minute). To perform the assay, coverslips containing HUVEC cells were placed on the bottom plate of the flow chamber so that a field of 5 × 25 mm was exposed. The prepared PMNs sample was perfused through the HUVEC monolayer over a 5-minute period to evaluate PMNs interactions. These analyses were made possible by a real-time camera system (Sony Exware HAD) connected to the microscope. By means of this assay is it possible to evaluate PMN rolling velocity and flux and PMN adhesion. Platelet-activating factor (1 μM, 1 hour) and tumor necrosis factor (10 ng/mL, 4 hour) were used as positive controls for leukocytes and HUVEC, respectively.

       Adhesion Molecule Expression, Interleukin-6 (IL-6), and Tumor Necrosis Factor Alpha (TNFα) Levels

      Adhesion molecules E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) and levels of the proinflammatory cytokines IL-6 and TNFα were measured in serum from subjects at baseline and after 12 weeks with a Luminex 200 flow analyzer (Millipore).

       Data Analysis

      SPSS 17.0 software (SPSS) was used to perform statistical analyses. Continuous variables for parametric and nonparametric data were expressed as mean ± standard deviation or as median and 25th and 75th percentiles, respectively. The bar graphs in the figure show mean ± standard deviation. The Kolmogorov-Smirnov test was applied to the data to determine whether the distribution of the variables was normal. A paired Student’s t test was used for comparisons between groups. A confidence interval of 95% was employed for all tests, and differences were considered significant when P < .05.

      Results

       Study Population Characteristics

      The present study analyzed 157 TGM on testosterone treatment, at baseline and after 12 weeks of treatment. As shown in Table 1, no differences were observed between anthropometric values of subjects at baseline and those after 12 weeks of treatment. In contrast, we found significant differences in endocrine variables after testosterone treatment, also shown in Table 1. In fact, a significant increase was observed in TT (P < .001), free androgenic index (P < .001), androstenedione (P < .01), and AIP (P < .01) and a significant decrease was seen in SHBG levels (P < .05) and HDL-c (P < .001).

       Adhesion Assay

      Analysis of the adhesion assay data revealed that testosterone treatment increased leukocyte-endothelium interactions by reducing the rolling velocity of PMNs (Fig. 1A, P < .01), significantly increasing rolling flux (Fig. 1B, P < .001) and, in turn, the adhesion of PMNs (Fig. 1C, P < .001).
      Figure thumbnail gr1
      Figure 1Analysis of polymorphonuclear leukocytes– (PMNs-) endothelium interactions in transgender men (TGM) after 12 weeks of testosterone treatment. (A) Velocity of PMNs measured as micrometers/second. (B) Number of PMNs rolling along the endothelial monolayer during a 1-minute period, measured as number of cells/minute. (C) Number of adhering PMNs in 1 mm2, measured as cell number/mm2. ∗∗P < .01 and ∗∗∗P < .001 in TGM before and after treatment.

       Levels of Adhesion Molecules, Cytokines, and TNFα

      When we analyzed proinflammatory cytokines, we found a significant increase in IL-6 and TNFα levels after testosterone treatment (Fig. 2A and 2B, P < .05 and P < .01, respectively), thus suggesting a proinflammatory effect of testosterone in TGM. Regarding soluble adhesion molecules, we observed an increase in E-selectin and VCAM-1 levels (Fig. 3A and 3B, both P < .05), but not in ICAM-1, the levels of which remained unaltered after testosterone treatment (Fig. 3C).
      Figure thumbnail gr2
      Figure 2Serum and cellular inflammatory markers in transgender men (TGM) before and after 12 weeks of testosterone treatment. Serum levels of (A) interleukin-6 and (B) tumor necrosis factor alpha. ∗P < .05 and ∗∗P < .01 in TGM before and after treatment.
      Figure thumbnail gr3
      Figure 3Soluble adhesion molecules in the serum of transgender men (TGM) before and after 12 weeks of testosterone treatment. Serum levels of (A) E-selectin, (B) vascular cell adhesion molecule-1, and (C) intercellular cell adhesion molecule-1. ∗P < .05 in TGM before and after treatment.

