Laboratory analysis and outcomes of pregnant women with asymptomatic COVID-19 in the peripartum period

Objective

In this study, we aim to assess the relationship between laboratory results and pregnancy outcomes of the asymptomatic preripartum women who have been diagnosed incidentally by universal COVID-19 screening upon admission for delivery.
 

Methods

This is a case-control study conducted between January 2021 and March 2022. The study group consisted of the peripartum women with positive PCR result for SARS CoV-2 without the COVID-19 symptoms.  Age and gestational age-matched peripartum women were included as the control group (1:3). The primary outcomes measures are inflammatory laboratory parameters (lymphocyte, neuthrophyl, eosinophyl, aspartate aminotransferase, alanin aminotransferase, lactate dehydrognase, neuthrophyl lymphocyte ratio, platelet lymphocte ratio, eosinophyl lymphocyte ratio, monocyte eosinophyl ratio) and their association with a positive PCR result for SARS CoV-2. Additional outcome measures were the associations between asymptomatic COVID-19 and pregnancy, maternal and neonatal outcomes.
 

Results

A total of 369 (95 in study, 274 in control group) women were analyzed. The ROC curve and multivariate logistic regression analysis have shown that increased monocyte-to-eosinophile ratio (MER, ≥14.7, aOR: 3.49 (95% CI 1.893-6.435)) and lactate dehydrogenase (LDH≥214 U/L, aOR: 15.869 (95% CI 8.529-29.524)) levels; and decreased lymphocyte count (≤1.7 103/mm3, aOR:1.8 (95% CI1.27-3.437)) were associated with COVID-19 in asymptomatic peripartum woman. Asymptomatic COVID-19 was associated with an increase in late preterm delivery (p<0.001).

Conclusion

Asymptomatic COVID-19 do not cause an increase in maternal and neonatal mortality and major morbidity in peripartum women. Increased monocyte-to-eosinophile ratio (MER, ≥14.7) and LDH (≥214) levels; and decreased lymphocyte count (≤1.7) are associated with COVID-19 in asymptomatic peripartum woman. 
 

Keywords

Asymptomatic COVID-19, pregnancy, pregnancy outcomes, monocyte-eosinophil ratio, lymphocyte count

Introduction

Pregnant women face physiological alterations that may cause different reactions to infectious diseases like COVID-19. Studies and reviews have been designed to understand the effect of the hematological parameters and biomarkers to point the severity of the SARS-CoV-2 disease, on non-pregnant individuals [1-5]. The importance of screening COVID-19 in asymptomatic peripartum women has been addressed since the pandemic, and screening upon admission for delivery is now a dilemma; some studies advising universal screening, and some do not [6, 7]. Nine out of 10 COVID-19 positive pregnant women were found to be asymptomatic [8]. Identifying the asymptomatic positive population in delivery ward would offer a great opportunity to protect newborns and other pregnant women. Incidental COVID-19 diagnosis and relationship between laboratory findings and pregnancy outcomes are also worth investigating.  
 
The purpose of this study is to assess if asymptomatic COVID-19 has any effect on pregnancy outcomes. In addition, we sought to determine which laboratory assays may be useful for predicting COVID-19 infection in asymptomatic pregnant women. 
 

Methods

Settings and Study participants
 
Our research was designed as a case control study, and enrollment period took place between January 2021 and March 2022 in Zeynep Kamil Women and Children’s Diseases Training and Research Hospital. Study sample is chosen from the women who gave birth and followed in this hospital after birth. The participants were at the age from 19 to 45, gestational age from 23 to 42, and having singleton pregnancy, and SARS CoV-2 PCR test without COVID-19 symptoms.
 
Deliveries before the week 22 of gestational age, stillbirth, pregnancy terminations and fetus with major anomalies, multiple pregnancies, the pregnant women who has not delivered yet, and the women having COVID-19 symptoms at the time of admission were excluded.
 
SARS CoV-2 PCR test implies Rdrp fragment targeted real time PCR. At the time of the enrollment period, without the indication for a test, all the pregnant women admitting to our delivery ward had the SARS CoV-2 PCR test for screening. Oropharyngeal and nasopharyngeal swab tests were the material for PCR test. Our laboratory was authorized by Turkish Health Ministry Public Health Division.
 
Sample size was calculated using OpenEpi (Version 3). For sample size calculation in our case-control study, Seyit et al.'s SARS CoV-2 test included laboratory parameters in positive and negative groups [lymphocyte (p = 0.0001), NLR (p = 0.0001) and PLR (p = 0.001)). values] were calculated based on the data of the articles in which they were evaluated [9]. Considering the relatively limited number of peripartum women with asymptomatic COVID-19 forming the case group, we thought it appropriate to include the case and control groups at a ratio of 1:3. To determine the difference between groups, we planned to include at least 53 participants in the case group and at least 159 participants in the control group, with 95% confidence level, 80% power, and 5% type 1 margin of error, plus 20% for missing data.
 
