High Neutrophils to Lymphocytes Ratio in Maternal Blood Serum as Risk Factor for Preterm Premature Rupture of Membrane

 

Nicholas Renata Lazarosony1*, I Wayan Artana Putra2, Ryan Saktika Mulyana3, I Made Darmayasa4, Ida Bagus Gde Fajar Manuaba5, I Gde Sastra Winata6, I Nyoman Gede Budiana7

Department of Obstetrics and Gynecology, Faculty of Medicine, Udayana University/Prof. dr. IGNG Ngoerah Hospital Denpasar, Bali, Indonesia

*email: 1*[email protected], 2[email protected], 3[email protected], 4[email protected], 5[email protected], 6 [email protected], 7[email protected]

 

Keywords

 

ABSTRACT

inflammation, neutrophil-to-lymphocyte ratio, preterm premature rupture of membranes

 

Neutrophil-to-lymphocyte ratio (NLR) has been extensively studied as a prognostic factor for various diseases based on systemic inflammation. Premature rupture of membranes (PROM) is an obstetric problem that does not only occur in term pregnancies but can also occur in preterm pregnancies. One of the main etiologies for premature rupture of membranes is inflammation. Knowing the difference in the NLR between preterm premature rupture of membranes (PPROM) and without PPROM is important to increase understanding of the crucial role of NLR in predicting the incidence of PPROM. This analytic case-control study compared NLR values in maternal blood serum between PPROM and without PPROM. This research was conducted in the emergency delivery room and obstetrics and gynecology outpatient clinic at Prof. dr. I.G.N.G. Ngoerah Hospital Denpasar from February to June 2022. A high NLR in maternal blood serum may be a risk factor for PPROM. Patients with a high NLR had a 4.5 times greater likelihood of experiencing PPROM than those with a low NLR (OR = 4.5; 95% CI = 1.4 � 13.83; p = 0.007). A high NLR in maternal blood serum is a marker of inflammation with an increased risk of 4.5 times for the occurrence of PPROM.

 

 

 

 

 

INTRODUCTION

Premature rupture of membranes (PROM) is an obstetrical problem still frequently encountered in daily practice. PROM occurs not only in term pregnancies but also in preterm pregnancies. One of the main etiologies for preterm rupture of membranes is inflammation. Under these conditions, an immune system-mediated response affects the number of leukocyte subtypes circulating in the circulation, in which the number of neutrophils increases while the number of lymphocytes decreases. The dynamics of the number of leukocyte subtypes is then calculated in the form of a ratio, a parameter known as the value of the neutrophil-to-lymphocyte ratio (NLR). Based on systemic inflammation, NLR has been extensively studied as a prognostic factor in various diseases.

Because PROM is a disease with inflammation as one of its pathophysiological bases, determining the difference in NLR between preterm premature rupture of membranes (PPROM) and without PPROM is important to increase understanding of the crucial role of NLR in predicting the incidence of PPROM.

 

 

METHOD

This study was an analytic case-control study that compared the value of the NLR in maternal blood serum between PPROM (case group) and without PROM (control group). This research was conducted in the emergency department and obstetrics and gynecology outpatient clinic at Prof. dr. I.G.N.G. Ngoerah Hospital Denpasar. The research was done from February 2022 to June 2022. The sample was pregnant women with a gestational age of 20 weeks to less than 37 weeks who came to the emergency delivery room and obstetrics and gynecology outpatient clinic at Prof. dr. I.G.N.G. Ngoerah Hospital Denpasar, who met the inclusion and exclusion criteria.

The research data were processed using the software IBM SPSS version 26.0. All data obtained in this study were analyzed descriptively based on age, parity, and gestational age, and the results were described in the table. The normality test was carried out with the Kolmogorov-Smirnov test. A homogeneity test was performed using Levene's test. The comparative test was performed using the independent t-test and the Mann-Whitney test. Risk factor analysis was carried out by correlating the incidence of PPROM with an increase in NLR.

