DOI: https://doi.org/10.21203/rs.3.rs-2684351/v1
To investigate the predictive values of maternal systemic inflammatory markers, such as the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), systemic immune-inflammatory index (SII), platelet mass index (PKI), and mean platelet volume (MPV), in treatment-requiring retinopathy of prematurity (TR-ROP).
The mothers of the 21 preterm infants who were followed up due to ROP but did not require treatment and the mothers of preterm infants who received ROP treatment (intravitreal injection) (19 patients) were included in the treatment group. The birth weights (BW) and gestational ages (GA) of the infants were recorded. A prenatal maternal complete blood count (CBC) analysis was performed within 3 days before birth. NLR, PLR, LMR, SII, PCI and MPV data were calculated and compared statistically from the complete blood count (CBC) samples of the mothers of the preterm infants who did or did not need ROP treatment. The results were evaluated by adjusting them with logistic regression analysis.
There was no significant difference between the groups in terms of BW (p = 0.108). The GA was significantly lower in the TR-ROP group compared to the control group (p = 0.04, 26.5 (24–33), 29 (27–32), respectively). Between TR-ROP and control groups, NLR (p = 0.02, 5.9 (3.2–12.9), 4.2 (0.9–11.8)), PLR (p = 0.02, 136.4 ± 27.6, 111.1 ± 37.1), LMR (p = 0.001, 2.06 (1.1–4.2), 3.01 (1.2–5.9)) and SII (p = 0.001) values were significantly different. In the TR-ROP group, when these values were corrected with GA in logistic regression analysis, NLR, PLR, and SII were not statistically significant (p = 0.11, p = 0.83, and p = 0.14), but there was an increase in LMR [p = 0.02, OR = 0.38 95% CI (0.16–0.88)].
The prenatal maternal LMR was found to have a statistically significant predictive value for TR-ROP. In the prenatal period, a maternal systemic inflammatory state may be a risk factor for ROP development in the premature baby. Prenatal maternal LMR may be a guide for infants with TR-ROP. Babies who are thought to be at higher risk of TR- ROP may be screened more frequently.
Premature infants with ROP have abnormal retinal capillary development. Preterm birth week and low birth weight are the two best-known risk factors in the pathogenesis of ROP. Many studies suggest that retinal neovascularization and fibrosis play a significant role in the development and progression of ROP [1, 2]. Inflammation has been associated with diabetic retinopathy (DRP), age-related macular degeneration (AMD), and ROP in recent studies [3–6]. Hematological markers and ratios have been studied as potential predictors of an increased inflammatory response in ROP [7]. Studies have investigated the relationship between the development of ROP and the systemic immune inflammation index (SII), lymphocyte-monocyte ratio (LMR), neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) have been reported. [7–11].
Studies on these inflammatory parameters in mothers of preterm infants are limited. In this study, we aimed to investigate the predictive values of systemic inflammatory markers, such as the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), systemic immune-inflammatory index (SII), platelet mass index (PKI), and mean platelet volume (MPV), in mothers of infants with TR-ROP in the prenatal period.
This study was conducted at the Ophthalmology Departments of Erciyes University Hospital and ethical approval was obtained from the local ethics committee (2023/116).
ROP screening was performed according to the latest ROP screening guideline published in Turkey in 2021 [12]. Preterm infants with a gestational age (GA) < 34 weeks or a birth weight (BW) ≤ 1700 g and preterm infants with a GA ≥ 34 weeks or BW > 1700 g who receiving cardiopulmonary support were included in the study.
