Maternal Hypertension and Early-Onset Neonatal Neutropenia

Abstract Objective: Maternal hypertension is considered a risk factor for early neonatal neutropenia. We sought to explore this relationship. Study Design: This retrospective cohort study compared initial neutrophil counts in infants born to mothers with preeclampsia with severe features (PSF) and infants born to normotensive mothers using Negative Binomial Regression (NBR) and logistic regression models. Results: Maternal hypertension negatively affected the early neonatal neutrophil count (adjusted NRB coefficient 0.4 [0.2, 0.6], p < 0.0001) but did not increase the risk of neutropenia (OR 2.07 [0.97, 4.41], p = 0.06). The initial neutrophil count and neutropenia risk were not different between PSF subgroups. Gestational age had the greatest impact on neutropenia risk (OR 0.72 [0.64, 0.81], p < 0.0001). Almost all neutropenia resolved within 48 h. Conclusion: Maternal hypertension negatively affects the early neonatal neutrophil count while not increasing the risk of neonatal neutropenia.


Introduction
The neutrophil is an important component of the innate immune system and plays a crucial role in host defense [1].Neutropenia occurs when the neutrophil count falls two standard deviations below the mean [2].Estimates of the frequency of neutrophilia in the general Neonatal Intensive Care Unit (NICU) population are around 8% [3][4][5]; however, the frequency can increase significantly based on the definition of neutropenia used and the population studied [6][7][8][9].The causes of neutropenia in the early newborn period are numerous, including congenital and postnatal infections, immune-mediated processes, birth asphyxia, bone marrow infiltration, and bone marrow failure syndromes [2].Prematurity and low birthweight have also been associated with early neutropenia [6,9].
Maternal hypertension has been recognized for several decades as a potential cause of early neonatal neutropenia [10][11][12], although this finding has not always been consistent [8,13,14].Our group recently examined the relationship between maternal hypertension and early-onset neonatal thrombocytopenia [15].We found that maternal severe preeclampsia does not directly affect an infant's initial platelet count once we controlled for confounding variables.With this in mind, we set out to better understand the relationship between maternal hypertension and the early neonatal neutrophil count.In this study, we sought to investigate [1] whether maternal hypertension is an independent risk factor for the development of early-onset neutropenia [2]; whether infants born to women diagnosed with two different hypertension phenotypes are at different risks of early-onset neutropenia [3]; whether time to neutrophil normalization following an episode of neutropenia differed between infants born to mothers with and without hypertension; and [4] whether other maternal or neonatal characteristics influence an infant's early neutrophil count.

