Sars-cov-2 Infection And Its Association With Maternal And Fetal Redox Status And Outcomes: A Prospective Clinical Study

1. Introduction

WHO data show that the number of patients with COVID-19 from the beginning of the pandemic to August 2024 exceeds 776 million with more than 6.95 million deaths. [1,2]

Even after the WHO declared the end of the pandemic on May 11, 2023, COVID-19 remains one of the biggest challenges facing the health system and world economy because of its rapid spreading and unpredictable course.

SARS-COV-2 is the pathogen responsible for the onset of COVID-19, belonging to the group of beta-type Coronaviridae. [3,4] Based on structure, it is an enveloped RNA virus that shares a genetic sequence with some SARS viruses identified in bats and pangolins, indicating a zoogenic origin. [5] SARS-COV2 components are RNA and four structural proteins: spike (S) protein, nucleocapsid (N) protein, envelope (E) protein, and membrane (M) protein with a specific role in the pathogenesis of the disease. [6]

SARS CoV-2 is transmitted from infected individuals or asymptomatic carriers through droplet infection or contact with contaminated surfaces. [1,4] When the virus encounters a potential host, it first binds to ACE2 receptors with the S protein, resulting in the fusion of the virus's and cells' membranes. [7,8,9] These receptors are found on tissue cells of the kidney, endothelium, heart, macrophage, and pneumocyte cells, but predominantly in type 2 alveolar epithelial cells, making them highly sensitive to the virus's effects. [10,11,12]

In addition to the ACE2 receptor, Type 2 Transmembrane Serine Protease (TMPRSS2) also plays a key role. This protease leads to the cleavage of the S protein and a change in shape that facilitates the fusion of the virus's and the host's membranes and allows the virus's RNA to penetrate the host cell and begin replication. [1]

The RNA of the virus is a template for the multiplication of viral RNA and proteins that are further released infecting new cells. The release of the virus into the bloodstream leads to an inflammatory reaction mediated by the release of cytokines and the consequent activation of the coagulation system. [10,13] Activation of monocytes as part of the acute phase response leads to damage to the endothelium, which is crucial for activating the procoagulant system within the body and forming immune-thrombosis. In addition to this mechanism, intrahospital venous thromboembolism is also responsible for the occurrence of complications in COVID-19 patients. [16,20–24] VTE occurs because of activation of Virkoch's thryas: activation of the endothelium, prothrombotic state, and reduced mobility of patients that is characteristic of pregnancy. [10,14,15,16,17,18]

Based on the severity of symptoms, COVID-19 can be asymptomatic, mild, moderate, severe, and critical. [19] The viral load, inflammation, and the state of the immune system also influence the severity of the clinical course. [1] Factors such as age, gender, associated diseases, and obesity are related to the severity of the disease and increased mortality. [20] The physiological changes that accompany pregnancy could theoretically contribute to the development of more severe forms of COVID-19 in this group of patients. [21] Data from the US Centers for Disease Control and Prevention show that pregnant women have the same risk of death, but an increased risk of developing complications that require hospitalization and admission to the ICU compared to patients who are not pregnant. [10]

The immune system changes along with the development of the fetus to provide the necessary immunotolerance and adequate protection for the mother. These changes are dynamic and follow the gestational age to enable the smooth development of the fetus, so in the first trimester pro-inflammatory mechanisms dominate, the second trimester is characterized by anti-inflammatory, and in the third, pro-inflammatory mechanisms again prevail. [22] During pregnancy, the Th1-induced immune response decreases in favor of the TH2 response, leading to increased production of anti-inflammatory cytokines (IL4, IL10). Activation of the immune response and interleukin-initiated cytokine storms are a key factor in developing severe forms of the disease, and it is assumed that patients infected in the first and third trimesters are at a higher risk of serious complications. [23,24]

Pregnancy is a hypercoagulable condition characterized by elevated levels of prothrombotic factors such as factors VII, VIII, X, XII, von Willebrand factor, and fibrinogen, as well as decreased protein S and altered fibrinolysis, especially in the peripartum period. [10,25] Given that these physiological changes in pregnant women have a fourfold risk of developing serious thromboembolic complications compared to the general population, SARS-COV2 infection can lead to serious thromboembolic complications. [26,27]

Direct cytotoxicity of the virus and endothelial injury led to inflammatory changes in the complex functioning of the pregnant woman's immune system. This system constantly changes to satisfy the demands of the growing embryo, and thus the time of infection can be of great importance for the prognosis of the course of the disease, maternal immune response, release from the virus, and perinatal outcome. [28]

The impact of viral infection on the fetus can be direct, transmitted through the placenta, and indirect - creating unfavorable conditions for the development of the fetus because of inflammation, micro-thrombosis, and hypercoagulation.

