1. Introduction

COVID-19 (Coronavirus disease 2019) is a disease which develops as a result of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) infection. It is an enveloped virus belonging to the family Coronaviridae, genus Betacoronavirus and subgenus Sarbecovirus, with its genome being positive-sense single-stranded RNA. Surface proteins are embedded in the viral envelope, giving the virion a crown-like shape, hence the name of the family. The SARS-CoV-2 virion contains four main structural proteins: surface protein (S), envelope protein (E), transmembrane glycoprotein (M) and nucleocapsid protein (N). Angiotensin-converting enzyme 2 (ACE2) is the specific cellular receptor of SARS-CoV-2. However, the process of viral penetration into the cell requires the participation of coreceptors, such as transmembrane serine protease 2 (TMPRSS2) and cathepsin L. CD147 is an additional coreceptor involved in the virus adsorption process. It is a protein inducing intercellular matrix metalloproteinases, but its presence is not necessary for successful cell infection ^{[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55]}[1]. The virus multiplies effectively in upper and lower airway epithelial cells, as well as in other types of cells presenting ACE2: e.g., in enterocytes, cardiomyocytes, pericytes, cells of the kidney, pancreas and testicles, and in the vascular endothelium and arterial smooth muscles. SARS-CoV-2 does not manifest a strong cytolytic effect against the infected cells, but it effectively stimulates local and systemic inflammatory response. Acute respiratory distress syndrome and progressive lung tissue damage seen in severe forms of COVID-19 primarily result from an intensified and improperly regulated inflammatory response, rather than from the direct cytolytic activity of the virus. The complex and still not fully elucidated mechanism of interaction between SARS-CoV-2 and the immune system is also most likely responsible for other syndromes associated with an infection with this virus, e.g., post-COVID syndrome, pediatric inflammatory multisystem syndrome, septic shock syndrome in adults, or chronic neurological syndromes ^[2].

The transmission of SARS-CoV-2 infection is primarily by airborne droplet. Other transmission routes (airborne-aerosol, fecal-oral) have been also confirmed, but play insignificant role. Direct contact with virus-contaminated respiratory secretions is a fairly important transmission route, which plays a role primarily in young children cases. The upper airway epithelium is predominantly the primary site of viral proliferation. However, cases of COVID-19 beginning with conjunctivitis, occurring before respiratory symptoms, were also reported. The infection of the newborn from the mother during the perinatal period is an exceptional situation. In addition, an unique route of transmission from the infected pregnant woman to the fetus, have been discussed.