 

 

RESULTS

This study involved 56 pregnant women using a case-control study design that compared the value of the NLR in maternal blood serum between PPROM and without PPROM. Table 1 compares the distribution based on age, parity, and gestational age. There was no significant difference in age between the two groups (p = 0.117). In the PPROM, the mean age of the maternal was 27.32 � 5.5 years, while in the maternal without PPROM, the average age was 29.75 � 6.7 years, with the lowest age being 17 years old and the oldest being 43 years old. There was no significant difference in parity status between the two groups (p = 0.149), where the mean parity in PPROM was 0.96 � 0.962, while in the maternal without PPROM was 1.54 � 1.37. There was no significant difference in gestational age between the two groups (p = 0.520).

 

Table 1

Mean Distribution by Age, Parity, and Gestational Age in Both Groups

Variable

Preterm PROM

(N = 28)

Preterm without PROM

(N = 28)

P-value

Mean

SD

Mean

SD

Age (years)

27.32

5.5

29.75

6.7

0.117

Parity

0.96

0.962

1.54

1.37

0.149

Gestational age

30.96

3.57

31.25

4.6

0.520

 

Determination of the cut-off value of the NLR in maternal blood serum, which was used as the limit value for the risk factor for PPROM, was obtained from a study conducted by Zhan et al. with a cut-off value of 4.59. The sensitivity and specificity of the cut-off were 43% and 87%, respectively (Zhan et al., 2018).

Table 2 shows that a high NLR in maternal blood serum may be a risk factor for PPROM. The analysis found that high NLR in maternal blood serum had a 4.5 times greater likelihood of experiencing PPROM (OR = 4.5, CI 95% = 1.4-13.83, p = 0.007).

 

 

 

Table 2

Maternal Serum Neutrophil-To-Lymphocyte Ratio as A Risk Factor For Preterm Premature Rupture Of The Membrane

NLR

With PPROM

Without PPROM

OR

CI 95%

P-value

High

18

8

4.5

1.4 � 13.83

0.007

Low

10

20

 

DISCUSSION

In this study, the mean age of the maternal without PPROM was 27.32 years, and the mean age of the maternal with PPROM was 29.75 years (p-value = 0.117). There was no significant difference in age between mothers with PPROM and those without PPROM. Mostly, the age of mothers with PPROM was 20 � 35, followed by > 35 years and < 20 years of age. The age of 20 � 35 years is the childbearing age for women. In a study by Torika et al., similar results were reported. In their report, the pregnant women with PROM in term and preterm pregnancies were mostly at the age of 20 � 35 years (Pradana, 2020).

The mean parity of women with PPROM was 0.96, and that of women without PPROM was 1.54 (p-value = 0.149). It indicated that there was no significant difference in parity between the two groups. Hackenhaar et al. stated that maternal with PROM were mostly found in primigravid (Hackenhaar, 2014). Another study by Movahedi et al. also showed that the highest incidence of PPROM occurred in primigravid (69.7%) (Movahedi et al., 2013). Previous research by Budijaya and Negara also showed that the incidence of PROM in the primigravid was 41.05% (Budijaya & Negara, 2016).

On the other hand, Manuaba and Varney stated that women who have given birth several times had a higher risk. Women who had experienced PROM in previous pregnancies and were too close in the birth period had more risk of PROM in subsequent pregnancies. Multiparities had a higher risk of occurrence of PPROM because of the faster cervix opening than Nulliparities. Therefore, PROM can occur earlier. The infection can cause biomechanical disturbance in the amniotic membranes by proteolytic formation. It makes the membranes rupture easier. In multiparities, due to a history of previous labor, the connective tissue is looser than in nulliparities due to the increased cervical damage. Therefore, there is no resistance to the amniotic membrane.