      Discussion

      The present study demonstrates that 12-week treatment with testosterone undecanoate increased leukocyte-endothelium interactions, adhesion molecules, and proinflammatory cytokines in a large cohort of TGM. This effect was evident in the decreased rolling velocity and increased PMN rolling and adhesion, as well as higher levels of IL-6, TNFα, E-selectin, and VCAM-1. In addition, we observed a significant increase of androgens that was accompanied by an increase of AIP and a decrease of HDL-c and SHBG.
      The literature contains different studies that demonstrate that testosterone esters can be administered to raise levels of testosterone in men with hypogonadism and in TGM, producing several desired physical effects and mental benefits, as well as undesired and undetermined effects related to high concentrations of this hormone (
      • Cupisti S.
      • Giltay E.J.
      • Gooren L.J.
      • Kronawitter D.
      • Oppelt P.G.
      • Beckmann M.W.
      • et al.
      The impact of testosterone administration to female-to-male transsexuals on insulin resistance and lipid parameters compared with women with polycystic ovary syndrome.
      ,
      • Buvat J.
      • Maggi M.
      • Guay A.
      • Torres L.O.
      Testosterone deficiency in men: systematic review and standard operating procedures for diagnosis and treatment.
      ,
      • Gulanski B.I.
      • Flannery C.A.
      • Peter P.R.
      • Leone C.A.
      • Stachenfeld N.S.
      Compromised endothelial function in transgender men taking testosterone.
      ). On the other hand, there are studies that have shown that androgen deprivation can affect physiological functions such as insulin sensitivity, levels of lipoproteins, sexual life, or orgasmic disorders (
      • Corona G.
      • Gacci M.
      • Baldi E.
      • Mancina R.
      • Forti G.
      • Maggi M.
      Androgen deprivation therapy in prostate cancer: focusing on sexual side effects.
      ). The goal of testosterone therapy is usually to achieve adequate serum testosterone concentrations in cisgender men. In the present study, mean testosterone levels reached 8.39 ng/mL, which is high according to the guidelines of the Endocrine Society regarding transgender people. The main advantages of these treatments are the achievement of relatively normal and stable testosterone levels and the least frequent dosing among intramuscular preparations.
      Furthermore, testosterone has been shown to activate and sensitize PMNs to high doses of glucose in patients with PCOS (with enhanced levels of testosterone) and other females treated with high doses of testosterone (
      • Gonzalez F.
      • Nair K.S.
      • Daniels J.K.
      • Basal E.
      • Schimke J.M.
      • Blair H.E.
      Hyperandrogenism sensitizes leukocytes to hyperglycemia to promote oxidative stress in lean reproductive-age women.
      ). In fact, different reports have demonstrated that PCOS is characterized by enhanced levels of ROS and mitochondrial impairment (
      • Victor V.M.
      • Rocha M.
      • Banuls C.
      • Alvarez A.
      • de Pablo C.
      • Sanchez-Serrano M.
      • et al.
      Induction of oxidative stress and human leukocyte/endothelial cell interactions in polycystic ovary syndrome patients with insulin resistance.
      ,
      • Murri M.
      • Luque-Ramirez M.
      • Insenser M.
      • Ojeda-Ojeda M.
      • Escobar-Morreale H.F.
      Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis.
      ). However, PCOS is a multifactorial condition, and direct comparisons are difficult.