The neonates had their first ophthalmological examination at 31 weeks' gestational age (GA) for those born before 27 weeks, and at 4 weeks' postnatal age for those born after 27 weeks. The infants were examined by the same ophthalmologist (OAP). The examination was performed according to the guidelines of the International Committee for the Classification of ROP (ICROP) [13]. Retinal examination was performed with a binocular indirect ophthalmoscope, + 20 diopter lens, pediatric speculum, and scleral depressor. All infants were examined 1 hour after the instillation of 1% phenylephrine and 0.5% tropicamide. Eyes of infants diagnosed with stage 3 or aggressive posterior ROP (APROP) were treated according to ETROP criteria [14]. Treated infants were defined as the TR-ROP group, and infants with ROP not requiring treatment were defined as the control group. None of the infants had stage 4 or 5 ROP. The same ophthalmologist (OAP) performed the intravitreal injections.
Prenatal 0–3 days complete blood cell (CBC) samples were analyzed from the mothers of these preterm infants. We used the hospital's electronic database to obtain these data. White blood cell (WBC), lymphocyte, neutrophil, monocyte, platelet and C-reactive protein (CRP) levels, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), systemic immune-inflammatory index (SII) (neutrophil x platelet / lymphocyte), platelet mass index (PKI) (platelet count x MPV), and mean platelet volume (MPV) were calculated and recorded. All results of the data were compared between groups.
The mothers of these infants were also divided into two groups according to the condition of their infants; (1) followed up for retinopathy of prematurity (ROP) but not treated, and (2) treated for ROP (intravitreal anti-VEGF injection). Inclusion criteria for infants in the TR-ROP group: less than 34 weeks’ gestational age (1), having regular ROP examination (2), receiving anti-VEGF therapy (3), regular follow-up visits (4), and having APROP or stage 3 ROP (5). Mothers who had babies with these criteria were included in the TR-ROP group. Criteria for inclusion of babies in the control group: less than 34 weeks of gestational age (1), having regular ROP examination (2), no treatment for ROP (3), being followed up in our clinic (4). Mothers with infants meeting these criteria were included in the control group. Mothers with clinical chorioamnionitis (1), premature rupture of membranes (2), gestational diabetes (3) and pre-eclampsia (4) were excluded from this study. In addition, mothers who received antenatal steroids (5) were not included in this study.
The Statistical Package for the Social Sciences version 22.0 application was used to analyze the data (SPSS, Chicago, IL, USA). The Shapiro–Wilk test was used to check the normality of the data. The analysis of numerical data with a normal distribution was done using parametric methods, while the analysis of numerical and categorical data without a normal distribution was done using non-parametric methods. The Mann-Whitney U test (for not normally distributed data) and the Student-T test (for normally distributed data) were used to determine whether the means of the two independent groups differed. The values of p < 0.05 were considered statistically significant. The significant independent risk variables associated with the presence of ROP were estimated using logistic regression. Statistical significance was defined as an exact P value < 0.05. For each potential risk factor, the adjusted odds ratio (OR) and 95% confidence interval (CI) were determined. The area under the curve (AUC) was calculated for each plot, and sensitivity, specificity, and cutoff values were determined based on the LMR value and expressed via receiver operating characteristic (ROC) curve analysis.
A total of 40 mothers of infants with ROP were included in the study. 19 premature infants with ROP were treated (intravitreal injection) and represented the TR-ROP group. 21 premature infants with ROP were followed up without any treatment and represented the control group. Infants in the TR group had stage 3 ROP in 15 patients and APROP in 4 patients. The control group had stage 1 or 2 ROP that eventually resolved without treatment. GA values, BW data, and the maternal age are shown in Table 1. There was no statistically significant difference between the groups in terms of maternal age and body weight. GA was lower in the TR-ROP groups (p = 0.04).
