As a new disease, many questions still need to be clarified about COVID-19. One of these refers to the possible different methods of transmission, besisdes the respiratory route; the other concern is the long-term impact on the health of SARS-COV-2 infected infant. With the transmission spread of the new coronavirus, the number of pregnant women with COVID-19 increased and the attention of medical community and scientists has turned to these particular patients.
The absence of data that could allow the direct association of vertical transmission of other knwon coronavirus such as SARS-CoVand MERS-CoV, together with the first case reports of non-infected infants born of COVID-19 diagnosed mothers supported the previous theory that noval coronavirus was not vertically transmited [6,9,11]. However, later case of clinical manifestation of COVID-19 in a neonate  called the attention to the possible vertical transmissionof SARS-CoV-2, even in a small percentage of cases compared to the number of infected mothers.
In order to determine a possible vertical transmission of SARS-CoV-2 from the mother to the fetus during pregnancy, we selected 3 pregnant women with confirmed laboratory diagnosis for COVID-19. It is considered to be vertical transmission not only during pregnancy, with the invasion of virus in placenta through hematogenous route, but also during delivery through transcervical route and postpartum infection through environmental exposure [5,12]. In order to exclude contamination by other routes than transplacental route, we selected three cases of pregnant women whose delivery occured by cesarean section.
Serological antibody-based test for SARS-CoV-2 was performed in the totality of pregnant women admitted to the HNMD for delivery as a screening test to identify those one who had COVID-19 or was in the course of the disease. Thus, it was identified one case with positive results for both IgM and IgG (case 3), which means current or recent infection for COVID-19 and two cases of positive IgM and negative IgG (case 1 and 2), wich indicates current infection . Antibodies seroconvertion can occur between less than 1 week and more than 6 weeks after the emergence of symptoms . In fact, obstetric patient case 3 related to had symptoms of headache, runny nose, nausea, myalgia, malaise, joint pain 2 months before the hospitalization date. In one case, serological test was positive for IgM even with no symptoms related by thepatient (case 2). This finding corroborates with another study that identified 13.7% asymptomatic obstetric patients through RT-PCR screening test for SARS-CoV-2, pointing out to the importance of carrying out screening diagnostic tests in order to identify infected patients and monitor them more carefully, as well as their infants .
Next step was the performing of RT-PCR test in the selected pregnant women. Positive results for SARS-CoV-2 was identified in case 2 and 3, indicating the presence of RNA of SARS-CoV-2 virus. Positive RT-PCR test can be observed in the incubation period, prior to the beginning of COVID-19 symptoms (in case 2, for example) and last until the resolution of symptoms (in case 3, for example) . SARS-CoV-2 was not detected in case 1, being compatible with positive IgM serological test and the oneset of symptoms two months before testing.
The probability of ocurrence of infectious vertical transmission through transplacental route increases with increased gestacional age  and the reults of serological and molecular tests performed in the 3 obstetric patients showed that they were infected in the third trimestre of pregancy. In 5 cases of fetal death, SARS-CoV-2 was detected in the amniotic fluid or placenta, and vertical transmission was attested to had occurred during the third trimester of gestation .
In order to verify the presenceof SARS-CoV-2 in the neonates of our study, RT-PCR was performed in the nasopharyngeal swabs colleted shortly after labor and before contact with their mothers and the results turned out to be negative. Although RT-PCR is considered the gold standard for diagnoses of Sars-CoV-2 infection, diagnostic efficiency of this test in newborns has not been stablished yet . This can be explained by te fact that the airways are not funcitional during intrauterine life and that proliferation of SARS-CoV-2 in the upper respiratory tract seems to be irrelevant for the infection of the fetus .
Because the hematogenous route is the most probably mechanism for vertical viral infection , serological test of the umbilical cord blood was performed and the results were different in the three cases. IgM and IgG antibodies for SARS-CoV-2 were negative in case 2 and IgG was detected in the newborn of the case 1, probably of maternal origin that was transferred to the fetus by the placenta. In case 3, positive results for both IgM and IgG in the cord blood immediately after birth is a highly indication that vertical infection occurred, since IgM is a macroglobulin that can not cross the placenta from mother to the fetus . The presence of IgM in the cord blood with negative result for the detection of SARS-CoV-2 in swab samples of the newborn is rare but can be observed , which confirms that RT-PCR analysis of nasopharingeal swab samples may not be a gold standard for diagnosisof COVID-19 in neonates .
Detecting of SARS-CoV-2 was also performed in amniotic fluid and umbilical cord blood samples of the 3 cases. A systematic review identified 51 amniotic fluid samples tested but none of them was positive for SARS-CoV-2 RNA . In our study, first RT-PCR was also not able to detect the virus, then nested-PCR was considered for these type of sample. In fact, CDC guideline atests RT-PCR analysis to be performed in upper respiratory swabs pecimens only. We could successfully detect SARS-CoV-2 in the 3 aminiotic fluid and cord blood samples by nested PCR.
This tecnique could nested polymerase chain reaction (nested PCR) normalyis used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR .The product of the first amplification reaction (all negative for SARS-CoV-2) was used as the template for the second PCR. Additionally, in this work we used the reverse primers for reverse transcription, generating a specific ssDNA for the region of interest. The use of nested PCR methodology allows to increase the sensitivity of the test up to 100 folds , thus allowing a differentiated research potential for viral infection swith a low number of copies of initial genetic material.
Macroscopically, the analysis of the placentas from cases 2 and 3 did not indicate morphological aspactes suggestive of abnormality, Except in relation to the case 1 sample that presented placenta increased for gestacional age.
Regarding the microscopic aspects, all the placentas revealed maternal and fetal vascular malperfusion, corroborating with a systematic review of histopathological lesions observed in third semester placentas of COVID-positive mothers . Maternal vascular malperfusion can be observed in placentas of SARS-CoV-2 infected mothers, even though the virus itself is not identified by RNA in situ hybridization . Histopathological analysis of the placentas showed some extension of old infaction and thrombi, lesions associated with coagulation. Vascular thrombotic disease may be associated with hypertension and preeclampsia , pathologies not observed in the mothers of this study. Another possibility is an associated inflammatory disease to be the cause of the vascular lesions, and SARS-Cov-2 can not be excluded. Also, the placetas from the three cases presented a mild fibrin deposit as well as foci of hemorrages and calcifications in the intervillous space. It is worth mentioning that only in a single study (case 2) it was noticed a mild focal acute inflammatory infiltrate (mild acute intervilositis), which may be related to a viral infection. Regarding the umbilical cord, in case 2 was observed extensive foci of hemorrage in the jelly and hemopoiesis. Notwithstanging, It is important to notice that so far, there is no specific pathology characteristic of SARS-CoV-2 that could be observed in the placenta .
We are still facing the uncertainties that follows the emergence of a novel virus. As the pandemic spreads around the world, more case reports of COVID-19 accumulate, which allow us to conclude that SARS-CoV-2 is a virus capable of cross the placenta and infect the fetus, even at low rates. We conducted different diagnostic tests that add value information in order to confirm vertical virus transmission via transplacental route. Also, the results of serological and PCR tests obtained of maternal and neonatal samples indicated that RT-PCR of infant nasopharingeal swab samples is not an option for detectionof SARS-CoV-2 vertical infection. Instead, amniotic fluid or umbilical cord blood are non invasive samples that can be analyzed by serological or virological tests.
So far, there is no standard protocol that states the best sample to collect or diagnostic test to run in order to determine the occurrence of vertical transmission  . The results here presented show that a single analysis test may not be enough for a diagostic conclusion and we strongly suggest to conduct different analysis in order to confirm a possible infection of the neonate.