Functional ovarian reserve in transgender men receiving testosterone therapy: evidence for preserved anti-Müllerian hormone and antral follicle count under prolonged treatment.

STUDY QUESTION: Is the functional ovarian reserve in transgender men affected by testosterone therapy? SUMMARY ANSWER: Serum anti-Müllerian Hormone (AMH) levels slightly decrease during testosterone treatment but remain within the normal range, suggesting preserved follicular ovarian reserve. WHAT IS KNOWN ALREADY: Few small studies have investigated the impact of gender-affirming treatment on reproduction in transgender men. Conflicting results were reached concerning ovarian morphology and AMH levels in this context. STUDY DESIGN, SIZE, DURATION: The study consisted of two arms. The first arm was a prospective pilot study, which enrolled 56 transgender men (median age 22.5 [interquartile range (IQR)-19-27.7] years), 27 of whom had polycystic ovary syndrome (PCOS), prior to the initiation of gender-affirming testosterone therapy. A structured assessment was conducted prior to, and at 3 and 12 months after treatment initiation. The second arm was a cross-sectional study that comprised 47 transgender men (median age 24 [IQR-20-31] years) who received testosterone for a median duration of 35 [IQR 13-62] months. The main outcome measures were serum AMH and antral follicle count (AFC) as indices of ovarian follicular reserve. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a tertiary center for transgender health. Gender-affirming therapy was administered according to standard practice. AFC was determined by pelvic (abdominal or transvaginal) ultrasound and blood collection for measurements of AMH, testosterone, estradiol, LH and FSH was performed at the designated time-points. MAIN RESULTS AND THE ROLE OF CHANCE: Prospective arm for the entire group we observed a decrease of 0.71 ng/ml in AMH levels between baseline and 12 months (P = 0.01). When expressed in age-specific percentiles, AMH went from the 47.37th to the 40.25th percentile at 12 months (P < 0.001). In a sub-group analysis, a decline of 9.52 points in age-specific percentile was seen in subjects with PCOS (P < 0.001), while no changes were detected in the non-PCOS group. Testosterone treatment did not affect AFC over time in the entire cohort. In the sub-group analysis, a mean decrease of 5.0 follicles was detected between baseline and the 12 months assessment (P = 0.047) only in subjects with PCOS. In the cross-sectional study, AMH inversely correlated with age but not with treatment duration. Notably AMH did not deviate from the 50th age-specific percentile. Finally, four men fathered biological children after being under testosterone treatment for up to 12 years. LIMITATIONS, REASONS FOR CAUTION: The limited sample size of the pilot study should be kept in mind. An additional limitation is the lack of a control group in the prospective study, as each participant served as his own control. Also, roughly 40% of the ultrasound examinations were performed transabdominally, potentially affecting the accuracy of the AFC measurements.As study participants were quite young, our reassuring data may not apply to older transgender men, either because of an age-related decline in ovarian reserve or to possible long-term effects of testosterone therapy. Furthermore, the chances for fertility preservation may be more limited in subjects with PCOS. WIDER IMPLICATIONS OF THE FINDINGS: This is an additional contribution to the emerging evidence that prolonged testosterone treatment may not be a major obstacle to later fertility potential in transgender men desirous of having children. Larger confirmatory studies, and particularly more with reproductive outcome data, are needed for evidence-based fertility counseling prior to treatment initiation in these subjects. STUDY FUNDING/COMPETING INTEREST(S): This study received no funding. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Insulin-degrading enzyme higher in subjects with metabolic syndrome.

Metabolic syndrome (MS) is comprised of a cluster of abnormalities in glucose, lipid, and vascular homeostasis, which is most commonly linked to abdominal obesity. MS heralds increased risk for development of diabetes and is linked to impairment in insulin signaling. Insulin-degrading enzyme (IDE) is one of the mechanisms through which insulin blood levels are maintained. It has been previously suggested that controlling IDE levels could provide yet another potential therapeutic approach in diabetes. Here we aim to investigate whether changes in serum IDE levels correlate with the severity of MS. Using a highly sensitive ELISA assay of active IDE in human serum, we found a strong correlation between circulating IDE levels and circulating levels of triglycerides, insulin, and c-peptide and an inverse correlation with HDL cholesterol (HDLc). Serum IDE levels were higher in MS subjects than in control subjects. Hence, circulating IDE may serve as a tool to identify subjects with abnormal insulin metabolism, possibly those with MS that are at risk to develop diabetes.

