Fetal hemoglobin in umbilical cord blood in preeclamptic and normotensive pregnancies: A cross-sectional comparative study

Preeclampsia (PE) is associated with increased fetal hemoglobin (HbF) in the maternal circulation but its source is unknown. To investigate whether excessive HbF is produced in the placenta or the fetus, the concentration of HbF (cHbF) in the arterial and venous umbilical cord blood (UCB) was compared in 15825 normotensive and 444 PE pregnancies. The effect of fetal gender on cHbF was also evaluated in both groups. Arterial and venous UCB sampled immediately after birth at 36–42 weeks of gestation were analyzed for total Hb concentration (ctHb) (g/L) and HbF% using a Radiometer blood gas analyzer. Non-parametric tests were used for statistical comparison and P values < 0.05 were considered significant. Our results indicated higher cHbF in venous compared to arterial UCB in both normotensive (118.90 vs 117.30) and PE (126.75 vs 120.12) groups. In PE compared to normotensive pregnancies, a significant increase was observed in arterial and venous ctHb (171.00 vs 166.00 and 168.00 vs 163.00, respectively) while cHbF was only significantly increased in venous UCB (126.75 vs 118.90). The pattern was similar in both genders. These results indicate a substantial placental contribution to HbF levels in UCB, which increases in PE and is independent of fetal gender, suggesting the elevated cHbF evident in PE results from placental dysfunction.


Introduction
Preeclampsia (PE) is a leading cause of fetal and maternal mortality and morbidity, affecting 3-8% of pregnancies worldwide [1][2][3]. PE evolves in two stages; the first stage is initiated by inadequate placentation and insufficient remodeling of the uterine spiral arteries [4]. In stage two, the clinical manifestations occur after 20 weeks of gestation. Several factors, including free fetal hemoglobin (HbF), leak from the placenta into the maternal circulation, inducing inflammation and oxidative stress (OS) causing widespread vascular endothelial damage, a hallmark of PE [5][6][7][8]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Preeclamptic placentas have increased HbF gene expression and accumulation of free HbF protein [9]. Free HbF has been shown to cause OS and damage the placenta barrier [10,11] due to its high redox potential [5,12]. As a consequence, leakage of HbF into the maternal circulation has been reported as early as the first trimester in pregnant women who later develop PE [11]. Furthermore, in term pregnancies, the level of HbF in the maternal plasma correlates with the severity of the disease [10]. Although its mechanism of contribution to the pathophysiology of PE has been studied and reviewed [13,14], the source of the free HbF has not been investigated.
The increased HbF in the maternal plasma has been demonstrated among PE women independent of fetal gender. Several studies have reported Hb levels among newborns to be dependent on fetal gestational age and gender [15,16] while increased Hb has been associated with fetal mortality [17]. Other studies have suggested gender-specificity in maternal adaptation to pregnancy [18], placental function and gene expression [19,20] and occurrence of PE [21,22].
The placenta plays various roles during fetal development; from mediating gas and nutrition exchange to producing several hormones and growth factors [23]. It is also a hematopoietic organ, bearing hematopoietic stem/progenitor cells (HSPCs) [24] and contributing to extramedullary fetal hematopoiesis and erythropoiesis [24][25][26][27][28][29]. Parameters measured in the venous UCB of the fetoplacental circulation can be ascribed to placental function [30].
The aim of the study was to evaluate if the increased synthesis and accumulation of free HbF previously observed in PE placentas is related to fetal gender and altered placental function. The placental contribution of HbF was estimated by comparing its concentration in venous and arterial umbilical cord blood (UCB) in PE vs normotensive pregnancies.

Blood analysis
Arterial and venous UCB gas analysis at birth has been a routine procedure at the Skåne University Hospital maternity units in Malmö and Lund for the last three decades. During the period 2001-2010, blood samples were obtained in 2-mL pre-heparinized syringes immediately after birth and analyzed within 15 minutes using Radiometer ABL 735 blood gas analyzers (Radiometer A/S, Copenhagen, Denmark). In addition to blood gases, the analyzer measures the pH by potentiometry as well as various compounds such as the total hemoglobin concentration (ctHb) (g/L) and HbF% by spectrophotometry at 37˚C. In order to obtain optical clarity, the blood gas analyzer hemolyzes the blood samples by ultrasonication, which prevents distinguishing free Hb in the plasma from the intracellular Hb released due to ultrasonication. As HbF and adult hemoglobin have different molecule structures and light absorbance spectra, they can be measured at different wavelengths. The ctHb includes fractions of oxy-, carboxy-, deoxy-and methemoglobin and the HbF concentration (cHbF) (g/L) can be derived from cHbF = HbF% × ctHb. All the obtained data was transferred to an electronic database including a report of analysis quality for each parameter and personal identification number for each analysis. The origin of sampling, i.e. "artery" or "vein", was also indicated for each sample at analysis. The placental contribution to the UCB Hb concentration was calculated by deducting the arterial value from that of the venous in paired samples, where both arterial and venous Hb values were available. These factors were indicated as VActHb and VAcHbF for tHb and HbF, respectively.