There are three modes of transmission of the virus to the fetus/neonate: direct/intrauterine transmission, intrapartum and postpartum transmission.

Oxidative stress is one of the key factors in the pathogenesis of COVID-19 infection. [29,30,31] Oxidative stress is an impaired homeostasis in the body between oxidants and antioxidants, which leads to further disruption of the redox system and damage to molecules. Free radicals are highly reactive molecules with an excess of one or more electrons that easily interact with other molecules and lead to disruptions in the physiologically tuned system of a healthy organism. Excessive production of free radicals: reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive chlorine species (CRS) lead to direct peroxidation of membranes, structural proteins, enzymes, and nucleic acids, and can be neutralized under physiological conditions by interaction with enzymatic and non-enzymatic antioxidants. In addition to endogenous (enzymatic and non-enzymatic antioxidants), there is also a group of exogenous (natural or synthetic antioxidants) that also have a role in binding unpaired electrons of free radicals and stabilizing them, and their cumulative effect is defined as total antioxidant capacity (TAS). [32,33,34,35]

During scientific attempts to determine the level of oxidative stress in the body, a whole panel of biochemical markers of OS were created, which, based on their origin, were divided into:

Molecules that interact with free radicals, such as:

Products of lipid peroxidation: malondialdehyde, acrolein, isoprostanes),

Protein oxidation products: (carbonyl protein, tyrosine group),

DNA oxidation products (8-hydroxy-2deoxyguanosine),

Protein stress reactions, new biomarkers (microRNAs)

Molecules that belong to the antioxidant protection system, such as

Enzymes (myeloperoxidase),

Glutathione level

Circulating antioxidants (CAT, GPX, SOD) [34,36,37,38]

Pregnancy itself represents a state of increased oxidative stress due to the intense metabolic and immunological alterations necessary for the complex process of growth and development of conception products. The main source of ROS in pregnancy is the placenta, where these molecules are key factors in the complex system responsible for intercellular signaling, transcription, adaptive homeostasis, apoptosis, and host defense through phagocytosis [39,40,41,42]. Viral infections affect the excitation of oxidative stress of the host through numerous mechanisms such as excessive cytokine production, activation of the complement system, release of lipid mediators, and lipid peroxidation during the interaction of the body's defense cells with viral pathogens. [43,44]

In addition to its nutritional, excretory, oxygenation, and hormonal function, the placenta has an important protective role and is the main barrier to bacterial and viral infections in the fetus. This role predisposes the placenta to pathological changes under the direct influence of the SARS-COV2 virus and the consequences of systemic disease and coagulation changes. Examining the changes in the placenta provides us with a unique opportunity to uncover the mechanisms by which the virus affects pregnancy and fetus. MVM encompasses a spectrum of macroscopic and microscopic changes in the placenta. These changes are the result of ischemic (hypoxic) damage and oxidative stress. [45] Macroscopic changes are placental hypoplasia (weight <10th percentile), narrowed umbilical cord (diameter less than 0.8 cm at term), infarction, and retroplacental hemorrhage. [46,47,48] Microscopic lesions of the placenta include: distal villi hypoplasia and accelerated villi maturation, [49,50,51]

(FVM) Fetal vascular malperfusions are specific lesions of the placenta that arise from the obstruction of fetal blood flow. They can originate from the umbilical cord (hyperkoyling of the umbilical cord), from hypercoagulability, hypoxia, and inflammation-mediated endothelial damage (acute chorioamnionitis with chorionic vasculitis or funisitis and chronic villitis). [48,52,53,54,55]

Chronic villitis can be a consequence of viral infections, but in most cases, the etiology has not been identified. According to the Amsterdam criterion, FVM is represented by the following findings: thrombosis, segmental avascular villi, villous stromal-vascular karyorexa, vascular intramural fibrin deposition, blood vessel obliteration/fibromuscular sclerosis and vascular ectasia. [48]

Previous observational studies dealing with the problem of MVM have shown that these changes were asymptomatic, but the cumulative effect was reflected in placental hypoperfusion leading to adverse perinatal outcome and fetal complications and is associated with preeclampsia and intrauterine growth retardation (IUGR). [56,57,58,59]. IUGR is a fetus that does not reach its genetic potential during intrauterine growth and development and is present in as many as 15% of pregnancies. [60]

FVM lesions are associated with abnormalities of the neonatal central nervous system, IUGR, intrauterine fetal death, and stillbirth.[55,61,62,63].