At the time of the enrollment period, routine SARS CoV-2 PCR tests were performed for every women in the delivery ward. Case group includes the patients who are admitted to delivery ward and whose PCR test is positive for SARS CoV-2 without the COVID-19 symptoms. Control group consisted of the patients with a negative SARS CoV-2 PCR test. Control group were chosen by systematic sampling method. When a participant was enrolled, three other women in the admission book were added as control group participants, from their admission numbers 3, 6, 9.
 
The patients’ laboratory findings, pregnancy and neonatal outcomes were obtained from the electronic health records. Sociodemographic, medical and obstetric (preterm delivery, preeclampsia, gestational diabetes mellitus, cholestasis) history of the participants were also assessed. In addition to sociodemographics, personal health, and obstetric information, the laboratory findings at the time of admission were evaluated. The definitions of the adverse pregnancy outcomes (gestational diabetes, preeclampsia, fetal growth restriction, intrahepatic cholestasis of pregnancy, preterm birth) were based on international criteria [10-14].
 
The study was approved by the Institutional Review Board (decision number 65, 03/2021). All procedures in our study were carried out per the 1964 Declaration of Helsinki and subsequent amendments.
 
The primary outcomes measures are inflammatory laboratory parameters (lymphocyte, neuthrophyl, eosinophyl, aspartate aminotransferase, alanin aminotransferase, lactate  dehydrognase, neuthrophyl lymphocyte ratio, platelet lymphocte ratio, eosinophyl lymphocyte ratio, monocyte eosinophyl ratio). Incidental SARS CoV-2 test positive patients’ laboratory findings and pregnancy, delivery, postpartum and neonatal outcomes were assessed. 
 
Statistical analysis was done using the Statistical Package for Social Sciences version 17 for Windows (SPSS Inc., Chicago, IL, USA). The conformity of the variables to the normal distribution was examined by visual (probability charts and histograms) and analytical methods (Kolmogorov Smirnov test). Descriptive statistics were given as median, range. Categorical variables were reported as percentages and frequencies. In comparison of quantitative data in paired groups, in the presence of normal distribution Student T test, in asymmetrical distribution, Mann Whitney U test were used. Qualitative data were compared with Chi square test and when the conditions did not meet Fishers Exact Chi square tests were used.   
 
Laboratory findings predicting SARS CoV-2 positivity were analyzed with reciever operating characteristic (ROC) curve analysis and threshold levels were calculated with Youden Index method. Specific thresholds, specificity, sensitivity, positive predictive value and negative predictive value were evaluated separately. To define factors predicting SARS CoV-2 positivity, multiple variable logistic regression analysis (backward stepwise method) was performed.  Association is reported by odds ratio (OR) and 95% confidence interval. P<0.05 was used as statistical difference level. 
 

Results

During the study period, there were 7322 deliveries in total; and among pregnant women who were admitted to labor ward without any symptoms or history of exposure, screened for COVID-19 per universal screening protocol, there were 325 positive PCR results (4.4%).  Of those who had a positive test result, 100 (30.7%) were asymptomatic, and included in the case group. PCR negative 300 women who were admitted to labor ward were randomly selected as controls. After excluding 17 multiple pregnancies, 4 fetuses with major congenital abnormalities and 7 intrauterine fetal demise cases, a total of 369 women were analyzed (Figure 1). 
 
Demographic and clinical characteristics of the groups are shown in Table 1. Baseline characteristics were similar between asymptomatic COVID-19 cases and controls, there were no statistically significant difference between groups in age, parity, ethnicity and presence of a chronic disease. Mean body mass index (BMI) in the case group was significantly higher (34.5 vs. 29.1, p 0.05). Vaccination rates were similar between the groups, only 28.4% of COVID-19 cases were fully vaccinated.
 
The pregnancy, maternal and neonatal outcomes of the groups are shown in Table 2. Frequency of adverse obstetric outcomes such as preeclampsia, gestational hypertension, fetal growth retardation (FGR), gestational diabetes mellitus (GDM), preterm birth, placental abruption and chorioamnionitis were similar between asymptomatic COVID-19 patients and controls. Delivery in late term gestation was observed significantly more in COVID-19 group compared to the control group (15.8% vs. 1.1%, p 0.001). There was no difference between groups in terms of mode of delivery. Length of hospital stay was significantly higher in COVID-19 group than the control group (2.3 days vs. 1.9 days, p 0.001). 
 