2. Transmission Routes of SARS-CoV-2 Infection in the Fetus and Newborn

As regards published studies, with case reports or a series of cases constituting the majority, only a few confirmed cases of intrauterine infection were presented ^[3][4][5][6]. By analogy with other viral vertical infections, it is believed that the transmission of SARS-CoV-2 may occur in one of three ways: (i) intrauterine, during maternal viremia, or by the ascending route, (ii) at birth, through contact with maternal genital secretions or feces, and (iii) postnatal, through contact with the mother, during breastfeeding, or through contact with other persons. In special situations associated with fetal hypoxia, the virus, as well as maternal IgM antibodies, may reach the fetus through the damaged syncytiotrophoblast barrier ^[7]. Blood-borne infection is a consequence of viremia in the pregnant woman. Starting from the uterine vessels, the viruses reach the intervillous spaces, and then, through the villi, they reach the fetal circulation. The phenomenon of viremia in pregnant women with symptomatic SARS-CoV-2 infection is rare. It occurs in about 1-2% of pregnant women with a symptomatic infection. In addition, viremia is usually transient and a small amount of virus is detected in the blood. The highest risk of viremia applies to people with severe COVID-19, or when the delta variant of the virus is the etiological factor of the infection ^[8][9]. Due to the surface expression of SARS-CoV-2 receptors, placental cells are potentially susceptible to infection. The presence of ACE2 was primarily demonstrated on the villous syncytiotrophoblast and, to a lesser extent, in cytotrophoblast cells, villous stromal macrophages (Hofbauer cells) and endothelial cells. At the same time, poor expression of TMPRSS2 was confirmed in the villous endothelium and syncytiotrophoblast ^[2]. Transplacental transmission of the virus is hindered by TMPRSS2 deficiency in the syncytiotrophoblast and the sporadic and brief presence of viruses in the maternal blood. These are factors limiting the mechanical transplacental transmission of the virus, which may be responsible for its low rates ^[10][11]. As an inflammatory process is the natural consequence of infection, the placenta of infected mothers was examined for the presence of inflammatory foci in order to confirm the phenomenon of intrauterine infection. Evidence of inflammation was very rarely found, while the presence of viral RNA in the material collected from the placenta was confirmed only in isolated cases of infection ^{[6][12][13][14][15]}. If inflammation was found in the placenta of women infected with SARS-CoV-2, it was characterized by Hofbauer cell hyperplasia, chronic villitis and the occurrence of intervillous fibrin deposits ^[16]. In addition to the transplacental transmission, the probability of which was assessed by detecting viral RNA in the placenta and umbilical cord blood, the participation of amniotic fluid as a potential source of infection, both: in utero and perinatally, were also discussed. However, amniotic fluid testing for SARS-CoV-2 RNA was rarely performed, confirming its presence in only a few cases, with no attempts being made to isolate the infectious virus from this material. Therefore, the data are insufficient to assess the role of this route of the transmission of infection ^[17][18].

Ascending route is another potential route of the vertical transmission of SARS-CoV-2. However, it was considered only theoretically, and no cases of fetal infection acquired in such a way have been described so far.

The humoral response directed against SARS-CoV-2 during pregnancy, as well as its impact on the vertical transmission of the virus, has not been well elucidated. In an attempt to understand this process, Israeli researchers conducted a prospective cohort study and performed serological tests in infected mothers and their newborns ^[19]. The highest level of anti-SARS-CoV-2 antibodies was shown to occur in women who gave birth 8 to 12 weeks after the infection. After that time the level of antibodies decreased. Twenty weeks after the confirmed SARS-CoV-2 infection, only IgG antibodies against S proteins were present, while IgM antibodies and IgG antibodies against the N protein disappeared. In the study group, viral RNA was detected in nasopharyngeal swabs in 3% of newborns, indicating a possible vertical transmission and, at the same time, IgG antibodies against the S protein were detected in up to 10% of newborns born to seronegative mothers. The mechanism of this phenomenon is unknown. However, a very rapid decrease in IgG levels in postpartum women or local IgG synthesis in the placenta have been suspected. Moreover, a faster decrease in protein S-specific IgG levels was demonstrated in pregnant women and in the postpartum period comparing to the control group of non-pregnant women. However, the baseline level of antibodies in the acute infection period was similar in both groups. This phenomenon may be explained by the pregnancy-specific shift in the balance of the Th1/Th2 immune response and the resulting immunosuppression. Regrettably, the collected material does not provide an unequivocal answer on the role of the maternal humoral response in the immunoprotection of the fetus against SARS-CoV-2 infection ^[19].

