Two types of donor sex comparison are commonly reported in the adult literature: single donor sex exposure and donor-recipient sex mismatch. For instance, transfusion with RBCs from female donors has been reported to be associated with greater mortality rates ^[1][2][3][26,32,33], an association also seen with donor-recipient sex-mismatched transfusion with female donor RBCs ^[4][5][27,34]. However, this inconsistency in the donor sex comparisons investigated and the inclusion of patients who have received RBCs from both male and female donors has limited interpretation of the effects attributable to a specific donor sex. Studying only patients who received one transfusion would simplify patient exposure classification as ‘sex-matched’ or ‘sex-mismatched’ while avoiding the confounding effect of number or transfusions received. This, however, results in inclusion of only less unwell patients in any analysis.

Criticism of the data generated from these studies has focused on residual confounders, resulting in model mis-specification ^[1][26], large proportions of missing data ^[3][33], and the introduction of selection bias through informative censoring ^[6][35]. Other possible explanations for inconsistency in the findings could be related to the fact that some include all types of blood transfusion exposures, including plasma, platelets, and RBCs ^[2][7][32,36], whereas others focus solely on RBC transfusions ^[1][3][4][8][26,27,33,37]. Finally, most of the adult literature uses mortality as the transfusion outcome, with less attention paid to the possibility of donor-related factors being associated with morbidity outcomes.

2. Inherent Differences in RBCs from Male and Female Donors

RBC transfusion packs from male and female donors are inherently different with haematological and redox parameters differing between male and female donors during storage. Female donors have a greater proportion of younger RBCs at the time of donation, a difference greatest during reproductive age, when more young RBCs enter the circulation to compensate for blood loss through menstruation ^[9][48]. As a result, cell deformability and oxygen carrying capacity are greatest in RBCs from female donors ^[10][11][49,50] however, RBC mechanical fragility is lower ^[12][51]. Conversely, RBC transfusion packs from male donors have higher hemoglobin content, resulting in larger increments in hemoglobin concentration ^[13][52], but undergo higher rates of storage hemolysis ^[10][13][49,52]. There are also sex-specific differences in RBC membrane properties during storage ^[14][53]. RBC membrane expression of CD47 falls significantly with time in RBCs from male donors while membrane lipid leakage is greater, suggesting conformational changes in the RBC membrane and phospholipid bilayer destabilization leading to greater RBC clearance ^[15][54]. Finally, female donor RBC units have greater preservation of intracellular antioxidant capacity, lower levels of intracellular reactive oxygen species, and decreased levels of oxidative hemolysis compared to those from male donors ^[16][17][55,56]. There is some evidence that these differences may result in sex specific responses in the transfusion recipient. For female transfusion recipients, the delivery of larger amounts of free haemoglobin with RBC transfusions from male donors may result in the haptoglobin scavenging capacity of the reticuloendothelial system being overwhelmed ^[18][57]. Excess free haemoglobin is also scavenged by endothelial derived nitric oxide, producing methaemoglobin, a reduction in nitric oxide bioavailability and endothelial dysfunction and oxidative injury ^[18][19][57,58]. This increase in methaemoglobin levels has also been shown to decrease tissue oxygen delivery with resultant at end organ hypoxia ^[20][59].