Population and data collection
This retrospective study utilized the same cohort of patients as our previously published work [15].In that study, patients were identified using an institution-specific dataset.Patient data was collected from the electronic medical record and entered into a REDCap database [16,17].Refer to our previous study for additional information on data collection [15].Both our previous work and this current study were approved by the site-specific and university Institutional Review Boards.
Like our previous study [15], infants admitted to our level III NICU during the study period (April 2018 to November 2020) were eligible for inclusion in this study.Exclusion criteria for this study included presence of a maternal hypertension diagnosis other than preeclampsia with severe features, absence of a CBC in the first 12 h of life, and presence of conditions known to affect the early neonatal neutrophil count not related to maternal hypertension, such as immune-mediated neutropenia, birth asphyxia, pre-or postnatal infection, or aneuploidy [2].The CBC cutoff time of 12 h was chosen intentionally since after 12 h of life, the neutrophil count begins to fall and reaches a steady state around 72 h of life [10].
As in our previous work [15], infants were classified into hypertension and control groups based on the presence of maternal preeclampsia with severe features.Infants born to mothers diagnosed with other hypertension subtypes (preeclampsia without severe features, gestational hypertension, and chronic hypertension) were not included in this study given the low number of these infants who had CBCs obtained in the first 12 h of life.Infants born to mothers diagnosed with preeclampsia with severe features constituted the hypertension group.These infants were subdivided into the blood pressure only (BPO) and end organ dysfunction (EOD) subgroups based on the presence or absence of signs of end organ dysfunction common in preeclampsia with severe features [18].Subgroup definitions were established in our original study.
Infants born to mothers with preeclampsia with severe features were matched 1:1 with infants born to normotensive mothers, and matching was based on gestational age (± one week).All subjects in our original study had a CBC within the first 24 h of life.In our present study, we only included infants with CBCs in the first 12 h of life, and therefore we eliminated 12 subjects from the control group and 22 subjects from the hypertension group who had their first CBC obtained between 12 and 24 h of life.As a result, the hypertension and control groups became slightly uneven; however, the groups remained similar in terms of gestational age.
All neutrophil counts were obtained via a manual white blood cell count differential with at least 100 cells counted by a pathologist or laboratory technologist.We defined early neutropenia as an absolute neutrophil count (mature plus band neutrophils) <1,500/µL.We chose this threshold as it is the definition most often used in older children and adults [19]; however, it remains unclear if this cutoff truly represents the point at which a neonate is at increased risk of infection [20].The critical neutrophil value at which infection risk is significantly increased remains elusive, and other authors have suggested alternative definitions of neonatal neutropenia, including <2,000/ µL [8,21], <1,100/µL [22], and <1,000/µL [14].A threshold of 1,500/µL is less than the lower boundary of the Manroe curve (1,800/µL) [10], but greater than the lower boundary (500/µL) and steady state neutropenia cutoff suggested by Mouzinho (1,100/ µL) [23].Neutrophil normalization was defined as the point in time at which the neutrophil count reached at least 1,500/µL.The immature to total neutrophil proportion (I:T proportion) was calculated for all neutropenic infants by dividing the sum of the immature neutrophils (band neutrophils and younger neutrophil forms) by the sum of all neutrophils (mature neutrophils plus immature neutrophil forms).Neutrophilia was defined as a single neutrophil count >15,000/µL, the definition proposed by Mouzinho et al [23].Rupture of membranes >18 h was considered prolonged, and small-for-gestational-age (SGA) was defined as a birthweight for gestational age <10% using the Fenton Preterm Growth Chart.

Statistical analysis
All statistical analyses were performed using SAS 9.4 for Windows (SAS Institute Inc., Cary, North Carolina).Descriptive statistics were computed including frequencies and proportions for categorical variables and means and standard deviations for continuous variables.Basic comparisons with respect to the groups were done via Student's t-test for continuous variables.For categorical variables, either Pearson's Chi-square or Fisher's exact test was used.Both Pearson and Spearman correlation coefficients were calculated to evaluate the linear and monotonic relationships between neutrophil counts and weeks gestational age.Multivariable Negative Binomial Regression (NBR) modeling was performed to model neutrophil counts for both the overall sample and hypertension subgroups.This model was preferred to address any over-dispersion in the data.We also assessed the potential risk factors leading to an initial neutrophil count <1,500/µL using multivariable Firth's logistic regression, which is a penalized likelihood-based method that solves the separation problem.Firth's logistic regression modeling was applied to the overall sample and hypertension subgroups.Finally, we performed the Wilcoxon Rank Sum test to compare time to neutrophil normalization between control and hypertension groups, as well as between BPO and EOD subgroups.

Results
Four hundred of the 1,322 infants (30.3%) admitted to the NICU during our study period were born to mothers with any type of hypertension.Of these 400 infants, 202 (50.5%) met inclusion criteria.The hypertension group had a significantly lower initial neutrophil count (4,787/µL vs. 5,894/µL, p = 0.006) and a greater proportion of infants with a first neutrophil count <1,500/µL (8.2% vs. 3.4%, p = 0.0012) compared to the control group.The groups were also different in terms of infant race, birthweight, proportion of SGA infants, proportion of infants born via Cesarean delivery, proportion of infants born to mothers with prolonged rupture of membranes, and first platelet count.The groups were similar with regards to other characteristics including gestational age and age at which the first CBC was obtained.Descriptive characteristics of the two groups are shown in Table 1.
The timing of follow up neutrophil measurements in initially neutropenic subjects varied widely, ranging from 6.8 to 498.8 h, although most follow up measurements were obtained in the first 24 h of life (43/48, 89.6%).The majority of neutropenic infants experienced normalization of the neutrophil count on the first follow up measurement (two measurements in total) (43/48, 89.6%).Three infants required two additional neutrophil measurements (three measurements in total) to reach neutrophil normalization, and one infant required three additional measurements (four measurements in total).One infant did not achieve a neutrophil count of at least 1,500/µL prior to discharge.Of the subjects who achieved neutrophil normalization, almost all reached this point by 48 h of life (45/47, 95.7%).