Interaction between beta-carotene and lipoxygenase in human skin.

beta-Carotene is widely used in skin care therapy. Its effects on skin are unclear, but actions on lipid peroxidation pathways may be an important element of any protection activities it exerts. This study examines the possible effects of Beta-carotene on enzymatic lipid peroxidation by lipoxygenase in human skin, using in vitro and ex vivo models. The effect of Beta-carotene on lipid peroxidation in human skin were studied in skin homogenates and in a semi-in vivo model of skin penetration, using [1-14C]-arachidonic acid or [1-14C]-linoleic acid as substrate. When relatively low concentrations (about 0.3 microM) of beta-carotene were added to epidermal homogenates, the major metabolites of arachidonic acid (12-hydroxy-cis-5,8,14, trans-10-eicosatetraenoic acid and 15-hydroxy-cis-5,8,11, trans-13-eicosatetraenoic acid) and of linoleic acid (13-hydroxy-cis-9, trans-11-octadeca dienoic acid and 9-hydroxy-trans-10, cis-12-octadeca dienoic acid) were significantly decreased. Following [1-14C]-linoleic acid penetration through the semi in vivo model layers, the skin surface was the main site in which the major linoleate product, 13-hydroxy-cis-9, trans-11-octadeca dienoic acid was detected. Furthermore, its level was inhibited by up to 80%, compared with the control, when beta-carotene was added to the system. The data presented in this study suggest possible interactions between beta-carotene and human epidermal lipoxygenase. Beta-carotene may effect lipid peroxidation in human skin, either as a free radical scavenger or as a specific lipoxygenase inhibitor.

Lipoxygenase activity in heart cells.

Arachidonic acid (AA) metabolism via the lipoxygenase (LOX) pathway in rat hearts and in cultured rat cardiomyocytes was investigated using 1-[14C]AA. LOX activity was detected in the microsomal fraction, in the high speed supernatant prepared from rat hearts and in rat cardiomyocyte supernatant. LOX products from all fractions comigrated in thin layer chromatography as 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE. Enzyme linked immunosorbent assay for 12-HETE showed its formation by the microsomal fraction, the ammonium sulfate (AS) pellet, and by rat cardiomyocyte supernatant, while radioimmunoassay for 15-HETE showed its formation only by the AS pellet. The properties of LOX in each fraction are reported here.

Lipopolysaccharide-induced oxidative stress in the liver: comparison between rat and rabbit.

In the present study the effect of LPS on biochemical systems involved in radical formation and scavenging processes in tissues from rabbit (LPS-sensitive) and rat (LPS-resistant) was investigated. The results obtained show a significant enhancement in the endogenous antioxidative enzyme system in rats as a result of LPS injection. In rats, 24 h after LPS injection, glutathione peroxidase (G-POX) and superoxide dismutase (SOD) activities were increased by 60% and 120%, respectively, compared to the control. However, in rabbits the increase in these activities was relatively mild. Moreover, NADPH-oxidase activity, which produces superoxide radical, was increased about twofold in rabbit, 15 h following LPS injection. In rats, injection of LPS did not result in any significant changes in the activity of this enzyme. In rats, a decrease in malonaldehyde (MDA) levels appeared after injection of LPS, while in contradistinction, the peroxidative levels of lipids in the rabbit's liver were increased about 3-fold. Injection of D-galactosamine (Gal-N) in combination with LPS significantly increased the sensitivity of rats to LPS characterized by a significant increase in NADPH-oxidase activity. This study indicates that one possible mechanism (among others) that may explain the relative sensitivity of rabbits compared to rat, may be related to the increase in the production of reactive oxygen substances (ROS) which is not accompanied by a concomitant increase of the protective antioxidative enzymes. Furthermore, the relative resistance of the rat was found to be related to an increase in the activity of the protective antioxidative systems following administration of LPS.

The effect of retinol and retinoic acids on lipoxygenase activity.