The majority of the reported cases of COVID-19 in newborns were associated with the infection of a pregnant woman during the third trimester of pregnancy. No information is available on the nature, course and consequences of the infection in the first and second trimester ^[6]. ACE2 expression on placental cells is thought to increase with gestational age, which is theoretically correlated with an increased risk of intrauterine transmission ^[4][5][20]. The presence of ACE2 was confirmed in the heart, liver, lungs and perivascular cells of the fetus. TMPRSS2 is also found in the cells of the heart. The expression of ACE2 in fetal liver cells increases during pregnancy, potentially making the fetus more susceptible to infection. These findings were confirmed by the dynamically increasing expression of ACE2 on the surface of the internal organ cells of murine nurslings, observed on days 1-3 after birth ^[21]. The presence of ACE2 receptors in the cells of the lung epithelium and pulmonary vascular endothelium may be important if the virus is found in the fetal blood. The effective penetration of the virus through the placental barrier may lead to its spread with the blood of the fetus, and potentially, to the development of multiple organ infections. It has been confirmed by observations made in the few cases of a confirmed intrauterine infection that occurred in the late pregnancy ^[22] and in the case report of an intrauterine infection, which led to natural miscarriage at 13 weeks of gestation ^[6]. In that case, viral RNA was detected both in the placenta and in the kidneys and lungs of the fetus. In addition, multisystem damage associated with generalized inflammation was confirmed. Recently, based on a multicenter analysis of 68 cases of pregnant women infected with SARS-CoV-2, and in whom the pregnancy ended with premature delivery or miscarriage, a common picture of histopathological lesions of the placenta has been described, including trophoblast necrosis, fibrin deposits and Hofbauer cell infiltration. Therefore, the introduction of the term "COVID-19-related placenta inflammation" has been postulated ^[15].

Intrauterine SARS-CoV-2 infection may thus be considered possible, but rare, if the following conditions are met: (i) adequately long-lasting and severe maternal viremia, (ii) the multiplication of the virus in placental cells, and (iii) the penetration of the virus into the fetal blood in an amount sufficient to infect fetal tissues. Indirectly, it may be concluded that the pathomechanism of such infection should be similar to other viral infections acquired via the transplacental route, i.e., it should carry a significant risk of a serious fetal compromise. This has been validated by the observations included in the reports of confirmed transplacental infection cases. ^[6][15][22].

The risk of infection of the newborn from the mother after birth, during nursing activities or during breastfeeding is another important issue. Direct contact between the mother, the medical personnel, and the child creates opportunities for the transmission of the virus via the airborne droplet route as well as through direct contact with hands or objects contaminated by respiratory secretions. An attempt to reduce this risk was made by issuing recommendations on the implementation of the sanitary regime during contact. Mothers have been advised to wear masks or keep a distance and hands desinfection before contact with the child. Currently, there is no clear answer to the question about the role of breastfeeding as a route of transmission of the virus. However, the available data indicate a minimal risk of infection. A single large study conducted to analyze breast milk collected from 66 mothers with confirmed SARS-CoV-2 infection showed the presence of viral RNA in 10.7% of the samples. However, the subgenomic RNA of the virus, which is a potential marker of infectivity, was not detected in the positive samples. Moreover, attempts to isolate the virus from cell cultures to determine the presence of infectious viral particles were also unsuccessful ^[23]. Therefore, the role of breast milk as a source of infection has not been confirmed so far. No viral RNA was found in a number of other cohort studies, including a series of several to 60 breast milk samples ^{[24][25][26][27]}.

When studying the protective role of breastfeeding, the level and dynamics of specific antibodies in IgA, IgG and IgM classes were assessed in breast milk. An increase in IgG antibody concentrations was confirmed from 41 to 206 days after the infection, as well as the presence of IgA and IgM antibodies, which indicates the protective role of feeding with maternal breast milk ^[24]. Currently, recommendations to maintain direct contact between the newborn and the infected mother and to encourage breastfeeding have been widely implemented, while observing the principles of the sanitary regime described above. Maintaining a close relationship between mother and child seems not only safe, but also desirable, due to the known benefits of breastfeeding, creating a bond between mother and child, and a positive impact on the mother's psyche.

