Are SARS-CoV-2 Antibodies Detectable in Human Milk After Vaccination Against COVID-19?

BACKGROUND: Human milk (HM) permits transfer of immunity against infections to infants via bioactive factors. The role of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in HM is poorly understood [1, 2]. This study evaluated SARS-CoV-2 antibodies in the HM of vaccinated healthcare workers (HCW). METHODS AND RESULTS: This prospective study of 122 HCWs was performed from February to April 2021 at the Hospital Universitario Nuestra Señora de Candelaria. Immunoglobulin G (IgG) against nucleocapsid protein and IgG, immunoglobulin M (IgM), and immunoglobulin A (IgA) antibodies against spike 1 protein receptor-binding domain against SARS-CoV-2 (anti-SARS-CoV-2 RBD-S1) were analyzed. Unvaccinated breastfeeding mothers without COVID-19 were the control group.The 98 vaccinated participants underwent serum and HM evaluation 14 days after receiving 2 doses of either BNT162b2 mRNA (94%) or mRNA-1273 (6%) coronavirus disease 2019 (COVID-19) vaccines. The mean SARS-CoV-2 RBD-S1 IgG serum concentration was 3379.64 binding antibody units (BAUs)/mL with neutralizing antibody titers >560.9 BAUs/mL. Serum SARS-CoV-2 antibodies in the 24 unvaccinated participants were negative. The HM from vaccinated participants had anti-SARS-CoV-2 RBD-S1 IgG with a mean of 12.19 BAUs/mL compared to 0.02 BAUs/mL (P < .001) in HM from unvaccinated participants. Anti-SARS-CoV-2 S1 IgA was noted in 89% of HM from vaccinated women; no anti-SARS-CoV-2 S1 IgM was detected.A positive correlation was reported between anti-SARS-CoV-2 RBD-S1 IgG in serum and HM (r = 0.36; P < .001). This association was stronger if breastfeeding had been <24 months (r = 0.67; P < .001) vs ≥24 months (r = 0.32; P = 0.19). In subgroup analysis, breastfeeding for >24 months and high serum anti-SARS-CoV-2 RBD-S1 IgG levels predicted high HM IgG levels. This was an independent association in both linear and multiple regression models. Compared with breastfeeding <24 months, lactation >24 months was associated with increased HM anti-SARS-CoV-2 RBD-S1 levels. COMMENTS: This study in breastfeeding HCWs showed that the HM antibody levels were higher in women who had been breastfeeding for >24 months prior to receiving 2 doses of COVID-19 vaccine compared to participants who had been breastfeeding <24 months. Limitations include lack of in vitro plaque reduction neutralization tests which is the gold standard for evaluating SARS-CoV-2 antibody deactivation effectiveness. The study was conducted at a single site and did not assess infant serology or clinical outcome.According to the authors, breastfeeding by vaccinated women during a pandemic when young children are ineligible for vaccination may be encouraged. These results support findings from other studies of vaccines, such as influenza, in which the HM of vaccinated women may confer protection to their infants [3]. The benefits of maternal immunization, including the duration of protection afforded by HM from maternal recipients of mRNA COVID-19 vaccines, are research areas deserving of additional exploration. Additionally, further understanding of the association of the duration of receipt of HM from vaccinated women on infant immune responses would be beneficial in understanding the potential for passive protection through nutrition.

Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques.

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.

BNT162b2 mRNA vaccine elicited antibody response in blood and milk of breastfeeding women.

The importance of breastmilk in postnatal life lies in the strong association between breastfeeding and the reduction in the risk of infection and infection-related infant mortality. However, data regarding the induction and dynamics of breastmilk antibodies following administration of the Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine is scarce, as pregnant and lactating women were not included in the initial vaccine clinical trials. Here, we investigate the dynamics of the vaccine-specific antibody response in breastmilk and serum in a prospective cohort of ten lactating women who received two doses of the mRNA vaccine. We show that the antibody response is rapid and highly synchronized between breastmilk and serum, reaching stabilization 14 days after the second dose. The response in breastmilk includes both IgG and IgA with neutralization capacity.

A Novel 29-Messenger RNA Host-Response Assay From Whole Blood Accurately Identifies Bacterial and Viral Infections in Patients Presenting to the Emergency Department With Suspected Infections: A Prospective Observational Study.