      It is well established that hypertension and atherosclerosis are characterized by recruitment of leukocytes by the arterial wall. To evaluate this process in our laboratory, we have designed an ex vivo model in which human PMNs flow over a monolayer of HUVEC cells with a shear stress similar to that observed in vivo (
      • Hernandez-Mijares A.
      • Rocha M.
      • Rovira-Llopis S.
      • Bañuls C.
      • Bellod L.
      • de Pablo C.
      • et al.
      Human leukocyte/endothelial cell interactions and mitochondrial dysfunction in type 2 diabetic patients and their association with silent myocardial ischemia.
      ). This method reproduces the process that precedes inflammation in vivo (rolling and adhesion) and that is critical to homeostasis and vascular cell integrity and function. When these interactions are enhanced, it is possible to develop vascular dysfunction and, consequently, cardiovascular diseases (
      • Krieglstein C.F.
      • Granger D.N.
      Adhesion molecules and their role in vascular disease.
      ). Our experimental system has been widely applied to visualize and analyze the multistep recruitment of PMNs in different conditions and allows the mechanisms of action implicated in this recruitment to be evaluated (
      • Rao R.M.
      • Yang L.
      • Garcia-Cardena G.
      • Luscinskas F.W.
      Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall.
      ). In the present study, we have observed how high levels of testosterone sustained over 12 weeks of treatment induced a significant increase in rolling flux and PMNs adhesion and a decrease in its rolling velocity in TGM. It is difficult to evaluate whether the magnitude of this change was clinically significant, as we did not establish ranges in a control population, but the levels of leukocyte-endothelium interactions (rolling flux and PMNs adhesion) after testosterone treatment were in similar ranges to those reported by a previous study of cisgender male type 2 diabetic patients with silent myocardial ischemia (
      • Hernandez-Mijares A.
      • Rocha M.
      • Rovira-Llopis S.
      • Bañuls C.
      • Bellod L.
      • de Pablo C.
      • et al.
      Human leukocyte/endothelial cell interactions and mitochondrial dysfunction in type 2 diabetic patients and their association with silent myocardial ischemia.
      ). In line with the potentially deleterious effects of testosterone on the blood vessels, this hormone has also been associated with endothelial dysfunction in women (
      • Liu P.Y.
      • Death A.K.
      • Handelsman D.J.
      Androgens and cardiovascular disease.
      ). In fact, the activation of androgen receptors in women can undermine NO release and NO responsiveness (
      • Usselman C.W.
      • Yarovinsky T.O.
      • Steele F.E.
      • Leone C.A.
      • Taylor H.S.
      • Bender J.R.
      • et al.
      Androgens drive microvascular endothelial dysfunction in women with polycystic ovary syndrome: role of the endothelin B receptor.
      ), therefore impairing vascular function.
      In addition, a previous study of patients with PCOS demonstrated that proinflammatory cytokines, oxidative stress, and NF-κB activation can contribute to endothelial impairment, thus highlighting that the impaired endothelial function in TGM is independent of obesity and blood lipids and is likely to be associated with inflammatory factors due to testosterone (
      • Krishna M.B.
      • Joseph A.
      • Thomas P.L.
      • Dsilva B.
      • Pillai S.M.
      • Laloraya M.