3. Consequences Implication of Pregnant Women SARS-CoV-2 Infection in the Fetus and the Newborn

SARS-CoV-2 infection in pregnant women may affect the course of pregnancy and the well-being of the fetus, increasing the risk of premature delivery. Placental infection may be a source of fetal infection. It also impairs gas exchange and the transport of nutrients, which may lead to hypoxia and malnutrition and, ultimately, to intrauterine fetal demise ^[8][28]. If the mere fact of infection is considered, without comprising the difference between symptomatic and asymptomatic infection, or the severity of the infection, the comparison between the general population of pregnant women infected with SARS-CoV-2 and healthy women revealed no statistically significant differences in the number of spontaneous miscarriages or an increased rates of birth defects in newborns ^[29][30][31]. In contrast, most (but not all) studies including groups of women with symptomatic forms of infection demonstrated an increased incidence of preterm deliveries and cesarean sections in the population with confirmed a SARS-CoV-2 infection. A large cohort study from the USA revealed that the number of premature deliveries in patients with COVID-19 symptoms was higher, i.e., 7.2%, while in uninfected patients it was 5.8% (OR=1.26, P<0.001) ^[32]. An increase in premature delivery ratio by 4% from 10.09% in 2020 to 10.48% in 2021 was recorded in the NCHS (National Center for Health Statistics) report ^[33]. A similar trend was observed by Katz et al. in their study. The number of premature deliveries was higher in women with a positive PCR test for SARS-CoV-2 and reached 14.8%, while in women who tested negative, a reported rate of premature deliveries was 10.2% ^[34]. Higher number of premature births may have been caused by: premature amniotic fluid drainage, fever, hypoxia, fetal tachycardia in the course of maternal infection, but also by an unconfirmed belief that an earlier delivery would improve the respiratory capacity of the mother with increased dyspnea ^[35]. Stress during the pandemic and changes in the health care systems could also have a significant impact on the results.

However, a retrospective cohort study conducted in Canada revealed no changes in the number of premature deliveries and stillbirths during the first twelve months of the COVID-19 pandemic compared to the previous observation period of 17.5 years ^[36]. Some authors also reported a decrease in the number of premature deliveries during the pandemic period ^[37][38][39]. The following factors may have been responsible for this trend: decreased physical activity during pregnancy, reduction of stress resulting from the difficulty of maintaining work-life balance, lower exposure to infections, fewer medical interventions, less frequent travel and less environmental pollution ^[40], and the improvement of standards in the field of hygiene and recreation. The lack of distinction between premature spontaneous and iatrogenic deliveries constitutes the limitation of numerous studies. A systematic review of 1,035 newborns of COVID-19 mothers showed that the incidence of perinatal infections was lower in newborns delivered vaginally (9/417, 2.16%) than by cesarean section (25/618, 4.05%) ^[41]. Another review showed that 72.7% (32/44) of newborns infected with SARS-CoV-2 were delivered via cesarean section ^[8]. The presented results contradicted the belief in the protective role of cesarean sections. Based on the data, changing delivery mode due to confirmed or suspected SARS-CoV-2 is currently not recommended ^[42]. Cesarean section should be performed as per standard obstetric indications or due to the severe condition of the mother ^[42][43]. It was also found that severe SARS-CoV-2 infection was associated with an increased risk of delivering by cesarean section (34% in asymptomatically infected women and 59.6% in case of a severe course), as well as premature delivery (11.9% and 41.8%, respectively) ^[10]. However, a correlation between the severe infection in a pregnant woman and the likelihood of transplacental transmission has not been clearly demonstrated ^[6][7]. A mild and moderate course of infection was not associated with a higher risk of perinatal complications as compared to asymptomatic patients ^[10].

The number of stillbirths in the USA was higher in mothers diagnosed with COVID-19 than in uninfected women between March 2020 and September 2021. An even higher risk of stillbirth occurred in case of infection with the delta variant of the virus, as well as in patients with concomitant conditions such as hypertension, sepsis, shock or respiratory failure ^[11][34].