OBJECTIVES: The rapid diagnosis of acute infections and sepsis remains a serious challenge. As a result of limitations in current diagnostics, guidelines recommend early antimicrobials for suspected sepsis patients to improve outcomes at a cost to antimicrobial stewardship. We aimed to develop and prospectively validate a new, 29-messenger RNA blood-based host-response classifier Inflammatix Bacterial Viral Non-Infected version 2 (IMX-BVN-2) to determine the likelihood of bacterial and viral infections. DESIGN: Prospective observational study. SETTING: Emergency Department, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Germany. PATIENTS: Three hundred twelve adult patients presenting to the emergency department with suspected acute infections or sepsis with at least one vital sign change. INTERVENTIONS: None (observational study only). MEASUREMENTS AND MAIN RESULTS: Gene expression levels from extracted whole blood RNA was quantified on a NanoString nCounter SPRINT (NanoString Technologies, Seattle, WA). Two predicted probability scores for the presence of bacterial and viral infection were calculated using the IMX-BVN-2 neural network classifier, which was trained on an independent development set. The IMX-BVN-2 bacterial score showed an area under the receiver operating curve for adjudicated bacterial versus ruled out bacterial infection of 0.90 (95% CI, 0.85-0.95) compared with 0.89 (95% CI, 0.84-0.94) for procalcitonin with procalcitonin being used in the adjudication. The IMX-BVN-2 viral score area under the receiver operating curve for adjudicated versus ruled out viral infection was 0.83 (95% CI, 0.77-0.89). CONCLUSIONS: IMX-BVN-2 demonstrated accuracy for detecting both viral infections and bacterial infections. This shows the potential of host-response tests as a novel and practical approach for determining the causes of infections, which could improve patient outcomes while upholding antimicrobial stewardship.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Vaspin, a novel adipokine in woman granulosa cells physiology and PCOS pathogenesis?

Vaspin is a novel adipokine mainly expressed in visceral adipose tissue and closely related to obesity and insulin-resistance. Currently, data about its ovarian expression are limited to animal models and its role in human reproduction is largely unexplored. Our study's aims were then to characterise vaspin expression in the human ovary and to study in vitro its effects on granulosa cells physiology. Secondly, we assessed vaspin and its receptor GRP78 variations in granulosa cells and follicular fluid of a cohort of 112 infertile women undergoing an in vitro fertilisation procedure and allocated to three groups, each including normal-weight and obese subjects: 34 PCOS patients, 33 women with isolated polycystic ovary morphology (ECHO group) and 45 controls. Vaspin and GRP78 expression in the ovary was assessed by immunohistochemistry, RT-qPCR and Western blot. Granulosa cells and follicular fluid were analysed by RT-qPCR and ELISA, respectively. In vitro, granulosa cells metabolism was studied after stimulation with recombinant human vaspin, with and without a siRNA directed against GRP78. Vaspin was highly expressed in the human ovary and concentration-dependently enhanced granulosa cells steroidogenesis, proliferation and viability through GRP78 (P < 0.0001). Vaspin levels in both granulosa cells and follicular fluid were significantly higher in obese women (P < 0.0001) and in the normal-weight ECHO group (P < 0.001), which also had the highest expression rates of GRP78 (P < 0.05). Although further investigation is needed, vaspin appears as a novel modulator of human granulosa cells physiology and possibly plays a role in PCOS pathogenesis, notably protecting from insulin-resistance induced complications.

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in ß Cells.

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet ß cells. This would require binding and entry of SARS-CoV-2 into ß cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single ß cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in ß cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects ß cells in vivo through ACE2 and TMPRSS2.

Dissolvable sugar barriers to enhance the sensitivity of nitrocellulose membrane lateral flow assay for COVID-19 nucleic acid.

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.

In silico analyses on the comparative sensing of SARS-CoV-2 mRNA by the intracellular TLRs of humans.

The coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already resulted in a huge setback to mankind in terms of millions of deaths, while the unavailability of an appropriate therapeutic strategy has made the scenario much more severe. Toll-like receptors (TLRs) are crucial mediators and regulators of host immunity and the role of human cell surface TLRs in SARS-CoV-2 induced inflammatory pathogenesis has been demonstrated recently. However, the functional significance of the human intracellular TLRs including TLR3, 7, 8, and 9 is yet unclear. Hitherto, the involvement of these intracellular TLRs in inducing pro-inflammatory responses in COVID-19 has been reported but the identity of the interacting viral RNA molecule(s) and the corresponding TLRs have not been explored. This study hopes to rationalize the comparative binding of the major SARS-CoV-2 mRNAs to the intracellular TLRs, considering the solvent-based force-fields operational in the cytosolic aqueous microenvironment that predominantly drives these interactions. Our in silico study on the binding of all mRNAs with the intracellular TLRs depicts that the mRNA of NSP10, S2, and E proteins of SARS-CoV-2 are possible virus-associated molecular patterns that bind to TLR3, TLR9, and TLR7, respectively, and trigger downstream cascade reactions. Intriguingly, binding of the viral mRNAs resulted in variable degrees of conformational changes in the ligand-binding domain of the TLRs ratifying the activation of the downstream inflammatory signaling cascade. Taken together, the current study is the maiden report to describe the role of TLR3, 7, and 9 in COVID-19 immunobiology and these could serve as useful targets for the conception of a therapeutic strategy against the pandemic.

Local Angiotensin-Converting Enzyme 2 Gene Expression in Kidney Allografts Is Not Affected by Renin-Angiotensin-Aldosterone Inhibitors.

BACKGROUND: Preclinical studies suggested that pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) by ACE inhibitors (ACEis) or angiotensin II receptor blockers (ARBs) may increase local angiotensin-converting enzyme 2 (ACE2) expression. METHODS: In this study, we evaluated the effect of ACEi or ARB treatment on expression of ACE2, ACE, and AGTR1 in 3-month protocol kidney allograft biopsies of stable patients using RT-qPCR (n = 48). Protein ACE2 expression was assessed using immunohistochemistry from paraffin sections. RESULTS: The therapy with RAAS blockers was not associated with increased ACE2, ACE, or ATGR1 expression in kidney allografts and also ACE2 protein immunohistochemistry did not reveal differences among groups. CONCLUSIONS: ACEis or ARBs in kidney transplant recipients do not affect local ACE2 expression. This observation supports long-term RAAS treatment in kidney transplant recipients, despite acute complications such as COVID-19 where ACE2 serves as the entry protein for infection.

Review of COVID-19 and male genital tract.

COVID-19 pandemic leads to health challenges globally, and its diverse aspects need to be uncovered. Multi-organ injuries have been reported by describing potential SARS-CoV-2 entrance routes: ACE2 and TMPRSS2. Since these cell surface receptors' expression has been disclosed within the male reproductive system, its susceptibility to being infected by SARS-CoV-2 has been summarised through this literature review. Expression of ACE2 and TMPRSS2 at RNA or protein level has been reported across various investigations indicates that the male genitalia potentially is vulnerable to SARS-CoV-2 infection. Presence of SARS-CoV-2 within semen samples and following direct viral damage, secondary inflammatory response causing orchitis or testicular discomfort and finally the amount of viral load leading testicular damage and immune response activation are among probable underlying mechanisms. Therefore, genital examination and laboratory tests should be considered to address the male reproductive tract complications and fertility issues.

Are urologists in trouble with SARS-CoV-2? Reflections and recommendations for specific interventions.

OBJECTIVE: To assess the risk of viral infection during urological surgeries due to the possible hazards in tissue, blood, urine and aerosolised particles generated during surgery, and thus to understand the risks and make recommendations for clinical practice. PATIENTS AND METHODS: We reviewed the available literature on urological and other surgical procedures in patients with virus infections, such as human papillomavirus, human immunodeficiency virus and hepatitis B, and current publications on coronavirus disease 2019 (COVID-19). RESULTS: Several possible pathways for viral transmission appear in the literature. Recently, groups have detected severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in the urine and faeces, even after negative pharyngeal swabs. In addition, viral RNA can be detected in the blood and several tissues. During surgery, viral particles are released, aerosol-borne and present a certain risk of transmission and infection. However, there is currently no evidence on the exact risk of infection from the agents mentioned above. It remains unclear whether or not viral particles in the urine, blood or faeces are infectious. CONCLUSIONS: Whether SARS-CoV-2 can be transmitted by aerosols remains controversial. Irrespective of this, standard surgical masks offer inadequate protection from SARS-CoV-2. Full personal protective equipment, including at least filtering facepiece-2 masks and safety goggles should be used. Aerosolised particles might remain for a long time in the operating theatre and contaminate other surfaces, e.g. floors or computer input devices. Therefore, scrupulous hygiene and disinfection of surfaces must be carried out. To prevent aerosolisation during laparoscopic interventions, the pneumoperitoneum should be evacuated with suction devices. The use of virus-proof high-efficiency particulate air filters is recommended. Local separation of anaesthesia/intubation and the operating theatre can reduce the danger of viral transmission. Lumbar anaesthesia should be considered especially in endourology. Based on current knowledge, COVID-19 is not a contraindication for acute urological surgery. However, if possible, as European guideline committees recommend, non-emergency urological interventions should be postponed until negative SARS-CoV-2 tests become available.