Study group
After the study was approved by the Regional Ethics Committee in Lund, Sweden (Dnr 2009/ 222), the laboratory data was paired with the clinical data from the regional Perinatal Revision South Register for the aforementioned period. Any analysis with quality check error was excluded and each blood analysis was validated for the vessel of the origin by the criterion that the venous pH should be at least 0.02 units higher than in the artery [31]. Out of a total number of 44,423 patients merged from Lund and Malmö databases, the final validated sample population consisted of 16,269 patients with ensured identifications, complete panels of maternal and fetal clinical data, and paired arterial and venous UCB pH determinations with ctHb and HbF% in the artery and/or vein. PE was defined as BP ! 140/90 mmHg plus proteinuria ! 300 mg/L and severe PE as BP ! 160/110 mmHg [32,33].

Statistical analyses
The Mann-Whitney U test and the Wilcoxon signed-rank test for matched pairs were used to compare the distribution of continuous variables among all and paired samples, respectively. Spearman's rank correlation (rho) were used to calculate the correlation between variables and the 95% confidence interval (CI) for the correlation value was determined by bootstrapping for 1000 samples. Finally, The Chi-Square test was employed for comparisons of categorical variables. Values are reported as median ± 95% CI. The statistics was performed using SPSS computer software (SPSS Statistics for Macintosh, Version 23.0. Armonk, IBM Corp., NY) and p values < 0.05 were considered statistically significant.

Results
The distribution of the blood samples based on maternal condition and fetal gender is presented in Table 1. Among the 16,269 UCB samples with cHbF data, 444 (2.72%) were diagnosed with PE; 393 (2.41%) with mild-moderate PE cases and 51 (0.31%) with severe PE patients. As the number of severe PE samples was considered too small, the two PE subgroups were merged and compared with normotensive pregnancies. The two groups were also examined for gestational age differences on HbF production. No major differences were observed in gestational age in the two groups (Median: 39; interquartile range: 38-40 weeks, in both groups). Comparing arterial and venous UCB ctHb and cHbF Statistically significant correlation was found between the arterial and venous values of ctHb (rho: 0.86, 95% CI: 0.85-0.88) and cHbF (0.83, 0.81-0.84) using Spearman's rank correlation (P = 0.01). In both normotensive and PE pregnancies, the median arterial ctHb was higher than the venous (relative increase: 1.8%, 95% CI; 1.8-2.4%) whereas the median cHbF was higher in the vein rather than the artery (2.0%, 2.0-2.1%); both differences were statistically significant (P < 0.001, exact median ± 95% CI values for each factor are indicated in Table 2).

Comparing normotensive vs PE values of the arterial and venous ctHb and cHbF
To investigate the influence of PE on the arterial and venous ctHb and cHbF, the distribution of all the values from normotensive and PE groups were compared using Mann-Whitney U test (Table 2). Compared to normotensive pregnancies, the UCB from the PE samples had statistically significantly higher arterial and venous ctHb (relative increase: 3.0%, 95% CI: 1.0-4.2% and 3.0%, 2.4-4.2%, respectively) (Fig 1A and 1B) and venous cHbF (6.0%, 5.0-8.0%) ( Fig 1E). There were no statistically significant differences in median arterial cHbF (Fig 1D), albeit estimated with some uncertainty as indicated by the 95% CI ( Table 2). The median and 95% CI values of VActHB or VAcHbF were not significantly different between PE and normotensive pregnancies either (Fig 1C and 1F).