In conclusion, the probability of congenital infection in the newborns of infected mothers assessed in single-center studies ranged from 0 to 5%, depending on the study center^{[4][8][19][35][44][45]}, whereas published meta-analyses assessed the rates of vertical infections at 1.6-3.2%, which was determined on the basis of a positive SARS-CoV-2 RNA test in nasopharyngeal swabs collected within 48 hours after infection ^[46][47][48]. Seemingly, the majority of the cases of infection occurred postnatally. Various definitions and criteria used in studies hinder the comparison of the available data. Initially, intrauterine infection was confirmed by detecting the genetic material of the virus in a nasopharyngeal swab collected on the first day after birth. It is currently known that RT-qPCR (reverse transcription quantitative real-time PCR) for upper airway samples may be positive both as a result of vertical infection and contamination of the sample during delivery, or as a result of early infection acquired postnatally from the mother or medical staff ^[49]. The possibility of establishing a common definition and introducing uniform systems for diagnosing and differentiating between transplacental, perinatal transmission and infection acquired later appeared after the publication of diagnostic criteria for congenital SARS-CoV-2 infection by the WHO ^[7][41] and Shah et al. ^[50]. The results of 47 studies were systematically analyzed using the formula by Shah et al. The analysis included 1,188 pregnant women with confirmed SARS-CoV-2 infection and 985 newborns. Vertical transmission was confirmed in 0.3%, transmission probable in 0.5%, and possible in 1.8% of newborns ^[8].

A large, prospective, cohort study conducted in the United Kingdom during the intensification of the pandemic (March, 1 – April 30, 2020) showed that confirmed SARS-CoV-2 infection was diagnosed in as many as 5.6 out of 10,000 live newborns, 42% of whom developed a severe form of infection, and 24% were born prematurely, 26% of newborns with COVID-19 were diagnosed prenatally, 3% were diagnosed with probable vertical infection, and 12% were suspected for infection acquired postnatally ^[51]. The data validated an opinion that COVID-19 infection in a newborn of a mother with an infection confirmed prenatally was possible, but unlikely. Early infection acquired postnatally is rare and its course is most commonly mild, which supports the recommendation not to separate the mother from the child ^[33][42][45]. In the first period of the pandemic, there was no uniform recommendation regarding the treatment of the newborn after birth. Depending on the center, the child was isolated from the sick mother or remained with her in the rooming-in system and was breastfed. The mother was informed about the risk of infection and recommended procedures to prevent the spread of infection. As in other infections, it was considered that breast milk was the source of antibodies specific for SARS-CoV-2, acting as a protective agent. There was no evidence of infectious virus in the breast milk of women with confirmed infection, regardless of whether the infection was asymptomatic or symptomatic ^[49]. Studies by Ronchi et al. ^[52] and Salvatore et al. ^[53] demonstrated that transmission from a sick mother to a breastfed newborn occurred very rarely (0-1.6%) when using the rooming-in system, and the infection in the newborn was mild. A multicenter study conducted in Sweden, in which 92% of all deliveries during the pandemic were analyzed, the newborns of SARS-CoV-2-positive mothers were diagnosed with respiratory disorders only slightly more often than newborns of healthy mothers (2.8% and 2%, respectively) ^[54]. The majority of newborns infected with SARS-CoV-2 had no symptoms, whereas mild infections were predominant in the symptomatic group. The symptoms which were most commonly reported in COVID-19 neonates included breathing disorders, fever and feeding difficulties ^[8]. The analysis of data from twelve countries including pregnant women with suspected SARS-CoV-2 infection and women with laboratory-confirmed infection showed that the number of early neonatal deaths remained unchanged compared to the number of deaths recorded before the pandemic period and it was reported 0.2-0.3 ^[42]. However, the long-term effects of intrauterine or perinatal infections have remained unknown. It is also unknown what impact the infection during this period will have on the future developmental outcomes of the child ^[33][52]. Long-term monitoring of children confirmed to have SARS-CoV-2 infection during the perinatal period seems to be justified.

4. Conclusions

SARS-CoV-2 RNA is detected in the placenta, umbilical cord blood, neonatal blood, amniotic fluid, genital secretions, and breast milk, but it is not equivalent to the presence of viral RNA of infectious virions. It is possible to develop an intrauterine, intrapartum or postnatal infection, but it occurs very rarely and is most commonly characterized by a clinical course. The exact mechanism of intrauterine infection is still not elucidated. The protective role of cesarean section and the isolation of the newborn from the infected mother after birth have not been confirmed. Due to lack of evidence for the possibility of transmission during breastfeeding, there is no justification to recommend formula feeding ^[55].