Some studies have proposed that the determination of different types of OS biomarkers from the umbilical cord, placenta, and maternal blood could have implications in the prediction of placental pathology and clinical symptomatology. [64,65]

Besides oxidative stress damage, a potential pregnancy hazard is the transmission of the virus to the fetus. There are a few possible maternal/fetal (neonatal) infection methods. Fetal infection can occur in utero by direct transplacental transmission. The other possible way is intrapartum infection (neonatal contact with cervical or vaginal secretions or blood), and the third way is after birth via breastfeeding or direct maternal-neonatal contact. [61]. Investigating routes of possible fetal or neonatal transmission should provide sufficient information for future decisions considering the obstetric way of delivery, neonatal care, isolation of neonates, or safety of breastfeeding. [66]

We hypothesized that:

• There is a statistically significant difference in the frequency of pathohistological changes in placental tissue in patients with confirmed SARS-COV2 infection during pregnancy compared to the control group.
• Vertical transmission of SARS CoV-2 from mother to fetus is possible.
• There is a statistically significant increase in maternal and umbilical cord blood biomarkers of oxidative stress in patients infected with SARS CoV-2 during pregnancy compared to the control group.
• There is a significantly elevated level of oxidative stress markers in patients with more pronounced pathohistological changes in placental tissue.
• There is a statistically significant difference in the neonatal outcome of newborns of mothers with COVID-19 compared to the control group.
• Certain clinical parameters such as gestational age, sex of the newborn, and maternal age significantly affect the level of oxidative stress markers in the newborn.

This study aims to:

1. To determine the types and frequency of pathohistological changes in placental tissue in patients in whom SARS-COV-2 infection was confirmed during pregnancy and compare them with the control group

2. Determine from the plasma and lysate of erythrocytes of the mother's blood immediately before delivery and the blood of the newborn from the umbilical cord the values of the parameters of the antioxidant protection system, as well as pro-oxidants: lipid peroxide index measured as TBARS, nitric oxide in the form of nitrite (NO2-), superoxide anion radical (O2-) and hydrogen peroxide (H2O2), catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH).

3. Determine the neonatal outcome: Apgar score, body weight of the newborn, admission to the intensive care unit, and length of stay in the neonatology department and ICU

2. Materials and Methods

2.1. Ethical Concerns

This study was designed as a case-series clinical study following the Good Clinical Practice and revised Helsinki Declaration. This study was approved by the Ethical Committee of Clinical Center Kragujevac number: 01/21-18. Written informed consent was obtained from all participants for research and publication before inclusion in the study.

2.2. Participants

This prospective clinical study included pregnant women who gave birth from the 24th to the 42nd week of gestation at the Ginecology Clinic at University Clinical Center Kragujevac, aged 18 to 43 years old, from Sumadia district, Republic of Serbia, followed in the period from the diagnosis of a positive SARS COV2 test to discharge from the obstetrics department after delivery and discharge of the newborn from the Department of Neonatology at University Clinical Center Kragujevac, Serbia.

2.3. Protocol of Study

Two cohorts were derived from the population of pregnant women who were delivered at the OB/GYN Clinic of the University Clinical Center in Kragujevac: women who had a positive result for SARS-COV2 (by RT-PCR method and/or rapid antigen test) during the pregnancy. The control cohort were woman who had a negative result on rapid antigen or RT-PCR SARS COV2 test on their admission day to the Obstetric Clinic for delivery, who were asymptomatic during hole pregnancy, and who had no IgG and IgM SARS COV2 antibodies detected on serological blood test. The main inclusion criteria were positive RT-PCR and/or rapid antigen tests for SARS-COV2 during the pregnancy. Exclusion criteria were positive personal history of thromboembolic diseases, thrombophilia, antiphospholipid syndrome, diabetes mellitus, systemic lupus erythematosus, hypertensive disorders in pregnancy, Rhesus factor incompatibility, ongoing anticoagulant therapy, and tobacco smoking during pregnancy.