The average gestational age in the PPROM and without PPROM was 30.96 and 31.25, respectively (p-value = 0.520). It showed no significant difference in gestational age between the two groups. Mostly, the gestational age was in the range of 30 � 36 weeks, followed by 24 � 29 weeks. This is by research by Locatelli et al., which stated that PPROM occurred in less than 1% in 24 � 27 weeks of gestational age, 2 � 5% in 28 � 33 weeks of gestational age, and 3 � 8% in 34 � 36 weeks of gestational age (Locatelli, 2012). However, other studies by Ozel et al. and Toprak et al. found no significant difference between gestational age and the occurrence of PPROM (Ozel et al., 2019; Toprak et al., 2017).

PPROM has a significant association with preterm delivery. Despite various etiologies, PROM is closely related to infection or inflammation. In cases of PPROM, there are increased levels of IL-6, IL-1β, and TNF-α. However, only a few cases are preceded by clinical signs and symptoms of acute infection. In contrast to clinical infection, subclinical infection is characterized by tissue infiltration by neutrophils, macrophages, and lymphocytes without clinically significant findings of infection. Such subacute infection can be proven by histological evidence of chorioamnionitis and positive amnion culture results (Melissa et al., 2018).

It is known that cytokines and chemokines produced by the early inflamed choriodecidua circulate into the maternal bloodstream, leading to changes in circulating leukocyte subtypes. Strong host immune response resulting from increased local production of proinflammatory cytokines and chemokines (such as IL-1β, IL-6, IL-10, TNF-α, G-CSF, prostaglandins, and leukotrienes) causes neutrophilia. On the other hand, lymphocytopenia is caused by inflammation-induced mechanisms such as impaired antigen presentation, activation of negative costimulatory signals, and production of immunosuppressive factors. They all lead to a significant decrease in T-helper lymphocytes in the early phase of the inflammation response.

In the presence of systemic disturbance such as trauma, infection, stress, or ischemic injury, the body response is regulated by the neuroendocrine and the innate immune system and mediated by the adaptive immune system (cellular and humoral). On the injury site, inflammation cells will recognize the site and recruit specific leukocyte subpopulations to the tissue to initiate the destructive process. It will lead to systemic inflammation characterized by fever, leukocytosis, increased acute phase proteins, and inflammation mediators (cytokines, chemokines). In this systemic inflammation response, the leukocyte subtypes that play an important role are monocytes, lymphocytes, and neutrophils. This response is characterized by an increase in circulating neutrophils and a decrease in lymphocytes.

Neutrophilia and lymphocytopenia are physiological responses of the innate immune system to various disorders and stressors, including systemic inflammation, malignancy, major trauma, and malnutrition. Several factors, including hormones, chemokines, and cytokines, induce the mechanisms that cause lymphocytopenia. They regulate the quantity and activity of lymphocytes and indicate the intensity of inflammation and the resistance and adaptability of the immune system. Furthermore, neutrophilia is caused by delaying neutrophil apoptosis and stimulation of stem cells by growth factors (G- CSF) (Mubark, 2015).

At the onset of inflammation, until it reaches its peak in the first 6 hours, there is an increase in the number of neutrophils. In acute inflammation, circulating neutrophils can rapidly increase 10-fold from 5000/μl to 30,000/μl. This increase is due to the migration of neutrophils from the spinal cord to the peripheral blood circulation and delays in the process of apoptosis (Bastek et al., 2012). In PPROM and imminent preterm delivery, cytokines released from the inflamed choriodecidual area can cause changes in the leukocyte subtypes. Lymphocytopenia is common in chronic inflammation due to increased lymphocyte stress and apoptosis (Akboga et al., 2015).

Several studies have examined the relationship between NLR ​​and the occurrence of PPROM. In 2019, Ozel et al. found that the NLR in the PROM was higher compared to the imminent premature delivery and control groups. The increase in NLR is also proportional to the increased risk of neonatal sepsis and C-reactive protein (CRP) levels, with a sensitivity and specificity of 69.7% and 72%, respectively (Ozel et al., 2019).