Lipoxygenase catalyzes the dioxygenation of polyenoic fatty acids such as linoleate and arachidonate. The aim of the present study was to examine the effect of retinol, all-trans-retinoic acid and 13-cis-retinoic acid on the activity of lipoxygenase-1 and lipoxygenase-2 towards linoleic acid. Lipoxygenase activity toward linoleic acid was followed by determining changes in absorption at 234 nm. All retinoids inhibited lipoxygenase-1 and lipoxygenase-2 activity. Lipoxygenase-2 activity towards linoleic acid was rapid at pH 6.5; all-trans-retinol (10 microM) caused a 50% inhibition in reaction rate. All-trans-retinol was oxidized in parallel with diene production by lipoxygenase-2. Lipoxygenase-2 activity on linoleic acid was competitively inhibited by all-trans-retinol and all-trans-retinoic acid; 13-cis-retinoic acid exhibited mixed type inhibition. Activity of lipoxygenase-1 towards linoleic acid at pH 9.0 was also inhibited by retinoic acids by 32-73%. All-trans-retinoic acid and 13-cis-retinoic acid inhibited lipoxygenase-1 activity competitively, whereas all-trans-retinol inhibited lipoxygenase-1 activity in a mixed manner. These findings suggest that retinoids may bind to the active site of the enzyme or simultaneously act as an antioxidant.

In vitro and in vivo effects of beta-carotene on rat epidermal lipoxygenases.

The in vitro and in vivo interaction between beta-carotene (BC) and lipoxygenase (LOX) was studied in rat skin. Significant in vitro inhibitory effects of BC on epidermal LOX activity were observed with both linoleic acid or arachidonic acid as substrate. Lineweaver-Burk plots for the inhibition of epidermal purified LOX indicated mixed competitive/non-competitive inhibition. In vivo effects of BC were examined in an ultraviolet A (UVA) irradiation model. Following UVA irradiation (200 Kjoule/m2) significant increases in LOX activity and malondialdehyde (MDA) values were found, whereas catalase activity was significantly decreased. Topical pretreatment of skin with BC prevented increases in LOX activity and MDA values 4 hr post-irradiation. Catalase activity was not affected by BC treatment. BC was more effective at preventing UVA induced lipid peroxidation at low then at high concentrations. Our present results indicate the protective potential of BC on in vivo UVA induced skin damage by reduction of non-enzymatic and enzymatic lipid peroxidation.

Effects of water-soluble antioxidant from spinach, NAO, on doxorubicin-induced heart injury.

Doxorubicin (DOX) produces clinically restorative responses in numerous human cancers, but its cardiotoxicity has limited its usefulness. Because reactive oxygen species may affect DOX-induced antitumor activity and cardiotoxicity, we evaluated the prophylactic effect of spinach natural antioxidant (NAO) on DOX-induced cardiotoxicity and oxidative stress in female Balb/c mice using histological, electron microscopical and biochemical parameters. Mice were treated with NAO for 7 days prior to and/or for 6 days after DOX administration. Pretreatment with NAO (cumulative dose: 130 mg/kg) did not hinder the effectiveness of DOX. Light and electron microscopy of DOX-treated heart revealed myocardial degeneration. When administered combined before and after DOX, NAO conferred the most significant cardiac protection. The effects of NAO on the lipid peroxidation product, malondialdehyde, and on H2O2/ hydroperoxides were examined on day 6 following DOX administration; levels of both were elevated in DOX-treated mice, compared to control. Pretreatment with NAO prevented these changes. Pretreatment with NAO before DOX administration decreased catalase and increased superoxide dismutase activities compared to the DOX group. Our results suggest usage of NAO in combination with DOX as a prophylactic strategy to protect heart muscle from DOX-induced cellular damage.

Effects of a typical acute oral calcium load on arterial properties and endothelial function in healthy subjects.