      Impaired arginine metabolism coupled to a defective redox conduit contributes to low plasma nitric oxide in polycystic ovary syndrome.
      ). Again, it is important to highlight that PCOS is a multifactorial condition, and direct comparisons are difficult.
      A recent meta-analysis revealed dyslipidemia at 3, 6, and 24 months in TGM treated with androgen (dihydrotestosterone) (
      • Maraka S.
      • Singh Ospina N.
      • Rodriguez-Gutierrez R.
      • Davidge-Pitts C.J.
      • Nippoldt T.B.
      • Prokop L.J.
      • et al.
      Sex steroids and cardiovascular outcomes in transgender individuals: a systematic review and meta-analysis.
      ). While hormone therapy with testosterone also induced an increase in blood pressure, insulin resistance, and dyslipidemia, the young TGM in question did not show signs of cardiovascular morbidity or mortality (
      • Streed Jr., C.G.
      • Harfouch O.
      • Marvel F.
      • Blumenthal R.S.
      • Martin S.S.
      • Mukherjee M.
      Cardiovascular disease among transgender adults receiving hormone therapy: a narrative review.
      ). In addition, in a study of a large cohort of TGM, Nota et al. (
      • Nota N.M.
      • Wiepjes C.M.
      • de Blok C.J.M.
      • Gooren L.J.G.
      • Kreukels B.P.C.
      • den Heijer M.
      Occurrence of acute cardiovascular events in transgender individuals receiving hormone therapy.
      ) demonstrated that TGM receiving testosterone were at a higher risk of myocardial infarction. However, although Getahun et al. (
      • Getahun D.
      • Nash R.
      • Flanders W.D.
      • Baird T.C.
      • Becerra-Culqui T.A.
      • Cromwell L.
      • et al.
      ) demonstrated the impact of cross-sex hormones on acute cardiovascular events (such as venous thromboembolism, ischemic stroke, and myocardial infarction) in transgender people, the evidence was insufficient to draw conclusions regarding risk among TGM. It is important to stress that no study has yet followed a large cohort of transgender people as they age and the risk of cardiovascular disease rises, so the long-term impact on cardiovascular disease remains unknown.
      Endothelial activation can also be assessed by measuring the soluble adhesion molecules E-selectin, VCAM-1, and ICAM-1. In general, circulating levels of adhesion molecules are elevated in healthy individuals in response to systemic inflammation (
      • Sela S.
      • Mazor R.
      • Amsalam M.
      • Yagil C.
      • Yagil Y.
      • Kristal B.
      Primed polymorphonuclear leukocytes, oxidative stress, and inflammation antecede hypertension in the sabra rat.
      ). In the current study, we have observed an increase in adhesion molecules in line with a rise in the number of leukocyte-endothelium interactions and selectins that mediate the initial tethering and subsequent rolling of PMNs. The elevated levels of adhesion molecules observed in the serum of TGM patients are of particular interest, as they provide strong evidence of an ongoing inflammatory process in the endothelium that could, theoretically, lead to cardiovascular diseases. Furthermore, we have observed an increase in proinflammatory levels of the cytokines TNF-α and IL-6 in our TGM population; this is relevant, as an enhanced release of TNF-α from PMNs after activation by ROS-induced oxidative stress may inhibit insulin signaling and impair glucose uptake (
      • Gonzalez F.
      • Rote N.S.
      • Minium J.
      • Kirwan J.P.
      In vitro evidence that hyperglycemia stimulates tumor necrosis factor-alpha release in obese women with polycystic ovary syndrome.
      ).