3. Transfusion Related Immunomodulation and the Influence of Donor Sex

RWesearchers and others have reported an increase in circulating plasma pro-inflammatory cytokines and markers of endothelial activation concentration between 2 and 48 h after single transfusion exposures in the weeks following preterm birth ^[21][22][43,44]. Furthermore, transfusion related pro-inflammatory immune responses increase in magnitude with repeat transfusion exposure in preterm newborns ^[23][17]. These responses may represent a biologically plausible pathway whereby RBC transfusion promotes immune cell activation and pro-inflammatory responses ^[22][44], processes that may result in adverse consequences for the transfusion recipient. How donor sex influences these immune responses is an area of growing interest, with immune cell activation by HLA antibodies ^[24][60], which are critical to induction and regulation of immune responses, identified as one potential candidate ^[25][61]. HLA antibodies are more common in women, with sensitization rates increasing significantly with a history of pregnancy of 20–50% ^[26][62]. Evidence linking donor derived HLA antibodies and donor sex to clinical outcomes centres on TRALI, where female donor plasma is a known risk factor ^[27][63]. In TRALI, HLA antibodies activate neutrophils in the pulmonary vasculature, releasing pro-inflammatory cytokines, including IFNγ, IL-6 and IL-8 ^[28][29][64,65], resulting in end-organ damage ^[30][66]. Whether these mechanisms are influenced by donor sex in neonatal transfusion recipients is currently unknown. Intriguingly, there is emerging evidence that preterm newborns may themselves exhibit sex-specific inflammatory responses to RBC transfusion ^[31][67]. In an analysis of 19 cytokines and inflammatory biomarkers measured pre- and post-transfusion in preterm newborns enrolled in the Transfusion of Prematures (TOP) trial ^[32][68], Benavides and colleagues reported significantly greater increases with each additional transfusion in monocyte chemoattractant protein (MCP-1) in females but not in males ^[31][67]. In addition, higher concentrations of MCP-1 were associated with worse neurodevelopmental outcomes determined by Bayley-III assessment. Future studies in considerably larger cohorts of newborns are clearly necessary to delineate any direct associations of inflammatory biomarkers and adverse outcomes. However, this data highlights the hurdles in understanding the complex interactions between donor sex, RBC transfusion and preterm newborn immunomodulatory responses.

4. Transfusion Related Endothelial Activation and the Influence of Donor Sex

Donor sex related immunomodulation, with increases in pro-inflammatory cytokines, also contributes to endothelial activation. This is a potential predictive marker for transfusion associated adverse outcomes and has recently been shown to be increased with sex mismatched RBC transfusion exposure ^[33][69]. In vitro data supports the release of markers of endothelial activation following the incubation of endothelial cells with blood product supernatant ^[34][35][70,71]. Clinical data reports increased ICAM-1, a marker of endothelial activation, and syndecan-1, which is released with the loss of endothelial glycocalyx integrity, following RBC transfusion ^[36][72]. The link between transfusion exposure and endothelial activation may be directly related to post-transfusion increases in pro-inflammatory cytokines ^[37][73], a response observed in the preterm newborn ^[21][22][43,44], or due to the presence of free heme and non-transferrin bound iron ^[38][39][74,75]. Recently, Alshalani and colleagues reported significantly greater post-transfusion concentrations of syndecan-1 and soluble thrombomodulin in adult intensive care patients who received a single sex-mismatched RBC transfusion compared to those who received sex-matched blood ^[33][69]. Critically, both syndecan-1 and soluble thrombomodulin are known predictors of in-hospital mortality ^[40][76]. Again, this clinical data has been generated from adult studies, with evidence in preterm newborns lacking. Extracellular vesicles, specifically microparticles, transfused along with the RBCs are biological response modifiers that may play a significant immunomodulatory role in the transfusion recipient ^[41][42][77,78]. They have been shown to accumulate with storage time and increase post-transfusion concentrations of inflammatory markers in the transfusion recipient ^[43][25]. This is likely through the direct interaction with monocytes, initiating a pro-inflammatory cascade, and subsequent interactions with T cells. Further, components of the complement cascade such as complement receptor 1 have also been identified in RBC microparticles ^[34][70]. Critically, microparticles result in the increased endothelial expression of ICAM-1 and E-Selectin and procoagulant activity ^[41][77]. However, no studies have specifically reported the influence of donor sex on extracellular vesicles and microparticles and recipient responses to transfusion exposure. In summary, there is increasing data supporting biologically plausible pathways by which donor sex related differences in RBC characteristics, immunomodulatory and endothelial responses to transfusion exposure could result in adverse outcomes. However, further exploration of potential biological mechanisms should be an important consideration of future planned studies.