Within the cohort of pregnant women who tested positive for the presence of SARS-COV2 during pregnancy concerning the time of gestation in which the infection occurred, three subgroups of patients will be distinguished in whom the impact of COVID-19 on placental tissue will be monitored in parallel:

1. Patients who have been diagnosed with SARS CoV2 in the first trimester of pregnancy (from pregnancy to the end of week 13 of gestation)

2. Patients who have been diagnosed with SARS CoV2 in the second trimester of pregnancy (from 14 to the end of week 27 of gestation)

3. Patients diagnosed with SARS CoV2 in the third trimester of pregnancy (28 to 42 weeks of gestation)

2.4. Sampling and Collecting Data

We have reviewed the electronic medical record for each subject and recorded demographic, clinical, obstetric, laboratory, and pathologic data. At that time, none of the patients in either group were vaccinated against COVID-19. Blood samples were collected from those two groups following delivery, namely: maternal peripheric blood and the umbilical cord blood for oxidative stress biomarkers test, placental tissue (a section of the entire thickness of the placenta with the membranes and one section of the umbilical cord for pathohistological analysis, fixed for 48 hours in a formalin solution to eliminate the infectivity).

2.5. Determination of Oxidative Stress Markers

We have collected venous blood from each participant, maternal peripheric blood, and umbilical cord blood for oxidative stress biomarkers testing in the next 3 minutes following delivery. Oxidative stress biomarkers were assessed spectrophotometrically from plasma samples (Shimadzu UV 1800, Japan), the index of lipid peroxidation, determined as thiobarbituric acid-reactive substances (TBARS), nitrites (NO₂-), levels of superoxide anion radical (O₂-) and hydrogen peroxide (H₂O₂).

2.5.1. Determination of TBARS

The plasma sample was incubated with 1% thiobarbituric acid in 0.05 NaOH for 15 minutes at 100 °C and measured as TBARS at 530 nm. As a control, distilled water was used as a blind probe. [67]

2.5.2. Nitrite Determination (NO2-)

Rapidly breaking down nitric oxide (NO) produces stable nitrite/nitrate compounds which were determined spectrophotometrically at 543 nm using Griess’s reagent according to Green`s method. Sodium nitrite was used as the reference standard to calculate the nitrite levels. [68]

2.5.3. Superoxide Anion Radical Determination (O2-)

Using the assay mixture that contains nitroblue tetrazolium, superoxide anion radical quantities were determined. The measurement was carried out at 530 nm on the wavelength. The blank control was performed using distilled water. [69]

2.5.4. Hydrogen Peroxide Determination (H2O2)

Horseradish peroxidase was utilized to accelerate the oxidation of phenol red by H2O2, which was used as a basis for the H2O2 measurement. 800 µl of freshly created phenol red solution was used to precipitate 200 µl of perfusate, and 10 µl of freshly made (1:20) horseradish peroxidase was added next. As the blank probe distilled water was utilized. At 610 nm, the level of H2O2 was detected.[70]

2.5.5. Catalase Activity Determination (CAT)

The analysis of catalase activity is carried out based on the Aebi method [71] .

First, we diluted the hemolysate with distilled water in a ratio of 1:7. 100 μl of the hemolysate sample was then mixed with an equal volume of ethanol along with 50μl of CAT buffer and 1000μl of 10 mM H₂O₂. The catalase activity was then determined spectrophotometrically at 230nm wavelength. [71]

2.5.6. Superoxide Dismutase Activity Determination (SOD)

The activity of superoxide dismutase is carried out by the Beutler method. In Eppendorf, we first mixed [72] 100μl of hemolysate with 1 ml of carbonate buffer, then processed the sample into a vortex and finally added 100μl of adrenaline. Spectrophotometry was performed at a wavelength of 470nm. [72,73]

2.5.7. Reduced Glutathione Concentration Determination (GSH)

The concentration of reduced glutathione (GSH) is determined by the Beutler method. This analysis is based on the oxidation reaction of GSH with GSH with 5,5-dithio-bis-6,2-nitrobenzoic acid. Spectrophotometric measurements were made at a wavelength of 412 nm. [72]

2.6. Pathohistological Analysis of Placental Tissue

Samples were also collected according to the recommendations of the Amsterdam Consensus [51]. Six samples were taken: first a sample of the amniotic sheath from the rupture site to the edge of the placenta; a sample of the cross-section of the umbilical cord, one from the fetal and the other about 5 cm from the uterine insertion; 3 more sections of the full-thickness placental disc cut with a scalpel, covering the uterine and fetal sides, taken from the central two-thirds of the surface of the placenta and one from the insertion site.