Discussion
This retrospective cohort study examined the relationship between maternal hypertension and the early neonatal neutrophil count.Our results suggest that infants without other neutropenia risk factors born to mothers with preeclampsia with severe features have a significantly lower first neutrophil count but are not at increased risk of an initial neutrophil count <1,500/µL in the first 12 h of life compared to infants born to mothers without hypertension.The two maternal hypertension subtypes we studied did not impart different risks of neonatal neutropenia or a lower neutrophil count.Gestational age appears to be the strongest predictor of both a decreased initial neutrophil count and an initial neutrophil count <1,500/µL.Given the wide variation in timing of follow up CBCs, we were unable to determine if there were significant differences in timing of neutropenia resolution between groups.
Manroe et al. first linked maternal hypertension with early neonatal neutropenia in 1979 (10).The authors found that 73/129 (56.6%) total neutrophil values in infants born to mothers with hypertension fell below the 5% of the reference ranges created by the authors.In the following decades, other researchers found a similar association between maternal hypertension and early neonatal neutropenia [9,11,12,[22][23][24][25][26][27][28][29]; however, some failed to observe this association [8,13,14].We found that the presence of maternal hypertension does not increase an infant's risk of developing neutropenia, and while maternal hypertension does lead to a lower initial neutrophil count, we are not confident that there is a meaningful clinical or immunologic difference between a neutrophil count of 4,787/µL and 5,894/µL.It seems unlikely that a neutrophil count of almost 4,800/µL would place an infant at a significantly increased risk of infection or other morbidity.Prolonged rupture of membranes was shown to positively impact the initial neutrophil count in our multivariate binomial regression model.Neutrophilia has been associated with infection [22], and we hypothesize that inflammation associated with prolonged rupture of membranes may result in an increased neutrophil count.Of note, the presence of prolonged rupture of membranes did not decrease the likelihood of neutropenia.
Previous research has suggested that more severe maternal hypertension is associated with a greater likelihood of neonatal neutropenia [9,12,27,30,31], which we did not observe when comparing our two hypertension subgroups.The hypertension subgroups had similar first neutrophil counts and proportion of infants with a neutrophil count <1,500/µL.It is possible that if we had included infants born to mothers with other hypertension types (gestational hypertension, chronic hypertension, and preeclampsia without severe features), we may have observed a statistical difference between hypertension groups; however, as previously mentioned, we lacked enough eligible patients born to mothers with other hypertension types.This type of comparison may be possible in future research via a prospective study using cord blood of infants born to mothers with all hypertension types and controls, similar to the method used by Bolat et al [28].
In both group and subgroup regression analyses, we found that gestational age affected the first neutrophil count and predisposed infants to neutropenia.The scatter plot in Figure 1 illustrates the direct relationship between gestational age and the initial neutrophil count.Compared to the entire cohort, a greater proportion of VLBW, ELBW, and extremely preterm infants had initial neutrophil counts <1,500/µL, <1,000/ µL, and <500/µL.Our findings concur with what other investigators have found.Mouzinho et al. observed that neutropenic neonates were smaller and younger, that gestational age was predictive of neutropenia in their logistic regression model, and that for every 100 gram decrease in birthweight, the risk of neutropenia increased 1.6-fold [9].
Diminished neutrophil production seems the most plausible explanation of the neutropenia in our hypertension cohort, and this has been supported by previous research in infants born to mothers with hypertension [12].We observed that neutropenic infants had significantly lower platelet counts compared to both the entire hypertension group and non-neutropenic infants, which has been shown in previous research [12,26,28,31,32].Considering neutropenia and thrombocytopenia frequently coexist and are strongly associated with gestational age and birthweight, we suspect these hematological anomalies are developmental and share, at least in part, a common etiology.