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR.

The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

Nano-curcumin therapy, a promising method in modulating inflammatory cytokines in COVID-19 patients.

BACKGROUND: As an ongoing worldwide health issue, Coronavirus disease 2019 (COVID-19) has been causing serious complications, including pneumonia, acute respiratory distress syndrome (ARDS), and multi-organ failure. However, there is no decisive treatment approach available for this disorder, which is primarily attributed to the large amount of inflammatory cytokine production. We aimed to identify the effects of Nano-curcumin on the modulation of inflammatory cytokines in COVID-19 patients. METHOD: Forty COVID-19 patients and 40 healthy controls were recruited and evaluated for inflammatory cytokine expression and secretion. Subsequently, COVID-19 patients were divided into two groups: 20 patients receiving Nano-curcumin and 20 patients as the placebo group. The mRNA expression and cytokine secretion levels of IL-1ß, IL-6, TNF-α and IL-18 were assessed by Real-time PCR and ELISA, respectively. RESULT: Our primary results indicated that the mRNA expression and cytokine secretion of IL-1ß, IL-6, TNF-α, and IL-18 were increased significantly in COVID-19 patients compared with healthy control group. After treatment with Nano-curcumin, a significant decrease in IL-6 expression and secretion in serum and in supernatant (P = 0.0003, 0.0038, and 0.0001, respectively) and IL-1ß gene expression and secretion level in serum and supernatant (P = 0.0017, 0.0082, and 0.0041, respectively) was observed. However, IL-18 mRNA expression and TNF-α concentration were not influenced by Nano-curcumin. CONCLUSION: Nano-curcumin, as an anti-inflammatory herbal based agent, may be able to modulate the increased rate of inflammatory cytokines especially IL-1ß and IL-6 mRNA expression and cytokine secretion in COVID-19 patients, which may cause an improvement in clinical manifestation and overall recovery.

Detection of SARS-COV-2 receptor ACE-2 mRNA in thyroid cells: a clue for COVID-19-related subacute thyroiditis.

PURPOSE: SARS-COV-2 is a pathogenic agent belonging to the coronavirus family, responsible for the current global world pandemic. Angiotensin-converting enzyme 2 (ACE-2) is the receptor for cellular entry of SARS-CoV-2. ACE-2 is a type I transmembrane metallo-carboxypeptidase involved in the Renin-Angiotensin pathway. By analyzing two independent databases, ACE-2 was identified in several human tissues including the thyroid. Although some cases of COVID-19-related subacute thyroiditis were recently described, direct proof for the expression of the ACE-2 mRNA in thyroid cells is still lacking. Aim of the present study was to investigate by RT-PCR whether the mRNA encoding for ACE-2 is present in human thyroid cells. METHODS: RT-PCR was performed on in vitro ex vivo study on thyroid tissue samples (15 patients undergoing thyroidectomy for benign thyroid nodules) and primary thyroid cell cultures. RESULTS: The ACE-2 mRNA was detected in all surgical thyroid tissue samples (n = 15). Compared with two reporter genes (GAPDH: 0.052 ± 0.0026 Cycles-1; ß-actin: 0.044 ± 0.0025 Cycles-1; ACE-2: 0.035 ± 0.0024 Cycles-1), the mean level of transcript expression for ACE-2 mRNA was abundant. The expression of ACE-2 mRNA in follicular cells was confirmed by analyzing primary cultures of thyroid cells, which expressed the ACE-2 mRNA at levels similar to tissues. CONCLUSIONS: The results of the present study demonstrate that the mRNA encoding for the ACE-2 receptor is expressed in thyroid follicular cells, making them a potential target for SARS-COV-2 entry. Future clinical studies in patients with COVID-19 will be required for increase our understanding of the thyroid repercussions of SARS-CoV-2 infection.

Expression of severe acute respiratory syndrome coronavirus 2 cell entry genes, angiotensin-converting enzyme 2 and transmembrane protease serine 2, in the placenta across gestation and at the maternal-fetal interface in pregnancies complicated by preterm birth or preeclampsia.