BACKGROUND/OBJECTIVES: Often recommended, calcium supplements have been incriminated as increasing the risk of cardiovascular events, whereas dietary calcium has generally been exonerated. As a first step to address the vascular safety of such dietary measures at the clinical nutritionist toolbox, we sought to determine and compare the acute effects of a typical oral calcium load, provided either as a supplement or as food, on vascular parameters assessed noninvasively in healthy subjects. SUBJECTS/METHODS: In this acute, cross-over, random-order intervention, 11 young and healthy vitamin D-sufficient volunteers (8 women/3 men, 33±6.1 years, body mass index 22.6±2.3 kg/m(2)), ingested 600 mg of calcium twice, once as calcium citrate and the other time from dairy products. Biochemical, vascular and hemodynamic parameters, before and 2 h after each challenge, were compared. Arterial stiffness was studied by measuring pulse wave velocity, augmentation index and large (C1) and small (C2) arterial compliance. Endothelial function was assessed by flow-mediated dilation (FMD). RESULTS: Despite effective calcium loading accompanied by a significant 60% parathyroid hormone level reduction on both occasions, there were no clinically significant changes in the vascular parameters neither in comparison with baseline, nor between the studies. A decrease in heart rate with no change in cardiac output was noticed after the supplement. CONCLUSIONS: An effective calcium load has no clinically significant untoward effect on the vascular properties of young healthy subjects, regardless of its source. Additional studies should determine whether this holds true for chronic calcium supplementation, particularly in subjects with a priori vascular impairment.

Arachidonic acid metabolism by perfused ram testis.

1. Arachidonic acid was metabolized by lipoxygenase and prostaglandin synthetase enzymes systems in the perfused ram testis. 2. The major product of the prostaglandin synthetase was 6-keto-PGF1 alpha (6KF). 3. Addition of testosterone resulted in a significant increase in the 6KF. 4. Arachidonic acid (AA) as well as testosterone penetrated the perfused testis. 5. Both 15-HPETE and 15-HETE, the products of the 15-lipoxygenase enzyme, were detected. 6. Addition of 0.1% BSA changed the pattern of the oxidized arachidonic acid metabolism.

Opposite changes with age in liver and muscle in the mitochondrial and soluble glucose-1,6-bisphosphate and 6-phosphogluconate dehydrogenase.

Glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was markedly decreased in liver of adult rats (2 months of age) as compared to young rats (1-2 weeks of age). This regulator was found to be present in both the mitochondrial and soluble fractions of liver. Its concentration in both these fractions was decreased with age. Concomitant to the decrease in Glc-1,6-P2, which is a potent inhibitor of 6-phosphogluconate dehydrogenase, the activity of this enzyme was markedly increased with age in both the mitochondrial and soluble fractions. However, the increase in this enzyme's activity was more pronounced in the mitochondrial fraction. The mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 as compared to the soluble enzyme, and this may explain the greater enhancement in its activity with age in this fraction. The tibialis anterior muscle exhibited changes with age opposite to those found in liver; Glc-1,6-P2 concentration, in both the mitochondrial and soluble fractions of muscle increased with age, and this increase was accompanied by a concomitant reduction in the activity of the mitochondrial and soluble 6-phosphogluconate dehydrogenase. Similar to liver, the mitochondrial enzyme was more affected by age, as it also exhibited a greater susceptibility to inhibition by Glc-1,6-P2.

Diagnosis of varicocele and postoperative evaluation using inguinal ultrasonography.

Ultrasonographic real-time imaging and measurements of spermatic veins in the inguinal canal were evaluated in two groups of patients: 20 young men with varicocele and 18 controls. Examinations were performed with the patient in an upright relaxed position, and performing the Valsalva maneuver. The increase in diameter of the main vein during Valsalva maneuver was considered to quantitatively represent venous reflux. The data obtained were correlated with pre- and postoperative clinical findings. This method of evaluation proved to be a useful noninvasive diagnostic modality for detecting varicocele and for assessing treatment results. On the basis of venous diameters and reflux values, a classification of varicocele is proposed. Such an objective method of grading would eliminate the subjective impression and interpretation factors of the examiner.

Age-dependent changes in glucose 1,6-bisphosphate levels and in the activities of glucose 1,6-bisphosphatase, and particulate hexokinase and 6-phosphogluconate dehydrogenase in rat skin.