Tissue processing: dehydration, rinsing, and impregnation is carried out by a tissue processor and then molded into paraffin blocks. Then, using a rotating microtome (Leica RM2135), we cut the tissue into samples 5 μm thick, at room temperature, which were then immersed in a water bath at a temperature of 400 C. The cross-sections processed in this way were applied to the subject glasses (Superfrost-OT Plus microscope slides) for microscopy.

2.6.1. Staining Hematoxylin-Eosin Technique

To de-paraffin the tissues, it was necessary to first subject the glasses to heating to + 56 ⁰ С and then to spontaneous cooling. After that, we immerse them in xylol consecutively twice for 5 minutes each. Once we have deparaffinated the tissues, we proceed to rehydrate the tissues by immersing the glasses in solutions with decreasing concentrations of ethyl alcohol and then evaporating them with distilled water. The next step was staining with Mayer solution (Sigma Aldric, USA) for 10 minutes, after rinsing with distilled water and staining with an alcohol solution of eosin (Sigma Aldric, USA) for the next 2 minutes. A dehydration process was then carried out, which involved submerging the glass with increasing concentrations of ethyl alcohol and then illuminating it with two xylol solutions, each for 5 minutes. [74] The tiles prepared in this way are protected by cover glass after treatment with a medium covering and dried at room temperature. The analysis was carried out independently by two experienced pathologists on an Olympus BX51 light microscope, Japan. In the case of inconsistent results, the attitude of the third pathologist was decisive.

2.7. Statistical Analysis

All data are presented in the form of tables and graphs. Statistical analysis was done using the descriptive (mean, standard deviations and errors, and analytical tests, frequency, range) and analytical tests (Student T-test, Chi-square test). The statistical threshold was set at 0.05. All analysis was done in SPSS version 26 for Apple. Inc.

3. Results

3.1. Basic Demographic and Anamnestic Data of Study Group

This study included 50 pregnant women divided into two groups, SARS-CoV-2 positive (COVID-19 group) and SARS-CoV-2 negative (control group). In the first group, the mean age was 30,61±4,72, and in the negative groups, the mean age was 31,41±4,65. The mean gestational week at delivery was 39,19±0,98 in SARS-CoV-2 positive women, and 39,42±1,26 in the group SARS-CoV-2 negative. Based on the severity of symptoms 41.38% of patients had mild symptoms, and 58.62% had moderate symptoms.

3.2. Characteristics of the Newborns at Delivery According to the Presence of SARS-CoV-2 Infection in Mothers

Table 1 presents the mean values of the newborn characteristics at delivery. Most of them were similar in both groups, except for the concentrations of IgM antibody, where we found that newborns from COVID group mothers had significantly higher levels of these inflammatory markers in comparison with the SARS-CoV-2 negative group. (Table 1)

3.3. Analysis of the Oxidative Stress Levels According to the Presence of SARS-CoV-2 Infection

According to the presence of the SARS-CoV-2 infection, the mean values of redox status parameters are presented in the form of Figure 1a–d and Figure 2a–c. The values of the superoxide anion radical and index of lipid peroxidation were significantly different in mothers concerning the presence of the SARS-CoV-2 infection, while the levels of the nitric oxide, index of lipid peroxidation, reduced glutathione, and superoxide dismutase were significantly different in the newborns depending on the SARS-CoV-2 infection (Figure 1 and Figure 2, Table 2). Other parameters were not significantly changed (Figure 1 and Figure 2, Table 2).

3.4. The Histopathologic Lesions in the Placenta Concerning the Presence of SARS-COV2 Infection and the Timing of Infection

The incidence of pathohistological changes of the FVM type in the COVID-19 group of pregnant women was 46%, while in the control group, they were represented in 18% of placentas, and the incidence of MVM in the COVID group was 32%, and in the experimental group 18%. According to the moment of infection in the first trimester, pathohistological changes were registered in only 3% of patients infected in the first trimester, while they were more prevalent when the infection occurred during the second and third trimesters, in 44.4% and 47% of patients of Covid group. Inflammatory lesions in placental tissue were detected in 14.81% of placentas in the COVID group and only 4.5% of subjects in the control group. 70% of all placental lesions were detected in a group of patients with moderate/severe presentation of COVID-19. Table 3 presents the main morphometrical Placental characteristics of mothers regarding the presence of COVID-19. The results are presented as the frequency of the observed entity in percentage (%) (Table 3).

