Signicance of placental swab in diagnosing vertical transmission in SARS-CoV-2 positive mothers.

Currently, there is limited date on the effects of COVID-19 on pregnancy and neonatal outcome. This literature review aims to investigate the possibility of fetal vertical transmission in COVID-19 positive pregnant mothers by diagnosing through placental swabs. Methods This literature review comprises 45 COVID-19 positive pregnant women whose placentas and neonates were also analysed by RT-PCR for the presence of SARS-CoV-2. 43 neonates were successfully delivered primarily via caesarean section out of 45 expectant mothers (96%). 2 mothers did not deliver due to severe preeclampsia and a miscarriage both occurring in the second trimester. 3 neonates tested positive for SARS-CoV-2 (7%). We report no neonatal mortality after birth and no maternal mortality. 8 female’s placentas tested positive for SARS-CoV-2 out of a total of 45 tested (18%). Of these 8, 2 cases of SARS-CoV-2 were identied in the maternal, neonatal and placental tissue. After reviewing conclude that there is no concrete evidence of vertical transmission occurring between and However, there are across the different papers used for this review and further research investigating the effects of COVID-19 on pregnant women by using RT-PCR to test the vaginal uid, and for at


Introduction
On March 11 2020, the World Health Organisation declared that the pneumonia outbreak of coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic [1]. Due to its highly transmissible nature, as of July 1 2020, there was a total of 10,446,353 con rmed cases of COVID-19, including 511,037 deaths across the ve continents [2]. With COVID-19's sustained spread across the globe, pregnant women are unfortunately not indiscriminate from contracting the virus. This may be attributed to the changes to the cardiorespiratory and immune system during pregnancy thereby increasing a woman's susceptibility to severe infection and hypoxic compromise [3].
Lopes de Sousa et al published a systematic review in June 2020 and concluded that there is no concrete evidence for vertical transmission of COVID-19 but acknowledged that signi cant knowledge gaps exist and they cannot rule out this possibility [4]. The presence of COVID-19 has been assessed in neonates born to COVID-19 positive mothers by examining the placenta, and carrying out nucleic acid testing on breast milk and vaginal mucus [5][6][7].
Two epidemics in the past two decades, namely severe acute respiratory syndrome (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2014 promote more serious complications than COVID-19 during pregnancy with approximately one third of infected pregnant women dying from the illness [3]. However, similarly to COVID-19, there have been no documented cases of vertical transmission seen in SARS or MERS to date [8].
Today, the effects of COVID-19 on pregnancy and neonatal outcome are being studied in real-time by researchers across the globe. This paper aims to review the current literature regarding the possibility of fetal vertical transmission in COVID-19 positive pregnant mothers by diagnosing through placental swabs.

Methods
Articles were searched using the following databases: Pubmed, ScienceDirect, Medline, Embase, Web of Science.
All studies in the review were selected using these databases, none were hand-selected. Studies relating to pregnant COVID-19 positive mothers and fetal vertical transmission and placental swabs were selected.
We followed the guidelines according to PRISMA, MOOSE, Cochrane Handbook of Systematic Reviews of Interventions.
Search terms used were: Pregnant COVID-19 positive mothers + fetal vertical transmission + placental swabs Inclusion criteria: Studies performed on above terminologies along with overlapping of terminologies from September 2019 to present.
Exclusion criteria: Studies performed prior to September 2019.
There are no con icts of interest.

Results
A review of studies focusing on pregnant COVID-19 positive mothers and placental swabs in the context of fetal vertical transmission has been performed. The eleven studies we reviewed are summarised in table one.
Neonatal Outcome 43 neonates were successfully delivered primarily via caesarean section out of 45 expectant mothers (96%). 3 neonates tested positive for SARS-CoV-2 (7%). We report no neonatal mortality after birth. Yu et al reported one neonate who was delivered via caesarean section testing positive at 36 hours after birth.
This neonate had mild shortness of breath and x-ray ndings of mild pulmonary infection. At 28 days after birth, the neonate was healthy and had no respiratory symptoms. SARS-CoV-2 was not identi ed in the mother's placental tissue though infection was con rmed in the mother's throat swab [11].
Patanè et al reported two neonates testing positive who both remained asymptomatic. One was vaginally delivered and tested positive immediately after birth, 24 hours later and 7 days later. After delivery, skin-toskin contact was not permitted but breastfeeding with a mask was allowed. The other positive neonate was delivered by emergency caesarean section due to a non-reassuring fetal status and tested positive only on day 7 after birth. There was no contact between the mother and neonate during this period as the newborn was immediately separated and transferred to the neonatal intensive care unit [14].