Evaluating gender-specific effect of PE on ctHb and cHbF
The effect of fetal gender on Hb values was studied separately in normotensive and PE groups using Mann-Witney U test. The arterial and venous UCB ctHb and cHbF were statistically significantly (P < 0.001) higher (1.2%, 1.8%, 1.7% and 2.3%, respectively) in male versus female infants from normotensive pregnancies and a similar pattern was observed among PE pregnancies. To elucidate gender-specific changes, intra-gender analysis was performed between normotensive and PE pregnancies. Arterial and venous ctHb and venous cHbF medians were statistically significantly (P < 0.02) higher in both male (2.9%, 3.6% and 7.8%, respectively) and female newborns (3.0%, 3.1% and 3.6%, respectively) affected by PE (Fig 2A, 2B and 2E). However, the differences between normotensive vs PE arterial cHbF, VActHB and VAcHbF medians (Fig 2D, 2C and 2F) were not statistically significant among male or female newborns.

Discussion
To our knowledge, this is the first study to recognize placental contributions to the UCB and distinguish it from the fetal endowment by comparing ctHb and cHbF in venous and arterial UCB from normotensive and PE pregnancies. The prevalence of PE in our database, which was collected over 10 years, was in agreement with previous data from Sweden. The number of severe PE cases was typically low for Sweden preventing us from including subgroups of PE.
The clinical relevance of our findings lies in the fact that arterial UCB provides an indication of cellular processes in the fetus, hence an indication of fetal health status. Our results showing significantly higher arterial ctHb may reflect active fetal erythropoiesis. This is in line with the present findings showing increased arterial UCB ctHb in PE and other studies reporting a higher proportion of nucleated reticulocytes [29,34,35] and younger population of red blood cells [36] in the fetuses from PE pregnancies that counteract the chronic hypoxic conditions, which induces erythropoietin synthesis and erythropoiesis [37,38].  Umbilical cord blood fetal hemoglobin in preeclampsia Our analyses showed significantly higher venous UCB cHbF compared to the arterial in both normotensive and PE cases. Interestingly, the venous UCB cHbF was significantly higher (5-8%) in PE compared to normotensive pregnancies. Even though a general biological variation is about 10%, the increased cHbF may play a role in the pathophysiology of PE. As the damage observed in the placental barrier in PE [10,11], may alter the fluid homeostasis, the increase in venous cHbF may inaccurately be attributed to fluid loss across the placenta and decreased venous plasma volume after placental passage. However, this is unlikely because the ctHb was lower in the venous UCB compared to arterial in both normotensive and PE pregnancies. As the venous, but not arterial, UCB represents contributions from the placenta, our results suggest a possible role for the placenta in altering the HbF levels. Interestingly, previous studies have described changes in the localization of placental hematopoietic stem/ progenitor cells (HSPCs) [39] and increased placental HbF mRNA and protein [9] in PE suggesting altered placental erythropoiesis as a plausible cause of the increased venous cHbF.
Gender-based differences (approximately 10%) in Hb values are clinically significant among adults [40], and the positive effect of testosterone in males [41][42][43] seems to be as important as the negative influence of menstruation in females [44] in intensifying this difference. Other studies have reported gender is an important factor affecting Hb concentration already during infancy [16,45]. We observed significantly higher arterial and venous ctHb in males than females that led us to perform intra-gender analyses between PE and normotensive groups. Both males and females demonstrated an increase in arterial and venous ctHb as well as venous cHbF, showing that there was no gender-specific pattern for Hb alteration in PE. Interestingly, a gender-specific pattern of increased risk of PE, abruptio placenta and pre-term birth has been reported in pregnancies with a male fetus [21,[46][47][48][49]. However, it has also been reported that a female fetus increases the risk of pre-term delivery (<37 gestational weeks) in PE pregnancies [22]. Inter-gender differences in Hb concentration may play a role in upregulating protective scavenger proteins such as hemopexin, haptoglobin and the rate limiting degradation enzyme heme-oxygenase 1, and thereby provide a protection against PE. Higher Hb in male fetuses could also be a risk factor per se by contributing to the elevated free Hb levels observed in PE. Further investigations are required to elucidate how these gender-specific differences in erythropoiesis may affect PE manifestations.
In conclusion, our findings confirm previous reports regarding increased Hb and erythrocytes in fetuses from PE pregnancies. Importantly, our results also show a significant increase in venous cHbF in the UCB, particularly in PE pregnancies. Accordingly, we propose a placental regulation of HbF that may be compromised in PE independent of fetal gender. Elucidating the exact underlying mechanisms requires further investigation on sample populations with higher number of severe PE cases where free HbF can be distinguished from its intracellular form.
Supporting information S1 Table. The raw data used in the study. All the raw data used in the study including the arterial and venous ctHb, HbF%, cHbF, maternal condition and its severity as well as fetal gestational age and gender is included in this table.