The levels of glucose 1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, changed in rat skin during growth: Glc-1,6-P2 increased during the first week of age, and thereafter was dramatically reduced during maturation. The activity of glucose 1,6-bisphosphatase, the enzyme that degradates Glc-1,6-P2, changed with age in an invert manner as compared to the changes in Glc-1,6-P2. These findings suggest that the age dependent changes in this enzyme's activity may account for the changes in intracellular Glc-1,6-P2 concentration. The age-related changes in Glc-1,6-P2 were accompanied by concomitant changes in the activities of particulate (mitochondrial) hexokinase and 6-phosphogluconate dehydrogenase, the two enzymes known to be inhibited by Glc-1,6-P2. The activities of both these enzymes in the soluble fraction were not changed with age. The particulate enzymes were more susceptible to inhibition by Glc-1,6-P2 than the soluble activities, which may explain why only the particulate, but not the soluble activities, correlated with the age-dependent changes in tissue Glc-1,6-P2. These results suggest that the changes in particulate hexokinase and 6-phosphogluconate dehydrogenase resulted from changes in intracellular concentration of Glc-1,6-P2. The marked reduction in Glc-1,6-P2 during maturation, accompanied by activation of mitochondrial hexokinase and 6-phosphogluconate dehydrogenase, may reflect an enhancement in skin metabolism during growth.

Spontaneous release of a factor with interleukin-3-like activity by human lymphocytes and monocytes.

The production of a factor with interleukin-3-like activity (IL-3-LA) by cultured human peripheral-blood mononuclear cells has been studied. Culture supernatants spontaneously derived from lymphocytes or monocytes stimulated the proliferative activity of 3 IL-3-dependent cell lines. Purification of this factor by gel filtration-high-pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing demonstrated the presence of a glycoprotein with a molecular weight of 25-30 kilodaltons and an isoelectric point of 7.6. The biochemical characteristics of the IL-3-LA derived from human monocytes and lymphocytes were very similar. The biological activity of the semipurified factor was tested on mononuclear cells from fetal cord blood. It was found that after 21 days 40% of the cells in the culture were mature basophils which released 15-18 ng histamine/10(6) cells.

Hepoxilin A3 is the endogenous lipid mediator opposing hypotonic swelling of intact human platelets.

When human blood platelets are exposed to hypotonic medium they swell first but, shortly thereafter, revert toward their original volume in a process termed regulatory volume decrease (RVD). RVD is the result of an enhanced efflux of K+ and Cl- ions and associated water. Platelet RVD is controlled by a short-lived lipoxygenase-derived product (LP). By using a combination of high-performance liquid chromatography, gas chromatography-mass spectrometry, and RVD reconstitution bioassay, we show that LP is identical with hepoxilin A3. In addition we demonstrate that authentic hepoxilin A3 possesses the same biological properties on RVD reconstitution as LP and that the activity of both compounds is amplified through epoxide hydrolase inhibition with 3,3,3-trichloropropene-1,2-oxide. Therefore, we report here that volume expansion causes the formation and release of hepoxilin A3 from intact human platelets and that this hepoxilin plays a major role in volume regulation.

Gonadotropin releasing hormone activates the lipoxygenase pathway in cultured pituitary cells: role in gonadotropin secretion and evidence for a novel autocrine/paracrine loop.

The formation and role of arachidonic acid (AA) and its metabolites during gonadotropin releasing hormone- (GnRH-) induced gonadotropin secretion were investigated in primary cultures of rat pituitary cells. Prelabeled cells ([3H]AA) responded to GnRH challenge with increased formation (about 2-fold) of the leukotrienes LTC4, LTD4, and LTE4 as well as 5- and 15-eicosatetraenoic acids (5- and 15-HETE) as identified by HPLC. Formation of leukotrienes and 15-HETE was further verified by specific radioimmunoassays. No significant increase in the formation of 12-HETE or of the cyclooxygenase products prostaglandin E (PGE) and thromboxane A2 by GnRH was noticed. Addition of physiological concentrations of LTC4 enhanced basal LH release, while subphysiological concentrations of LTC4 (10(-15)-10(-12) M) inhibited GnRH-induced LH release by about 35% (p less than 0.02). Using specific lipoxygenase inhibitors L-656,224 and MK 886, we found inhibition of GnRH-induced LH release by about 40% at concentrations known to specifically inhibit the 5-lipoxygenase pathway. The peptidoleukotriene receptor antagonist ICI 198,615 inhibited LTC4- and LTE4-induced LH release and surprisingly also the effect of GnRH on LH release by 40%. The data strongly suggest a role for AA and its lipoxygenase metabolites in the on/off reactions of GnRH upon LH release. The data also present a novel amplification cycle in which newly formed leukotrienes become first messengers and establish an autocrine/paracrine loop.