       Conclusion

      Although gender-affirming hormones are considered medically necessary, the present research demonstrates that testosterone can induce an increase in leukocyte-endothelium interactions, adhesion molecules, and proinflammatory cytokines in TGM. Future research concerning endothelial impairment and inflammation may determine the physiological mechanism involved in this effect, an effect that should be taken into account to monitor cardiovascular risk in these individuals.

      Acknowledgments

      The authors thank Brian Normanly, University of Valencia-CIBERehd, for his editorial assistance; and Rosa Falcón for her technical support.

      References

        • Irwig M.S.
        Testosterone therapy for transgender men.
        Lancet Diabetes Endocrinol. 2017; 5: 301-311
        • Dizon D.S.
        • Tejada-Berges T.
        • Koelliker S.
        • Steinhoff M.
        • Granai C.O.
        Ovarian cancer associated with testosterone supplementation in a female-to-male transsexual patient.
        Gynecol Obstet Invest. 2006; 62: 226-228
        • Krishna M.B.
        • Joseph A.
        • Thomas P.L.
        • Dsilva B.
        • Pillai S.M.
        • Laloraya M.
        Impaired arginine metabolism coupled to a defective redox conduit contributes to low plasma nitric oxide in polycystic ovary syndrome.
        Cell Physiol Biochem. 2017; 43: 1880-1892
        • McCrohon J.A.
        • Jessup W.
        • Handelsman D.J.
        • Celermajer D.S.
        Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1.
        Circulation. 1999; 99: 2317-2322
        • Stanhewicz A.E.
        • Wenner M.M.
        • Stachenfeld N.S.
        Sex differences in endothelial function important to vascular health and overall cardiovascular disease risk across the lifespan.
        Am J Physiol Heart Circ Physiol. 2018; 315: H1569-H1588
        • Getahun D.
        • Nash R.
        • Flanders W.D.
        • Baird T.C.
        • Becerra-Culqui T.A.
        • Cromwell L.
        • et al.
        Ann Intern Med. 2018; 169: 205-213
        • Paik S.G.
        • Michelis M.A.
        • Kim Y.T.
        • Shin S.
        Induction of insulin-dependent diabetes by streptozotocin: inhibition by estrogens and potentiation by androgens.
        Diabetes. 1982; 31: 724-729
        • Holmes S.
        • Singh M.
        • Su C.
        • Cunningham R.L.
        Effects of oxidative stress and testosterone on pro-inflammatory signaling in a female rat dopaminergic neuronal cell line.
        Endocrinology. 2016; 157: 2824-2835
        • Darbandi M.
        • Darbandi S.
        • Agarwal A.
        • Sengupta P.
        • Durairajanayagam D.
        • Henkel R.
        • et al.
        Reactive oxygen species and male reproductive hormones.
        Reprod Biol Endocrinol. 2018; 16: 87-100
        • Gonzalez F.
        • Rote N.S.
        • Minium J.
        • Kirwan J.P.
        Reactive oxygen species-induced oxidative stress in the development of insulin resistance and hyperandrogenism in polycystic ovary syndrome.
        J Clin Endocrinol Metab. 2006; 91: 336-340
        • Zuo T.
        • Zhu M.
        • Xu W.
        Roles of oxidative stress in polycystic ovary syndrome and cancers.
        Oxid Med Cell Longev. 2016; 20168589318
        • Victor V.M.
        • Rocha M.
        • Banuls C.
        • Rovira-Llopis S.
        • Gomez M.
        • Hernandez-Mijares A.
        Mitochondrial impairment and oxidative stress in leukocytes after testosterone administration to female-to-male transsexuals.
        J Sex Med. 2014; 11: 454-461
        • Victor V.M.
        • Rocha M.
        • Banuls C.
        • Alvarez A.
        • de Pablo C.
        • Sanchez-Serrano M.
        • et al.
        Induction of oxidative stress and human leukocyte/endothelial cell interactions in polycystic ovary syndrome patients with insulin resistance.
        J Clin Endocrinol Metab. 2011; 96: 3115-3122
        • Kim F.
        • Tysseling K.A.
        • Rice J.
        • Gallis B.
        • Haji L.
        • Giachelli C.M.
        • et al.
        Activation of IKKbeta by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells.
        J Mol Cell Cardiol. 2005; 39: 327-334
        • Ley K.
        • Laudanna C.
        • Cybulsky M.I.
        • Nourshargh S.
        Getting to the site of inflammation: the leukocyte adhesion cascade updated.