Figure 3. The representative pathohistological findings in placental tissue samples among the study population; 3a distal villous hypoplasia-long villi with wide intervillous space; 3b the vascular ectasia-enlarged luminal diameter of the chorionic vessel; 3c Chorioamnionitis-numerous polymorphonuclear leukocytes infiltrate edematous fetal membranes; 3d-Distal villous hypoplasia; 3e-Avascular villus-lose of vessels with preservation of trophoblast; 3f-Villus vessel thrombosis-organized intraluminal Thrombus; Magnification 100x.

Figure 3. The representative pathohistological findings in placental tissue samples among the study population; 3a distal villous hypoplasia-long villi with wide intervillous space; 3b the vascular ectasia-enlarged luminal diameter of the chorionic vessel; 3c Chorioamnionitis-numerous polymorphonuclear leukocytes infiltrate edematous fetal membranes; 3d-Distal villous hypoplasia; 3e-Avascular villus-lose of vessels with preservation of trophoblast; 3f-Villus vessel thrombosis-organized intraluminal Thrombus; Magnification 100x.

3.5. Neonatal Outcome

In the COVID-19 group, 62% of newborns were girls, 38% were boys, and in the control group, 41% were girls and 59% were boys.

The mode of delivery was vaginal birth in 68%, and C-section in 32% in the COVID-19 group, and vaginal birth in 45%, and C-section in 55% in the control group. Newborns of mothers from the Covid group had a similar mid-length of hospitalization- 4.37±1.1 days, as newborns from the control group with a mid-length of hospitalization of 4.22±2.72 days.

The length of newborn stay in neonatal ICU was 3.51±1.08 days in the COVID group and 2.22±1.39 days in the Control group.

Newborns from COVID-positive mothers were tested with nasopharyngeal swabs for RT-PCR SARS-COV2 test in the first 24 hours post-delivery. Only one positive result was registered in newborns.). 42,8% of newborns from COVID group mothers had positive SARS-COV-2 IgM antibody results, in the control group there were no positive SARS-COV-2 IgM antibody results.

4. Discussion

This prospective clinical study aimed to analyze the differences between basic, clinical, anamnestic, and biochemical data among pregnant women who were affected by COVID-19 or not. The special purpose of this research lies in the evaluation of the effects of the SARS-CoV-2 infection on fetal and newborn characteristics. Based on previous findings and literature data, for the first time, this study will evaluate the pathohistological changes in placental tissue among COVID-19 women who gave birth.

In the COVID group predominant mode of delivery was vaginal birth, except in a group of pregnant women with active COVID infection during delivery. Although the C-section birth rate was high (32%) it was significantly lower than previous studies showed. [75,76,77]. The leading indications for C-section in the COVID group were previous delivery completed by C-section, fetal distress during labor, and malpresentation, similar to the control group. Our study showed that there was no statistically significant increase in the number of cesarean sections in patients with COVID-19 during pregnancy. Studies dealing with this issue had contradictory results, some have shown an increased risk of operative termination of labor in COVID-19 patients [78,79,80], while others have shown that the mode of delivery does not depend on COVID-19 infection in pregnancy. [81]

No significant differences in neonatal outcomes were found between COVID-19 and the control group regarding birth weight, length, and head circumference. The hospitalization length for newborns was similar in both groups, with similar neonatal ICU hospitalization length 3.51±1.08 days in the COVID group and 2.22±1.39 days in the Control group.