| TR-ROP Group (n:19) | Control Group (n:21) | p | |
|---|---|---|---|
| Maternal Age | 31.2 ± 5 | 28.7 ± 5 | 0.156 |
| GA (wk) | 28 (24–33) | 30 (27–32) | 0.04* |
| BW (gr) | 1176.9 ± 353.9 | 1357 ± 343.1 | 0.108 |
| GA Gestational age, BW Birth weight, TR treatment-requiring, ROP retinopathy of prematurity, *statistically significant | |||
Maternal CBC results are summarized in Table 2. Lymphocyte count was lower in the TR-ROP group than in the control group (1.75 ± 0.5 and 2.36 ± 0.86 respectively, p = 0.01). There was no statistically significant difference between the TR-ROP group and the control group in neutrophil count, platelet count, monocyte count, MPV, and CRP. The NLR, PLR, and SII were increased and the LMR was decreased in the TR-ROP group compared to the control group (p = 0.02, p = 0.02, p = 0.05 and p = 0.001 respectively). PMI was comparable between groups (p = 0.260).
| Maternal hemogram parameters | TR-ROP Group n:19 | Control Group n:21 | p |
|---|---|---|---|
| CRP (µg/ml) | 12.1 (2.3-123.7) | 6.43 (0.49–48.3) | 0.345 |
| WBC (x103/mcL) | 12.46 (1.06–29.13) | 13.64 (8.15–21.77) | 0.893 |
| Platelets count (x103/mcL) | 227 (158–358) | 234 (170–386) | 0.649 |
| Neutrophils count (x103/mcL) | 9.06 (6.7-25.58) | 9.19 (1.56–18.88) | 0.405 |
| Lymphocytes count (x103/mcL) | 1.69 ± 0.5 | 2.23 ± 0.89 | 0.01* |
| Monocytes (x103/mcL) | 0.84 ± 0.24 | 0.71 ± 0.27 | 0.127 |
| MPV (fL) | 10.6 (9.4–12.6) | 10.7 (9.4–12.7) | 0.08 |
| NLR | 5.9 (3.2–12.9) | 4.2 (0.9–11.8) | 0.02* |
| LMR | 2.06 (1.1–4.2) | 3.01 (1.2–5.9) | 0.001* |
| SII | 1279058 (826576–4625070) | 1040023 (219121–2401754) | 0.05 |
| PLR | 143.8 ± 26.3 | 123.1 ± 36.2 | 0.02* |
| PMI | 2466 ± 570 | 2703 ± 688 | 0.260 |
| WBC White blood cell, NLR neutrophil-to-lymphocyte ratio, LMR lymphocyte-to-monocyte ratio, PLR platelet-to-lymphocyte ratio, CRP C-Reactive protein, SII Systemic immune-inflammatory index (Neutrophil x Platelet /Lymphocyte), PMI Platelet mass index (Platelet count x MPV), MPV Mean platelet volume, TR treatment-requiring, ROP retinopathy of prematurity *statistically significant The mean was used to express normally distributed data (standard deviation), and the median (min-max) was used to express non-normally distributed data. | |||
Table 3 shows the variable logistic regression analysis of NLR, PLR, SII, and LMR. The NLR, PLR, and SII were not statistically significant when these values were corrected by logistic regression analysis according to the week of birth for TR-ROP (p = 0.11, p = 0.83, and p = 0.14). However, only LMR was found to be a significant independent predictor for TR-ROP among all variables examined. (p = 0.02, OR = 0.38, 95% CI (0.16–0.88)).
| OR | %95 CI | p | ||
|---|---|---|---|---|
| lower | upper | |||
| NLR | 1.19 | 0.95 | 1.48 | 0.11 |
| LMR | 0.38 | 0.16 | 0.88 | 0.02* |
| SII | 1.00 | 1.00 | 1.00 | 0.13 |
| PLR | 1.01 | 0.99 | 1.04 | 0.83 |
| NLR neutrophil-to-lymphocyte ratio, LMR lymphocyte-to-monocyte ratio, PLR platelet-to-lymphocyte ratio, SII Systemic immune-inflammatory index (Neutrophil x Platelet /Lymphocyte), OR Odds ratio, 95% CI 95% confidence interval, *statistically significant | ||||
ROC analysis was performed for maternal LMR and is shown in Table 4 and Fig. 1. The ROC analysis showed a cut-off value of 2.807, a sensitivity of 0.73, and a specificity of 0.71, with an area under the curve (AUC) of 0.807 (0.66–0.94) (95% confidence interval).
