Oxidative Stress and Reproductive Capacity in Iron Overload Thalassemia Major Women
Authors: Singer Sylvia T , Vichinsky Elliott , Cedars Marcelle , Gildengorin Ginny , Suh Jung H , Lal Ashutosh 
Published: October 2019

Abstract
Abstract 2155
Despite progress in chelation regimens, excess iron still results in deleterious effect on the reproductive system of thalassemia major (TM) patients; subsequently compromised fertility is common in TM men and women. We have recently assessed the reproductive potential and its relation to iron overload in TM women as measured by the current methods for ovarian reserve testing (ORT) demonstrating a reduced ovarian antral follicle count (AFC) and corresponding low anti mullerian hormone (AMH) levels (Blood PMID 21757620). In the current study we further explored a possible link between iron overload and oxidative stress on fertility potential in thalassemia. Increase in reactive oxygen species (ROS) production and decrease in antioxidant defense mechanisms are thought to be major causes for accelerated follicle aging over time. Excess redox active non-protein bound iron acts as potent catalyst for highly deleterious ROS, causing oxidative damage to plasma lipids, DNA and proteins, and is therefore likely to further accelerate follicle aging and loss of reproductive potential. However, such an effect in TM women has not been demonstrated. Extensive oxidation of intracellular Glutathione (GSH) affects the structural integrity of cells. The ratio of GSH to its oxidized disulfide (GSH/GSSG) has been shown to be a sensitive measure of oxidative stress in biological systems and was noted to be compromised in thalassemia.
We studied 26 TM women (median age 28y); obtaining total non-transferrin bound iron (NTBI) and its low-molecular component, labile plasma iron (LPI). Liver iron concentration (LIC) was measured by superconducting quantum interference device (SQUID) biosusceptometer system. Fourteen women had a trans-vaginal ultrasound (TVUS) for assessment of AFC and for ovarian volume measurements. Plasma GSH, GSSG and GSH precursors were obtained from the TM women and from 12 age-matched healthy non-smoking women, all fasting, were assayed by liquid chromatography–electrospray positive ionization–tandem mass spectrometry (LC-MS/MS) as previously described.
GSH concentration was ∼25% lower in TM compared to normal controls, mostly due to low concentration of free GSH; 3.88±2.8 (median 2.9) and 5.74±1.4 (median 5.86), respectively. This resulted in a plasma GSH/GSSG ratio drop by 45% from the control ratio of 79.8 to a ratio of 43.4 in the thalassemia cohort (p=0.04). Plasma GSSG levels remained similar in both groups; 0.1±0.07μM in controls and 0.15±0.1μM in thalassemia women. TM women had an increase in precursors of GSH compare to controls (Table 1), suggesting that key metabolic pathways to augment synthesis of GSH may be up-regulated as a compensatory response to increased oxidative stress. GSH/GSSG, an oxidative measure, significantly correlated with AFC and with ovarian volume (R=0.57, p<0.05 and R=0.77, p=0.003, respectively). LIC (mean 14.5±11mg/gr dry wt, range 3.2–42) and GSH levels inversely correlated (R= −0.47 p<0.05). NTBI and LPI were elevated in the TM (as previously shown) but have not correlated with GSH/GSSG ratio. However, AFC (median 6.5, range 0–20) showed a trend of reverse correlation with NTBI and LPI (R=−0.21 and − 0.34, respectively).
Although the current finding of lower levels of GSH and GSH/GSSG ratio in TM women does not provide a direct cause-and-effect relationship between GSH redox and fertility in thalassemia, impaired GSH redox homeostasis in the context of iron overload may be a key precipitating factor involved in accelerated follicular aging experienced by female thalassemia patients. Ovarian tissue in TM women is likely vulnerable to the iron-induced oxidative damage and the low antioxidant protection; a direct deleterious effect beyond the ovarian quiescence due to low gonadotropine stimulation. Oxidative stress may have a major effect on the maturation and growth of oocytes, resulting in further shrinking of ovarian tissue. This data suggests a potential role for antioxidants and improved nutritional supplementation as a therapeutic approach for TM women in particular during their reproductive years.
Altered Glutathione (GSH) Precursors in Plasma of Thalassemia Patients Compared with Healthy ControlsGSH precursors (mM)Controls (n=15)Thalassemia women (n=23)pGlutamine297 ± 67440 ± 1760.005Cysteine7.4 ± 3.510 ± 3.80.03Cystathionine0.67 ± 0.20.82 ± 0.8ns

Disclosures:
No relevant conflicts of interest to declare.