All patients with active COVID newborns were tested with a SARS-COV-2 RT PCR test, only one was positive. Our study found that 42.8% of newborns from the COVID group mothers had positive IgM antibody findings. Initial studies focused on direct transmission showed that there was no convincing evidence of this mode of infection, however, at the end of the pandemic, worldwide pandemic meta-analyses showed that about 8.8% of newborns of COVID-19 positive mothers at birth had a positive SARS-COV2 PCR or serological test obtained after taking a nasopharyngeal swab. [82,83] The method of determining direct transmission to the fetus is also a subject of scientific doubt. In adults, the respiratory tract is the primary site of infection and the highest concentration of the virus, and the analysis of samples is performed through a nasopharyngeal or oropharyngeal swab, in the newborn, in addition to this method, is proposed to do the serological determination of the concentration of specific IgM antibodies to SARS-COV2, concerning the unknown site of primary infection. [84,85] IgM antibodies as part of the immune response to a specific viral infection, unlike IgG antibodies, cannot cross the placental barrier and can be useful in determining potential direct transmission to the fetus, but the weakness of this method is the existence of a cross-reaction and a false positive result, and the PCR test of the nasopharyngeal/oropharyngeal mucosa is the gold standard for determining the presence of SARS-COV2 infection in the newborns. [86] Studies focused on direct transmission have also used RNA Scope and In situ hybridization in scinciciotrophoblast cells, but the main problem with interpreting the results is the inconsistency of the results of the available tests. The sensitivity of the RT-PCR test is highest from the bronchial lavage sample (93%), and significantly lower from the nasopharyngeal swab and feces (63% and 29%, respectively), but as invasive, this method is not suitable for testing pregnant women and newborns. [87]

During this interaction of virus and host, studies have shown an increase in the activity of myeloperoxidase, NADPH synthetase, and inducible NO synthetase as well as changes in the levels of enzymatic and non-enzymatic antioxidants such as superoxide dismutase (SOD), glutathione peroxidase and catalase, and vitamin C, vitamin E, carotenoids, and glutathione flavonoids. [88,89,90,91,92] If the overproduction of ROS exceeds the antioxidant capacity of the placenta, tissue damage may occur. [43,93]

Placental ischemia, resulting from SARS-COV-2 infection, or systemic hypoxia and inflammation, leads to a disturbance in the balance of ROS and antioxidant protection and the onset of oxidative stress. OS is responsible for damage to proteins, lipids, and DNA. The placenta has mechanisms to counteract oxidative stress to reduce tissue damage, such as the production of NO, a potent vasodilator and antioxidant [65]. In addition to NO, CO, and H2S also play a significant gasotransmitter and vasodilator role [94].

Our study showed significantly increased TBARS levels in mothers of the COVID-19 group (p=0,002) compared with mothers of the control group, probably result of lipid damage caused by the SARS-COV-2 virus. Levels of O₂- are significantly higher in our study in control group, which could be a consequence of the mode of delivery. SC was more frequent mode of delivery in the control group 55%, and 32% in the COVID group. Also leading indication for C-section in the control group was fetal nonreassuring status and previous C-section. Levels of NO were significantly lower in newborns of the COVID-19 group in our study, indicating that the placenta of COVID mothers could not sufficiently counterbalance the produktion of ROS after SARS-COV-2 infection. Increased levelas of SOD in COVID-19 group newborns suggested that placentas developed an oxidative damage defense system. The method of delivery could significantly affect the elevated level of biomarkers OS in the newborn, where these values are significantly increased in prolonged vaginal delivery and emergency cesarean section, as well as in stimulated oxytocin delivery. [32,95,96,97] The effect of the oxidative stress of the mother on the oxidative stress of the fetus depends on the antioxidant capacity of the newborn, which is determined genetically, depending on the sex of the fetus, the maturity of the fetus, and the age of the mother.[32]

Determination of individual biomarkers of OS cannot predict the level of pathological changes within the placenta and potential fetal/neonatal complications. Pregnancy and placental growth and development are accompanied by numerous changes due to the balance of the ROS and the antioxidant protection system, which is also involved in the complex mechanisms of cell signaling. The development of the placenta begins with the insertion of a fertilized blastocyst and the primary mode of transport through the placenta is diffusion conditions of low partial pressure of O2, which is necessary for normal growth and development of the embryo and the prevention of the formation of ROS.[98] As pregnancy progresses from the periphery of the placenta to the center, there is a gradual opening of the occluding blood vessels and a gradual increase in the partial pressure of oxygen within the intervillous space due to the continuous flow of oxygenated blood. This process takes place at the end of the first trimester and is responsible for the shift in the partial pressure of oxygen in the intervillous space. [99] When there is a disturbance at any level of placentation, the result is a sudden increase in oxygen pressure within the intervillous space and the consequent formation of ROS. [100]A gradual increase in O2 pressure allows the placenta to adapt to the new conditions of ROS formation by developing an antioxidant protection system (primarily by producing GPx and CAT). [101] To counteract the effect of ROS (O₂ ^-, NO, H2O2, ONOO^-) in the cells of the cytotrophoblast and stroma, a protection system is developed that includes the enzymes MnSOD, CuSOD, ZnSOD, however, when the level of ROS production exceeds the antioxidative capacity of the placenta, protein, lipids, and DNA damage occurs with consecutive cell damage and death. [101,102].