Our study offers a number of clinical insights.Given that most infants born to mothers with preeclampsia with severe features were neither neutropenic nor at increased risk of neutropenia, it appears unnecessary to obtain a screening neutrophil count on all infants born to hypertensive mothers.In general, the neutropenia we observed resolved quickly and without specific intervention.If a neutrophil count is obtained for other reasons and neutropenia is noted, it seems reasonable to repeat neutrophil measurements until a normal value is achieved.If neutropenia persists or appears later in life, alternative diagnoses should be considered such as infection or immune-mediated processes.Most neutropenic infants in our study received antibiotics, especially VLBW infants.A short course of antibiotics while awaiting the results of a blood culture is a reasonable approach considering early neutropenia is seen in infants with bacterial infections [22].Previous research has suggested that maternal hypertension is a risk factor for neonatal infection [28].In our cohort, only one infant developed late-onset sepsis (infection occurring after 72 h of life).This infant was born to a normotensive mother and was not neutropenic.We cannot conclude if preeclampsia alters the risk of late-onset neonatal infection given the limited number of infants with culture-proven late-onset sepsis in our cohort.
Our study had limitations.We only evaluated a single neutrophil value at less than 12 h of life, and therefore it is possible that we missed neutropenia that was present before or after a neutrophil count was obtained.Additionally, the timing of neutropenia resolution could not reliably be ascertained given the wide variability in the timing of follow up neutrophil measurements.Comparing early neutrophil counts is challenging given the dynamic nature of a newborn's neutrophil count over the first days of life [10,23,33].Seventy-three percent of neutrophil counts (303/414) in this study were obtained before two hours of life which mitigated against some bias introduced due to differences in the timing of neutrophil count acquisition.We did not collect data on which infants received antenatal corticosteroids which have been shown to affect the early neutrophil count [34,35]; however, since antenatal corticosteroids are nearly universal in our preterm infant population, we feel justified in not accounting for the possible effect of steroids on the early neutrophil count.Our findings may not be applicable in all clinical settings.As Christensen et al. observed, altitude affects the early neutrophil count [14].Our study was conducted at sea level, and a similar study conducted at significant elevation may yield different results.Our study examined a single population at a single NICU which may not be indicative of other NICU populations.Finally, it is important to acknowledge that the cohort of subjects in this study were originally recruited for a different research study.
In summary, maternal hypertension negatively affects the early neonatal neutrophil count but does not place an infant at increased risk of neutropenia.Preeclampsia with severe features with signs of end organ dysfunction does not infer greater risk of a lower first neutrophil count or neutropenia compared to preeclampsia with severe features diagnosed by blood pressure parameters only.Gestational age has the largest impact on an infant's first neutrophil count and risk of neutropenia, with less mature infants being more severely affected.The mechanism leading to neutropenia in our cohort is most likely a transient decrease in neutrophil production.When other causes of neutropenia are not present, early neonatal neutropenia tends to resolve quickly and without intervention.This study deepens the understanding of a commonly encountered condition in the NICU.We hope our work helps clinicians feel more confident when interpreting early neonatal neutrophil counts.

Figure 1 .
Figure 1.Scatter plot showing the relationship between the initial neutrophil count and weeks gestational age.overall Pearson correlation = 0.39 (p < 0.001).triangles represent hypertension group subjects; circles represent control group subjects.

Table 1 .
demographics of control and hypertension groups.
a n (%); Mean (Sd) | b SGA small for gestational age | c range.

Table 2 .
negative binomial regression model predicting the initial neutrophil count in control and hypertension groups.

Table 3 .
Firth's logistic regression model predicting an initial neutrophil count <1,500/µl in control and hypertension groups.
a or odds ratio | ci confidence interval.