Laboratory Investigations
Serial RT-PCR identi ed SARS-CoV-2 in 8 female's placentas out of a total of 45 tested (18%). Of these 8, Patanè et al's study is the rst and only study we have identi ed which reports 2 cases of SARS-CoV-2 in the maternal, neonatal and placental tissue. Both of these mothers displayed signs and symptoms of SARS-CoV-2 [14]. Pen eld et al reported 3 positive placentas in women with severe to critical SARS-CoV-2 [13]. Interestingly, SARS-CoV-2 was also identi ed in the placentas of both mothers who did not deliver their neonate due to severe preeclampsia and a miscarriage [15,17]. We report one case of a positive placenta in a woman with asymptomatic SARS-CoV-2 who presented to the hospital due to a breech presentation [16]. Table 1. Summary of studies relating to vertical fetal transmission in COVID-19 positive pregnant mothers by diagnosing through placental swabs [9][10][11][12][13][14][15][16][17][18][19]. and amniotic fluid, umbilical cord blood, placenta, and mother's breast milk.
Timing of placenta analysis: On delivery day.
Vertical transmission: Inconclusive evidence. Variables tested: Neonate's nasopharyngeal swab and placenta. Results: Case 1: Vaginally delivered at 37 weeks gestation and tested positive from nasopharyngeal swab obtained immediately after birth, at 24 hours and 7 days. Neonate remained asymptomatic. Skin-to-skin contact was not permitted but breastfeeding was allowed.
Case 2: Delivered via emergency caesarean section at 35 weeks gestation and tested positive from nasopharyngeal swab on day 7 with no contact between mother and neonate during this period. Test at birth was negative.
Timing of placenta analysis: At birth.
Vertical transmission: First study to identify SARS-CoV-2 in the mother, neonate and fetal side of placental tissues via RT-PCR. Delivery: Caesarean section.
Variables tested: Neonate's nasopharyngeal swab, plasma serum and whole blood, placenta, vaginal mucus, mother's breast milk and umbilical cord blood.
Timing of placenta analysis: Unspecified.
Timing of placenta analysis: Unspecified.

Discussion
Baud et al and Hosier et al's studies describe two women suffering an adverse outcome during their pregnancy, notably miscarriage and severe preeclampsia respectively [17,15]. The miscarriage occurred in a symptomatic 28 year old woman at 19 weeks gestation. Baud et al concluded that the miscarriage appeared to be related to placental infection with SARS-CoV-2 which demonstrated mixed in ammatory in ltrates composed of neutrophils and monocytes on histological examination. Contamination during delivery was deemed unlikely given that all swabs from the foetus including the axillae, meconium, mouth and fetal blood were negative [17].
Wong et al reported a 57% miscarriage rate in a study of 12 pregnant women conducted during the 2002 SARS epidemic. They attributed this to acute or chronic placental insu ciency caused by severe maternal respiratory failure and hypoxemia thereby reducing uterine placental ow [20].
Hosier et al's study showed a 35 year old COVID-19 positive woman who presented at 22 weeks gestation with severe preeclampsia. This patient chose to terminate her pregnancy due to her heightened risk of maternal morbidity and mortality. High levels of SARS-CoV-2 were identi ed in the placenta and the invasion of intervillous macrophages was also seen on histology. Hosier et al concluded that COVID-19 may have contributed to placental in ammation resulting in early-onset preeclampsia and worsening maternal disease. It is important to note, however, that this patient was previously diagnosed with gestational hypertension which is a risk factor for her later developing preeclampsia in this pregnancy. No de nitive evidence for fetal infection was described [15].
Shanes et al examined 16 placentas in COVID-19 positive women. The placentas showed features of maternal vascular malperfusion, most prominently decidual arteriopathy and increased incidence of chorangiosis. Although, placental swabs were not performed which makes it unclear whether it was a local phenomenon or a systemic phenomenon [21]. Similarly, Hosier et al showed similar risk factors for maternal vascular malperfusion, i.e. gestational hypertension and preeclampsia [15]. This may tentatively imply a link between COVID-19 and severe preeclampsia.
Mulvey et al analysed ve COVID-19 positive mothers' placentas and concluded a thrombotic fetal vascular malformation phenomenon along with probable placental thrombosis lead to placental insu ciency. All ve newborns were successfully delivered which may be attributed to the mothers developing COVID-19 closer to full term. Mulvey et al hypothesize that infection earlier in the gestational course may have more serious consequences such as placental insu ciency with associated miscarriages or low birth weight infants [22]. This may be why the two adverse outcomes in our study described by Hosier et al and Baud et al occurred during the second trimester.
Of the 11 studies we analysed for our literature review, Patanè et al's is the only study which supports the possibility of vertical transmission in utero on the basis of their nding of positive SARS-CoV-2 infection in the maternal, neonatal and placental tissue. One neonate tested positive at birth, 24 hours and on day 7 and the other only tested positive on day 7. Both of these women's placentas exhibited signs of chronic intervillositis accompanied by the presence of macrophages in the villous and intervillous space [14].
Dong et al showed elevated IgM antibody levels 2 hours after birth in a neonate born to a mother with COVID-19 suggesting that vertical transmission is possible, even if it is uncommon. This neonate also had elevated cytokines and a leucocytosis. IgM antibodies do not cross the placenta and normally appear 3 to 7 days after infection suggesting the infection occurred in utero. Confusingly, this infant repeatedly tested negative for SARS-CoV-2 on the nasopharyngeal swabs [23]. It is important to consider the accuracy of nasopharyngeal and oral swab RT-PCR assays for SARS-CoV-2 which is deemed to have a sensitivity of between 56% and 83% [24].
The major limitation of our study is the small sample size of only 45 COVID-19 positive pregnant women and thus we cannot conclusively rule out the possibility of vertical transmission, though we deem it unlikely. However, we can consider that COVID-19 may affect the placental tissue due to the detection of the virus in certain cases.

Conclusion
After reviewing multiple studies and investigating the nature of placental physiology in SARS-CoV-2 positive mothers we conclude that there is no concrete evidence of vertical transmission occurring between mother and infant.
However, due to the novelty of the pandemic this small number of studies represent low levels of evidence due to the inconsistencies across the different studies reported. As the cases continue to rise worldwide, we expect the evidence to become more concrete on this topic with the development of more robust case control studies and long-term follow-up with the mothers and children.
Our literature review highlights the urgent need for a large scale study to be designed investigating the effects of COVID-19 on pregnant women by using RT-PCR to test the mother, placenta, vaginal uid, breast milk and infant for SARS-CoV-2 at various stages of transmission