Determination of the cut-off value of the NLR in maternal blood serum as a risk factor for the occurrence of PPROM was obtained from a study conducted by Zhan et al. (2018). The cut-off value was 4.59, with a sensitivity and specificity of 43% and 87%, respectively (Zhan et al., 2018). The results of this study indicated that a high NLR in maternal blood serum may be a risk factor for PPROM. It was found that 18 samples had a high NLR, while 10 samples had a low NLR in the PPROM. On the other hand, eight samples had a high NLR, while the other 20 samples had a low NLR in maternal without PPRO (p-value = 0.007). It indicated a significant association between a high NLR in maternal blood serum and the occurrence of PPROM. The odds ratio was 4.5 (95% CI: 1.4 � 13.83), which means that materials with high NLR in the blood serum ​​have a 4.5 times greater chance of experiencing PPROM (p = 0.007).

This is done by Toprak et al., which showed a significant increase in NLR in the PPROM compared to spontaneous preterm delivery (Toprak et al., 2017). A year earlier, Akkar et al. reported that NLR was significantly increased in preterm delivery compared to the term delivery (Akkar et al., 2016). Furthermore, Daglar et al. showed that NLR ​​was significantly higher in women with preterm delivery with PROM.

The analysis found that high NLR in maternal blood serum had a 4.5 times greater likelihood of experiencing PPROM (OR = 4.5; CI 95% = 1.4 � 13.83; p = 0.007). As a comparison, the researchers also analyzed the levels of white blood cells (WBC) in maternal blood serum, which were examined using previous research data that had also been conducted (Zhan et al., 2018) with a cut-off value of 9.63 with a sensitivity and specificity of 58% and 83%, respectively. It showed that the WBC count, % neutrophils, absolute count of neutrophils, and NLR in PROM were higher than the normal group.

In this study, the outcome of preterm pregnancy with or without PROM was not affected by high or low levels of WBC (OR 1.00; 95% CI 0.255 � 3.926; p < 0.05). Balciuniene et al. presented 137 adults with PROM before 34 weeks of gestational age, showing much greater WBC count and neutrophils in the PROM group with histological findings of chorioamnionitis. WBC, CRP, and NLR levels were higher in PROM with histological chorioamnionitis (p-value = 0.001). WBC, CRP, and NLR levels predicted HCA in the area under the curve (AUC) of 0.81, 0.81, and 0.89, respectively. Even though the AUC of the NLR was statistically larger than that of the WBC, there was no marked difference between the AUC of the NLR and the CRP.

Kim et al. stated that the ability of NLR to predict preterm labor is second to cervical length. Gezer et al. reported that high NLR ​​at the time of hospitalization was an independent risk factor for preterm delivery with previous PROM in women between 34 - and 37 weeks of gestational age. In a similar study, Ozel et al. found high NLR ​​in PPROM. Lakshmi et al. concluded that NLR monitoring can be carried out during the second and early third trimesters as a routine practice among high-risk mothers because it can significantly assist in the early prediction of PPROM and help minimize the bad outcomes of maternal and neonate.

In the presence of inflammation in the tissue, inflammatory cells recruit leukocytes to the site of infection. Depending on the strength and intensity of the inflammation and the resistance and adaptability of the immune system, neutrophilia and lymphocytopenia develop and are maintained (Wesche et al., 2005). Increasing NLR stimulates neutrophil progenitor cells and lymphocyte apoptosis by various hormones and cytokines (Zahorec, 2001; Roth & Pircher, 2004). In the study of Gezer et al. (2018), NLR > 6.2 on the admission proved useful as a cut-off point for predicting preterm birth with a sensitivity and specificity of 65.1% and 62.5%, respectively.

 

 

CONCLUSION

It can be concluded that a high NLR in maternal blood serum is a marker of inflammation with a higher risk of 4.5 times for the occurrence of PPROM. NLR, a marker of the body's response related to innate immune response, is thought to occur also in various stressful events in pregnancy. In this case, it is related to the inflammation response that occurs in the PPROM. NLR is a relatively cheap, easy, and simple examination. It is quite reliable in measuring the index of the systemic inflammation response. Therefore, it has been widely studied as a predictor and prognostic factor for the severity of several diseases, including the occurrence of PROM.

 

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