BACKGROUND: Although there is some evidence that severe acute respiratory syndrome coronavirus 2 can invade the human placenta, limited data exist on the gestational age-dependent expression profile of the severe acute respiratory syndrome coronavirus 2 cell entry mediators, angiotensin-converting enzyme 2 and transmembrane protease serine 2, at the human maternal-fetal interface. There is also no information as to whether the expression of these mediators is altered in pregnancies complicated by preeclampsia or preterm birth. This is important because the expression of decidual and placental angiotensin-converting enzyme 2 and transmembrane protease serine 2 across gestation may affect the susceptibility of pregnancies to vertical transmission of severe acute respiratory syndrome coronavirus 2. OBJECTIVE: This study aimed to investigate the expression pattern of specific severe acute respiratory syndrome coronavirus 2 cell entry genes, angiotensin-converting enzyme 2 and transmembrane protease serine 2, in the placenta across human pregnancy and in paired samples of decidua and placenta in pregnancies complicated by preterm birth or preeclampsia compared with those in term uncomplicated pregnancies. STUDY DESIGN: In this study, 2 separate cohorts of patients, totaling 87 pregnancies, were included. The first cohort was composed of placentae from first- (7-9 weeks), second- (16-18 weeks), and third-trimester preterm (26-31 weeks) and third-trimester term (38-41 weeks) pregnancies (n=5/group), whereas the second independent cohort included matched decidua and placentae from pregnancies from term uncomplicated pregnancies (37-41 weeks' gestation; n=14) and pregnancies complicated by preterm birth (26-37 weeks' gestation; n=11) or preeclampsia (25-37 weeks' gestation; n=42). Samples were subjected to quantitative polymerase chain reaction and next-generation sequencing or RNA sequencing for next-generation RNA sequencing for angiotensin-converting enzyme 2 and transmembrane protease serine 2 mRNA expression quantification, respectively. RESULTS: In the first cohort, angiotensin-converting enzyme 2 and transmembrane protease serine 2, exhibited a gestational age-dependent expression profile, that is, angiotensin-converting enzyme 2 and transmembrane protease serine 2 mRNA was higher (P<.05) in the first-trimester placenta than in second-trimester, preterm birth, and term placentae (P<.05) and exhibited a negative correlation with gestational age (P<.05). In the second cohort, RNA sequencing demonstrated very low or undetectable expression levels of angiotensin-converting enzyme 2 in preterm birth, preeclampsia, and term decidua and in placentae from late gestation. In contrast, transmembrane protease serine 2 was expressed in both decidual and placental samples but did not change in pregnancies complicated by either preterm birth or preeclampsia. CONCLUSION: The increased expression of these severe acute respiratory syndrome coronavirus 2 cell entry-associated genes in the placenta in the first trimester of pregnancy compared with those in later stages of pregnancy suggests the possibility of differential susceptibility to placental entry to severe acute respiratory syndrome coronavirus 2 across pregnancy. Even though there is some evidence of increased rates of preterm birth associated with severe acute respiratory syndrome coronavirus 2 infection, we found no increase in mRNA expression of angiotensin-converting enzyme 2 or transmembrane protease serine 2 at the maternal-fetal interface.

Quantification of mRNA Expression Using Single-Molecule Nanopore Sensing.

RNA quantification methods are broadly used in life science research and in clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for RNA quantification. However, in cases of rare transcripts or inhibiting contaminants in the sample, an extensive amplification could bias the copy number estimation, leading to quantification errors and false diagnosis. Single-molecule techniques may bypass amplification but commonly rely on fluorescence detection and probe hybridization, which introduces noise and limits multiplexing. Here, we introduce reverse transcription quantitative nanopore sensing (RT-qNP), an RNA quantification method that involves synthesis and single-molecule detection of gene-specific cDNAs without the need for purification or amplification. RT-qNP allows us to accurately quantify the relative expression of metastasis-associated genes MACC1 and S100A4 in nonmetastasizing and metastasizing human cell lines, even at levels for which RT-qPCR quantification produces uncertain results. We further demonstrate the versatility of the method by adapting it to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA against a human reference gene. This internal reference circumvents the need for producing a calibration curve for each measurement, an imminent requirement in RT-qPCR experiments. In summary, we describe a general method to process complicated biological samples with minimal losses, adequate for direct nanopore sensing. Thus, harnessing the sensitivity of label-free single-molecule counting, RT-qNP can potentially detect minute expression levels of RNA biomarkers or viral infection in the early stages of disease and provide accurate amplification-free quantification.