Sex hormone intake in female blood donors: impact on haemolysis during cold storage and regulation of erythrocyte calcium influx by progesterone

Abstract

Background: Sex hormone intake in blood donors may affect the quality of red blood cell (RBC) products via modulation of RBC function and predisposition to haemolysis during cold storage. The aims of this study were to evaluate the association between female sex hormone intake and RBC storage outcomes, and to examine possible mechanisms by which sex hormones interact with RBCs.

Materials and methods: Sex hormone intake by race/ethnicity and menopausal status, and association analyses between hormone intake and donor scores of storage, osmotic or oxidative haemolysis, were evaluated in 6,636 female donors who participated in the National Heart, Lung and Blood Institute's RBC-Omics study. A calcium fluorophore, Fluo-3AM, was used to define RBC calcium influx in response to exogenous sex hormones or transient receptor potential cation (TRPC) channel drugs.

Results: Sex hormone intake was more prevalent in premenopausal women from all racial groups (18-31%) than in postmenopausal women (4-8%). Hormone intake was significantly (p<0.0001) associated with reduced storage haemolysis in all females, reduced osmotic haemolysis in postmenopausal donors (23.1±10.2% vs 26.8±12.0% in controls, p<0.001), and enhanced susceptibility to oxidative haemolysis in premenopausal women. In vitro, supraphysiological levels of progesterone (10 μmol/L), but not 17β-oestradiol or testosterone, inhibited calcium influx into RBC and was associated with lower spontaneous haemolysis after 30 days of cold storage (0.95±0.18% vs 1.85±0.35% in controls, p<0.0001) or in response to a TRPC6 activator.

Conclusions: Sex hormone intake in female donors is associated with changes in RBC predisposition to haemolysis. Menstrual status and the type of hormone preparation may contribute to differences in haemolytic responses of female RBCs to osmotic and oxidative stress. Progesterone modulates calcium influx into RBC via a mechanism that may involve interactions with membrane TRPC6 channels.

Figure 1

Distribution of donor menstrual status by age of the donors in the RBC-Omics study. Bar graphs represent the numbers of pre- and postmenopausal female donors at each decade. Menstrual status was self-reported by female donors at the time of recruitment into the RBC-Omics study, as detailed in the “Materials and methods” section. Of the 6,636 female donors, 3,966 were defined as premenopausal and 2,583 as postmenopausal. Partial grey bars represent overlap between pre- and postmenopausal women at specific age groups.

Figure 2

Effect of female sex hormone intake on red blood cell predisposition to storage or stress-induced haemolysis. Leucocyte-reduced red blood cell concentrates from 6,549 pre- and postmenopausal female donors enrolled in the RBC-Omics study were stored for 39–42 days (at 1–6 °C) and then evaluated for spontaneous storage, osmotic or oxidative haemolysis. Each panel compares the levels of haemolysis between women who responded yes (Y) or no (N) to hormone supplements (Hormone Sup). Comparisons were made between all female donors or by menstrual status. (A) Percent storage haemolysis. (B) Percent osmotic haemolysis. (C) Percent AAPH-induced oxidative haemolysis. Box plots demonstrate the median and interquartile range (IQR) of the three haemolysis measurements in all female, premenopausal and postmenopausal donors, separated by hormone supplement use. *p<0.05, **p<0.01, ***p<0.001 by the Student’s t-test. AAPH: 2,2’-azobis-2-methyl-propanimidamide, dihydrochloride.

Figure 3

Effect of female sex hormones on red blood cell calcium influx. Human red blood cells (RBC) from healthy volunteers (n=3) were labelled with a fluorescent calcium probe (Fluo-3AM) and treated with selected concentrations of progesterone, 17β-oestradiol or norgestimate (a progesterone contraceptive analogue) or dimethyl sulfoxide (DMSO 0.1%, vehicle control). Kinetic curves represent the rates of RBC calcium influx during incubation for 60 min (37 °C, mild agitation). (A) Calcium influx in the presence of progesterone. Asterisks represent significant differences (p<0.0001) between results for DMSO and 10 μmol/L (*) or 20 μmol/L (**) progesterone. (B). Calcium influx in the presence of 17β-oestradiol. (C) Comparison of RBC calcium influx kinetics between cells treated with progesterone or norgestimate (each at 10 μmol/L). Asterisks represent significant differences between DMSO and progesterone (*p=0.033) or norgestimate (**p=0.003). AU: arbitrary units.

Figure 4

Red blood cell storage in the presence of female sex hormones. Human red blood cells (RBCs) from healthy volunteers (n=7) were treated with dimethyl sulfoxide (DMSO 0.1%, vehicle control), progesterone (10 μmol/L) or 17β-oestradiol (10 μmol/L) and stored at 1–6 °C for 30 days as described in the “Material and methods” section. Percent storage haemolysis was determined on days 1 and 30. ****p<0.0001 by one-way analysis of variance at day 30.

Figure 5

Effect of TRPC6 drugs on red blood cell calcium influx. Human red blood cells (RBCs) from healthy volunteers (n=3) were labelled with a fluorescent calcium probe (Fluo-3AM) and treated with SKF-96365 (a multiple transient receptor potential cation [TRPC] channel inhibitor that blocks TRPC3/6/7; 25 μmol/L) or dimethyl sulfoxide (DMSO 0.2%, vehicle control). After 12 min incubation (37 °C, mild agitation), Hyp9 (a selective TRPC6 activator; 25 μmol/L) was injected and samples were incubated (same conditions) for an additional 60 min. Kinetic curves represent the rates of RBC calcium influx during the incubation period. AU: arbitrary units.

Figure 6

Effect of TRPC3/6 inhibitors or progesterone on Hyp9-induced red blood cell calcium influx. (A,B) Red blood cells (RBCs) from five healthy volunteers were labelled with a fluorescent calcium probe (Fluo-3AM) and treated with SKF-96365 (a multiple transient receptor potential cation [TRPC] channel inhibitor that blocks TRPC3/6/7), Pyr3 (a selective TRPC3 inhibitor), progesterone or dimethyl sulfoxide (DMSO 0.2%, vehicle control) in the presence or absence of Hyp9 (a selective TRPC6 activator); all drugs at 25 μmol/L. Fluo-3AM fluorescence was recorded at 10 min (A) and 90 min (B) of incubation (37 °C, mild agitation). Box and whisker plots (median + range). ****p<0.0001 DMSO+Hyp9 compared with all other treatments. **p<0.0001 Pyr3+Hyp9 compared with all other treatments except DMSO+Hyp9 (p=0.99); p values obtained by one-way analysis of variance and Tukey’s correction for multiple comparisons. (C) RBCs from a healthy volunteer were labelled with Fluo-3AM and treated with progesterone, 17β-oestradiol, testosterone (all at 20 μmol/L) or DMSO (0.1%, vehicle control). After 15 min incubation (37 °C, mild agitation), Hyp9 was injected and samples were incubated (same conditions) for an additional 75 min. Kinetic curves represent the rates of RBC calcium influx during the incubation period. Error bars represent the standard error of the mean (n=12 replicates). AU: arbitrary units.