        Nat Rev Immunol. 2007; 7: 678-689
        • Cupisti S.
        • Giltay E.J.
        • Gooren L.J.
        • Kronawitter D.
        • Oppelt P.G.
        • Beckmann M.W.
        • et al.
        The impact of testosterone administration to female-to-male transsexuals on insulin resistance and lipid parameters compared with women with polycystic ovary syndrome.
        Fertil Steril. 2010; 94: 2647-2653
        • Buvat J.
        • Maggi M.
        • Guay A.
        • Torres L.O.
        Testosterone deficiency in men: systematic review and standard operating procedures for diagnosis and treatment.
        J Sex Med. 2013; 10: 245-284
        • Gulanski B.I.
        • Flannery C.A.
        • Peter P.R.
        • Leone C.A.
        • Stachenfeld N.S.
        Compromised endothelial function in transgender men taking testosterone.
        Clin Endocrinol (Oxf). 2020; 92: 138-144
        • Corona G.
        • Gacci M.
        • Baldi E.
        • Mancina R.
        • Forti G.
        • Maggi M.
        Androgen deprivation therapy in prostate cancer: focusing on sexual side effects.
        J Sex Med. 2012; 9: 887-902
        • Gonzalez F.
        • Nair K.S.
        • Daniels J.K.
        • Basal E.
        • Schimke J.M.
        • Blair H.E.
        Hyperandrogenism sensitizes leukocytes to hyperglycemia to promote oxidative stress in lean reproductive-age women.
        J Clin Endocrinol Metab. 2012; 97: 2836-2843
        • Murri M.
        • Luque-Ramirez M.
        • Insenser M.
        • Ojeda-Ojeda M.
        • Escobar-Morreale H.F.
        Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis.
        Hum Reprod Update. 2013; 19: 268-288
        • Hernandez-Mijares A.
        • Rocha M.
        • Rovira-Llopis S.
        • Bañuls C.
        • Bellod L.
        • de Pablo C.
        • et al.
        Human leukocyte/endothelial cell interactions and mitochondrial dysfunction in type 2 diabetic patients and their association with silent myocardial ischemia.
        Diabetes Care. 2013; 36: 1695-1702
        • Krieglstein C.F.
        • Granger D.N.
        Adhesion molecules and their role in vascular disease.
        Am J Hypertens. 2001; 14: 44S-54S
        • Rao R.M.
        • Yang L.
        • Garcia-Cardena G.
        • Luscinskas F.W.
        Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall.
        Circ Res. 2007; 101: 234-247
        • Liu P.Y.
        • Death A.K.
        • Handelsman D.J.
        Androgens and cardiovascular disease.
        Endocr Rev. 2003; 24: 313-340
        • Usselman C.W.
        • Yarovinsky T.O.
        • Steele F.E.
        • Leone C.A.
        • Taylor H.S.
        • Bender J.R.
        • et al.
        Androgens drive microvascular endothelial dysfunction in women with polycystic ovary syndrome: role of the endothelin B receptor.
        J Physiol. 2019; 597: 2853-2865
        • Maraka S.
        • Singh Ospina N.
        • Rodriguez-Gutierrez R.
        • Davidge-Pitts C.J.
        • Nippoldt T.B.
        • Prokop L.J.
        • et al.
        Sex steroids and cardiovascular outcomes in transgender individuals: a systematic review and meta-analysis.
        J Clin Endocrinol Metab. 2017; 102: 3914-3923
        • Streed Jr., C.G.
        • Harfouch O.
        • Marvel F.
        • Blumenthal R.S.
        • Martin S.S.
        • Mukherjee M.
        Cardiovascular disease among transgender adults receiving hormone therapy: a narrative review.
        Ann Intern Med. 2017; 167: 256-257
        • Nota N.M.
        • Wiepjes C.M.
        • de Blok C.J.M.
        • Gooren L.J.G.
        • Kreukels B.P.C.
        • den Heijer M.
        Occurrence of acute cardiovascular events in transgender individuals receiving hormone therapy.
        Circulation. 2019; 139: 1461-1462
        • Sela S.
        • Mazor R.
        • Amsalam M.
        • Yagil C.
        • Yagil Y.
        • Kristal B.
        Primed polymorphonuclear leukocytes, oxidative stress, and inflammation antecede hypertension in the sabra rat.
        Hypertension. 2004; 44: 764-769
        • Gonzalez F.
        • Rote N.S.
        • Minium J.
        • Kirwan J.P.
        In vitro evidence that hyperglycemia stimulates tumor necrosis factor-alpha release in obese women with polycystic ovary syndrome.
        J Endocrinol. 2006; 188: 521-529