The determination of individual biomarkers of oxidative stress in placental pathology has no scientific or clinical significance, but a comparative examination at birth showed a positive correlation between elevated values of OS biomarkers in the mother's blood and blood from the umbilical cord of the newborn.[103] Studies focused on the effect of oxidative stress on placental tissue depending on the timing of infection in pregnancy have shown that changes within the scinciotroblast dominate the first trimester compared to the cytotrophoblast. This is reflected in a decrease in the surface area of microvilli and a decrease in mitochondria, with a very low amount of antioxidants detected. [104] A factor responsible for the onset of oxidative stress during the second and third trimesters is the intermittent flow of maternal blood within the villus, which produces ischemic reperfusion damage[105]

Some studies have detected the frequent presence of pathohistological changes by type of maternal vascular malperfusions (MVM), while others have reported changes by type of fetal vascular malperfusions (FVM), diagnosed according to the Amsterdam criterion based on the side of the placenta on which they occurred. [62,77,106,107,108] In addition to vascular changes, some studies have also proven the presence of inflammatory histopathological changes within the placentas of patients infected with COVID. [109]. Several studies have examined macroscopic and microscopic histopathological changes within the placental tissue in patients infected during pregnancy, but the results are incoherent [110,111,112,113,114,115,116]. Some studies even demonstrated no specific placental pathological findings in COVID patients regardless of the timing or severity of the disease. [117,118] The weaknesses of these studies are represented by the small number of samples, the inconsistency in the classification of histopathological changes, confounding factors such as chronic diseases in pregnancy and lifestyle habits that can have a significant impact on the result, as well as the choice of the control group.

Our study showed that placental pathology findings differ significantly between COVID-19 and the control group. We found FVM in 46% of the placentas of patients in the COVID group and 18% placentas of patients in the control group. MVM lesions were present in 32% of the placentas of the COVID group and in 18% of patients in the control group. Previous studies showed unequivocal results of placental findings, some demonstrated FVM in 30% and the other only 8% of placentas examined.[107,119] The most frequent findings in placental pathology of COVID patients in our study were: the obliteration of blood vessels, avascular villi, retroplacental hemorrhage, and accelerated villus maturation. Other study's most frequent pathophysiological findings were fibrin deposition, micro classification, thrombus, avascular villi, infraction, and villous edema. [120]

Given that the growth and development of the placenta is a dynamic process controlled by genetics and immunology, the moment when COVID-19 infection occurs during pregnancy can be an important factor in histopathological changes and maternal and fetal outcomes. Some studies showed that the timing of infection is crucial for developing pathophysiological placental lesions [62]

Our study's limitations include the small number of participants and the fact that it was unblinded for pathologists, so there is potential bias.

5. Conclusions

Based on the previous observation, in the summary we can conclude the following issues:

1. The types and frequency of pathohistological changes in placental tissue in patients in whom SARS-CoV-2 infection was confirmed during pregnancy differs from the control group. FVM is found to be the most frequent placental lesion in the COVID-19 group;

2. Plasma and lysate of erythrocytes of the mother's blood immediately before delivery and the blood of the newborn from the umbilical cord the values of the parameters of the antioxidant protection system, as well as pro-oxidants: lipid peroxide index measured as TBARS, nitric oxide in the form of nitrite (NO₂-), superoxide anion radical (O₂-) superoxide dismutase (SOD), reduced glutathione (GSH) significantly deferred between COVID-19 and control group;

3. Neonatal outcome: Apgar score, body weight of the newborn, admission to the intensive care unit, and length of stay in the neonatology department and ICU did not differ significantly between newborns from mothers of COVID-19 and the control group. COVID-19 patients do not have higher obstetrics potential for C-section delivery.

COVID-19 pregnancies exhibited an increase in histopathological abnormalities of the placenta, namely inflammatory changes of unknown etiology, as well as disturbed redox status of the mothers and newborns. Future studies investigating this specific influence of SARS-CoV-2 on placental tissue and newborn outcomes are needed, with more participants, with longer follow-ups for newborns, following neonatal and child development.