| Maternal parameters | AUC (%95) | cut off | p | Sensitivity % | Specificity % |
|---|---|---|---|---|---|
| LMR | 0.807 (0.669–0.94) | 2.807 | 0.001* | 73.7 | 71.4 |
| MPV | 0.484 (0.301-0,667) | 21106.7 | 0.860 | 47.4 | 47.6 |
| SII | 0.318 (0.153-0,483) | 1184603.6 | 0.05 | 36.8 | 38.1 |
| PLR | 0.296 (0.132-0,459) | 114,92 | 0.27 | 36.8 | 38.1 |
| NLR | 0.291 (0.131-0,450) | 4,994 | 0.24 | 36.8 | 38.1 |
| PIM | 0.594 (0,416-0,772) | 2489,75 | 0.310 | 52.6 | 52.4 |
| AUC (%95) area under the curve with 95% confidence intervals, NLR neutrophil-to-lymphocyte ratio, LMR lymphocyte-to-monocyte ratio, PLR platelet-to-lymphocyte ratio, SII Systemic immune-inflammatory index (Neutrophil x Platelet /Lymphocyte), PMI Platelet mass index (Platelet count x MPV), MPV Mean platelet volume *statistically significant | |||||
ROP is defined as a multifactorial disease, and its pathogenesis consists of two phases. In the first phase, retinal vascularization is inhibited due to postnatal hyperoxia. In the second phase, the retina becomes hypoxic due to a retinal vascularization disorder. Afterward, neovascularization begins as a result of increased levels of mediators such as VEGF, erythropoietin, and IGF-1 [15]. Studies also show that there is a “pre-phase” due to placental inflammation that develops in the mother during the intrauterine period [11]. Studies have shown that both neonatal and maternal systemic inflammatory responses are important in the etiology of ROP [2, 16].
So far, inflammatory markers from complete blood counts have been suggested as potential markers of inflammation in a variety of diseases, including bronchopulmonary dysplasia and kidney diseases [17, 18]. With the increasing importance of the role of inflammation in ROP, studies in this area have increased in recent years. However, in the early period of a newborn's life, many factors such as birth stress, early or late cord clamping, normoblasts, and antenatal steroid use may cause inconsistent results in the pediatric complete blood count [19, 20]. Prenatal maternal inflammation may also influence the pathogenesis of ROP. Therefore, in this study, we investigated whether maternal inflammatory markers could predict TR-ROP.
In the study of Celik et al. [10], the complete blood count parameters of preterm infants who received ROP treatment and those who did not develop ROP and their mothers (1 day before birth) were evaluated. While maternal WBC was found to be significantly higher in the TR-ROP group, maternal NLR, LMR, PLR, and PCI were not significantly different. (maternal WBC AUC = 0.69). In addition, infant WBC was found to be significantly lower in the TR-ROP group. Woo et al. [21] found clinical chorioamnionitis (excluding histological chorioamnionitis) and increased maternal WBC to be significant independent risk factors for the development of ROP at any stage. We found a significant reduction in maternal lymphocyte count in the TR-ROP. However, we think that GA may affect the results when interpreting neonatal CBC results. Previous studies have reported that neutrophil abnormalities and lymphocyte maturation are altered in GA [22, 23]. When the lymphocyte counts adjusted for GA were evaluated, there was no significant difference between the groups (p = 0.06).