Worsening of COVID-19 clinical picture was rare, with only one patient requiring supplemental oxygen and eventually mechanical ventilation. However, a total of 23 patients were admitted to the intensive care unit (ICU) after delivery, 18 of whom had preeclampsia. Need for ICU was not significantly different between the groups.
 
We analyzed and compared complete blood count (CBC) parameters and biomarkers of inflammation derived from CBC such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ration (PLR), eosinophil-to-lymphocyte ratio (ELR) and monocyte-to-eosinophil ratio (MER), as well as alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) levels (Table 3). PLR, MER, AST and LDH were significantly increased in patients with COVID-19 (p=0.003, p 0.001, p=0.002 and p 0.001, respectively). In contrast, leukocyte, neutrophil, eosinophil and lymphocyte count was significantly lower (p 0.001, p=0.005, p 0.001 and p 0.001 respectively). The ROC curve analysis and optimal cut-off values of these parameters for prediction of positive PCR results are shown in Figure 2 and Table 4. Variables included in multivariate analysis were age, body mass index and gestational age at the time of delivery and laboratory parameters that were found to be significantly different between groups in univariate analysis (eosinophil, lymphocyte, PLR, MER, AST and LDH). Multivariate logistic regression analysis showed that MER, LDH and lymphocyte count were significantly associated with positive SARS CoV-2 PCR result (Table 5).
 
C- reactive protein (CRP), D-Dimer and procalcitonine levels were only ordered for COVID-19 patients, therefore mean values of these parameters are not reported for the control group (Table 3). When these parameters are dichotomized as high or low according to standardized cut-off values of our lab, CRP and procalcitonine were high in the majority of asymptomatic COVID-19 patients (CRP in 97.9% and procalcitonine in 67.4%).
 

Discussion

Since the pandemic, obstetric and neonatal outcomes of asymptomatic COVID-19 cases remain unclarified. Our study shows that maternal and neonatal mortality, and major morbidity was not increased in women with COVID-19 who are asymptomatic. Also, MER, lymphocyte count, and LDH levels- which are simple, readily available blood assays– have a role in predicting asymptomatic COVID-19. 
 
One aim of our study was to predict COVID-19 in asymptomatic women in admitted to labor ward in a cost-effective and timely manner, since women in labor might present themselves late in labor, thus the need for quick results upon admission. Even when COVID-19 is asymptomatic, the high infectivity to pregnant and postpartum women, as well as the high morbidity in this population relative to the general population, should not be overlooked[15]. When there is no opportunity to perform PCR testing on asymptomatic COVID-19 cases or there is insufficient time to obtain PCR results, these lab tests can be obtained promptly, affordably, and without difficulty. We found that some basic lab parameters can predict COVID-19, albeit weakly.
 
Some peripheral blood inflammation biomarkers is the kind of information that can be obtained from the most basic laboratory test result, CBC. At the same time CRP and LDH can also be helpful for defining COVID-19 positive patients and predicting their clinical phenotype. In our study MER and LDH were found to be useful for predicting COVID-19 PCR positive patients. Seyit et al., investigated the similar hypothesis in emergency department among non-pregnant adults and found that, CRP, LDH, PLR and NLR levels were higher in COVID-19 patients [9].  Covali et al., stated that components of the CBC did not differ in the term pregnancies with COVID-19 other than the increase in the MCHC and decrease in WBC, neuthrophyl and lymphocyte count [16]. A systematic review and meta-analysis published in 2021, revealed that the most common laboratory findings were elevated CRP and lymphocytopenia (57% and 50% in COVID-19 cases, respectively)[17]. Shang et al. pointed out that NLR was the best determinant of all biomarkers to predict the severity of the disease [18]. 
 
We compared pregnancy and neonatal outcomes between women with or without COVID-19. Mainly, outcomes did not differ between groups as discussed in the previous work [19-23]. Son et al. analyzed the results of  838,489 women giving birth during pre-COVID and COVID-19 period; and pregnancy related adverse outcomes were found to be similar in two groups, there were no significant differences in the frequency of preterm birth, stillbirth, pregnancy related hypertensive disorders, placental abruption and postpartum hemorrhage [24]. One meta-analysis has shown that preterm birth rates are higher in COVID-19 population[17]. In a similar manner to our study, Koç et al. investigated hematological parameters and perinatal outcomes of COVID-19 and they also revealed that asymptomatic COVID-19 was not associated with adverse perinatal outcomes [25]. Hill et al. also pointed out that asymptomatic patients screened positive at the time of delivery, were not at risk of adverse maternal and neonatal outcomes [26]. These studies reflect heterogenous groups of pregnant women with COVID-19, in terms of being symptomatic or asymptomatic; yet our findings were consistent with the existing literature. However, even though our results do not show any increase in maternal mortality and major morbidity, two cases of venous thromboembolism and one case that needed mechanical ventilation- both of whom were aymptomatic upon admission is remarkable. 
 