Novel inflammatory prognostic markers generated from the peripheral blood of infant, including the NLR, PKI, LMR, and PLR, have been researched for their prognostic significance in the development of ROP [7, 8, 24]. The prognostic effects of PLR, NLR, and LMR on ROP were investigated, and only LMR showed a significant difference in multivariate analysis. Similar LMR results were obtained in the hemogram study of infants with ROP by Hu et al [7]. Therefore, it can be said that both maternal and infant LMR values are more significant than NLR and PLR in terms of diagnostic value in ROP. In contrast to the study by HU et al., all infants in our study had ROP. The significant difference in infants with TR-ROP shows that LMR is also an effective parameter in the prognosis of ROP.
The SII is a new immune marker [25]. The SII was calculated by the formula = neutrophil x platelet/lymphocyte. Akdogan et al. [8] reported the only study in the literature on the relationship between SII and ROP, and our study is the first on the relationship between maternal SII and ROP relationship. Akdogan et al. reported that infants with ROP had a higher SII value than infants without ROP in the 1-month period, and they stated that for ROP, SII could be an independent predictor for ROP. In our results, we did not find a significant difference in maternal SII between the groups. We concluded that maternal SII values are not of sufficiently important in predicting the diagnosis and prognosis of ROP.
In the study of Korkmaz et al. [9], they compared premature infants who received laser photocoagulation therapy and premature infants who did not receive treatment, and they found no difference in PMI values between the groups in the first phase of ROP, but they found a significant difference between the study groups in the second phase of ROP. Their result showed that PMI levels in the second phase of ROP may be important in the follow-up of ROP. They stated that PMI levels may also increase in parallel with the increase in VEGF in the second phase of ROP, as platelets play a role in the transport, storage, and release of VEGF. Our study is the first to show an association between maternal PMI and ROP. The fact that there was no difference in maternal PMI between the groups suggests that maternal PMI levels are not as significant as infant PMI levels in TR-ROP.
Mean platelet volume (MPV) is a parameter that indicates platelet activity, and MPV values have also been studied in the development of ROP. Ozkaya [26] reported that there was no difference between the groups in terms of other platelet parameters such as PMI, MPV, platelet count, and platelet distribution width (PDW). Similar results were obtained in our study, and there was no difference between the groups in maternal; MPV, platelet count, or PMI values.
This study showed that LMR values during the prenatal period can be a guide for the treatment of ROP development. Considering the significantly different results of LMR in infants with ROP in previous studies, our results support the importance of LMR in terms of the diagnosis and prognosis of ROP. The association of maternal SII, PMI, and MPV parameters with TR-ROP is also shown for the first time in this study.
This study has several limitations. The first limitation of this study is its retrospective nature. Second, the limited sample size in the study.
Prenatal maternal LMR was found to have a statistically significant predictive value for TR-ROP. In the prenatal period, a maternal systemic inflammatory condition may be a risk factor for ROP development. Prenatal maternal LMR may be a guide for infants with TR-ROP. Babies who are thought to be at higher risk of TR-ROP may be screened more frequently.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. The authors have no relevant financial interest to disclose.
Author Contributions
Conceptualization; [Osman Ahmet Polat, Cagatay Karaca], Data curation; [Osman Ahmet Polat, Furkan Ozer], Formal analysis; [Osman Ahmet Polat, Furkan Ozer], Investigation; [Osman Ahmet Polat, Furkan Ozer], Methodology; [Osman Ahmet Polat, Furkan Ozer], Project administration; [Osman Ahmet Polat, Cagatay Karaca], Statistic Software; [Osman Ahmet Polat], Supervision; [Osman Ahmet Polat, Cagatay Karaca], Validation; [Osman Ahmet Polat, Furkan Ozer] Visualization; [Osman Ahmet Polat, Furkan Ozer], Writing - original draft; [Furkan Ozer, Osman Ahmet Polat], Writing – review & editing; [Osman Ahmet Polat, Cagatay Karaca, Furkan Ozer], all authors read and approved the final manuscript
Ethics approval and informed consent
The study protocol was approved by the Erciyes University Local Ethics Committee (2023/116). Written informed consent was obtained from all individual participants.
Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.