Even though vaccination had widespread availability during our study period, 61% of COVID-19 positive patients and 70% of negative ones were not vaccinated. This may be attributable to pregnant women’s reluctancy to try new drugs and/or vaccines. Mediu et al. addressed NLR changes based on vaccination status but did not find any difference between groups [27].
 
Remarkably, we discovered a statistically significant difference between groups for late-term delivery. According to institutional protocol, fetal surveillance is conducted twice per week in late-term pregnancies. We hypothesized that these women are more vulnerable to the risk of being COVID-19 positive due to their increased exposure to hospital environment.
 
Our research has strengths. There are insufficient high-quality and consistent studies evaluating the effect of asymptomatic COVID-19 disease on maternal and neonatal outcomes during the peripartum period, which was the purpose of our study. Data from hospitals where all pregnant women admitted to labor ward had routine PCR testing revealed that 88% of those who tested positive were asymptomatic [28]. This implies that the cases documented in the literature are most likely a small proportion of total cases, and results extracted from those should be interpreted carefully keeping in mind that clinical significance of asymptomatic infection during pregnancy is still unclear. In addition, there is insufficient research on laboratory parameters and disease estimation in asymptomatic COVID-19 cases. Our study is one of the first to evaluate the maternal and neonatal outcomes and laboratory parameters of peripartum women whose SARS-CoV-2 infection was confirmed by PCR test positivity during the COVID-19 outbreak. The findings of this study allow for the identification and possible early isolation of women suspected of having peripartum asymptomatic COVID-19, for whom we no longer routinely screen, and who will be referred for PCR testing to diagnose the disease.
 
There are also limitations of our study. Due to the retrospective nature of our study, the data are based on electronic health records. The fact that our research was conducted in a limited time period and in a single center brings a further limitation on the generalizability of our results to the population. Since our study was carried out in a tertiary center in the region where high-risk pregnant women (women with diabetes, hypertension etc.) are referred, the confounding factors that may result from additional morbidities should be kept in mind. Since the diagnostic value of the PCR test was approximately 50% in our study, we may not have been able to correctly distinguish those with a negative test even though they were sick.
 
Due to the fact that we no longer conduct routine SARS CoV-2 PCR testing upon admission to the labor ward, evaluating routine labs ordered may provide insight into the possibility of COVID-19 infection. Increased monocyte-to-eosinophile ratio (MER, ≥14.7) and LDH(≥214)  level; and decreased lymphocyte count (≤1.7) was associated with COVID-19 in asymptomatic peripartum woman. This type of information might aid in preventing the disease's spread in the vulnerable population of peripartum women by giving us the chance of early isolation.
 
Although we found that asymptomatic cases of COVID-19 did not cause an increase in maternal and neonatal mortality and major morbidity in peripartum asymptomatic women, we believe that a possible type 2 error should not be disregarded and that this issue should be revisited with larger samples and prospective studies. 
 

Conclusion

Asymptomatic COVID-19 do not cause an increase in maternal and neonatal mortality and major morbidity in peripartum women. Increased monocyte-to-eosinophile ratio (MER, ≥14.7) and LDH (≥214 U/L) levels; and decreased lymphocyte count (≤1.7 103/mm3) are associated with COVID-19 in asymptomatic peripartum woman. 
 

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	File/Dsecription
	Table 1 Sociodemographic and clinical charactheristics of peripartum women with and without asymptomatic COVID 19
	Table 2 Comparison of pregnancy, maternal and neonatal outcomes between women with and without asymptomatic COVID-19
	Table 3 Laboratory charactheristics of women with and without COVID-19
	Table 4: ROC analysis of laboratory charactheristics in women with COVID-19 Abbreviations: AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value; PLR, platelet-to-lymphocyte ratio; MER, monocyte-to-eosinophil ratio; AST, aspartate transaminase; LDH, lactate dehydrogenase
	Figure-1 Flow diagram
	Figure-2 The ROC curve of eosinophil, platelet-to-lymphocyte ratio (PLR), monocyte-to-eosinophil ratio (MER), lymphocyte, aspartate transaminase (AST) and lactate dehydrogenase (LDH), for prediction of positive PCR result for SARS-CoV-2
	Table 5. Multivariate logistic regression analysis for prediction of positive PCR result for SARS-CoV-2 Abbreviations: aOR, adjusted odds ratio; MER, monocyte-to-eosinophil ratio; LDH, lactate dehydrogenase. * Adjusted for maternal age, body mass index and gestational age at the time of birth