Droplet‐digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females

Tagliapietra, Andrea; Rotondo, John Charles; Bononi, Ilaria; Mazzoni, Elisa; Magagnoli, Federica; Otòn-Gonzalez, Lucia; Contini, Carlo; Vesce, Fortunato; Tognon, Mauro; Martini, Fernanda

doi:10.1002/jcp.29213

Cited by 12 publications

(27 citation statements)

References 38 publications

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“…Indeed, BD, EP, and LP samples belonged to healthy subjects, being blood analysis parameters in the normal index range. Blood samples from pregnant women were collected before surgical intervention from women underwent voluntary abortion (EP), and during the routinely controls of gestation (LP) ( Tagliapietra et al, 2019 , 2020 ). The County Ethical Committee of the University Hospital of Ferrara approved this study in accordance with the Declaration of Helsinki.…”

Section: Methodsmentioning

confidence: 99%

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women

Rotondo

Otòn-Gonzalez

Selvatici

et al. 2020

Front. Cell Dev. Biol.

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SERine Protein INhibitor-A1 (SERPINA1) is an inducible blood cell gene coding for alpha1-antitrypsin (AAT), a plasma protease inhibitor whose circulating levels are raised during inflammation, infection and advanced pregnancy. DNA methylation has been suggested to play a role in SERPINA1 gene expression regulation in peripheral blood mononuclear cells (PBMCs). The methylation status of SERPINA1 in PBMCs is unknown. The aim of this study was to evaluate the methylation profile of the SERPINA1 promoter in PBMC. To this purpose PBMCs and serum were collected from healthy subjects (HS) (n = 75), including blood donors (BD) (n = 25), pregnant women at early pregnancy (EP) (n = 25), i.e., within the first trimester, and pregnant women at late pregnancy (LP) (n = 25), i.e., at the third trimester. DNA from PBMCs was treated with sodium bisulfite and PCR amplified for SERPINA1 gene promoter, followed by sequencing analyses. AAT serum levels were determined by ELISA test. SERPINA1 was found hypermethylated in 58.7% of HS. The prevalence of SERPINA1 hypermethylation was significantly higher in BD (68%) and EP (88%) than in LP (20%) (p < 0.01). The median serum AAT concentration was 1.07, 0.63, and 3.15 mg/ml in BD, EP, and LP, respectively (p < 0.05, BD and EP vs LP). This study indicates, for the first time, that SERPINA1 gene promoter is differentially methylated in PBMCs from HS. Likely, modulation of the methylation may be a novel epigenetic regulator mechanism of AAT expression in the PBMC of HS. Therefore, SERPINA1 gene promoter methylation may represent an epigenetic biomarker of PBMCs in healthy subjects.

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Section: Methodsmentioning

confidence: 99%

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women

Rotondo

Otòn-Gonzalez

Selvatici

et al. 2020

Front. Cell Dev. Biol.

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“…Spontaneous abortion (SA) is the unintentional loss of the embryo or fetus before the 20th week of gestation [ 1 ]. Spontaneous embryo/fetus loss is considered the most common adverse complication during pregnancy [ 2 , 3 , 4 ]. The estimated SA occurrence, which varies according to the study population in question, is about 12–15% of clinical gestations [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

“…The relationship between SA and pathogenic infection agents is yet to be fully elucidated [ 2 , 3 , 4 , 18 ]. Infectious agents are considered SA risk factors, being potentially involved in about 40% of SA events [ 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

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Investigation on Spontaneous Abortion and Human Papillomavirus Infection

et al. 2020

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Viral infections are considered to be risk factors for spontaneous abortion (SA). Conflicting results have been reported on the association between Human Papillomavirus (HPV) and SA. HPV DNA was investigated in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from women who experienced SA (n = 80, cases) and women who underwent a voluntary interruption of pregnancy (VI; n = 80, controls) by qualitative PCR and quantitative droplet digital PCR (ddPCR). Viral genotyping was performed using real-time PCR in HPV-positive samples. Specific IgG antibodies against HPV16 were investigated in sera from SA (n = 80) and VI (n = 80) females using indirect ELISA assays. None of the DNA samples from SA subjects was HPV-positive (0/80), whilst HPV DNA was detected in 2.5% of VI women (p > 0.05), with a mean viral DNA load of 7.12 copy/cell. VI samples (n = 2) were found to be positive for the HPV45 genotype. The ddPCR assay revealed a higher number of HPV-positive samples. HPV DNA was detected in 3.7% and 5% of SA and VI chorionic tissues, respectively, with mean viral DNA loads of 0.13 copy/cell in SA and 1.79 copy/cell in VI (p >0.05) samples. All DNA samples from the PBMCs of SA and VI females tested HPV-negative by both PCR and ddPCR. The overall prevalence of serum anti-HPV16 IgG antibodies was 37.5% in SA and 30% in VI (p > 0.05) women. For the first time, HPV DNA was detected and quantitatively analyzed using ddPCR in chorionic villi tissues and PBMCs from SA and VI women. Circulating IgG antibodies against HPV16 were detected in sera from SA and VI females. Our results suggest that HPV infection in chorionic villi may be a rare event. Accordingly, it is likely that HPV has no significant role in SA.

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“…Each qPCR experiment was carried out using the recombinant plasmid vector containing the complete HPV16 genome (NC_001526.4), used as positive control. A standard curve was employed using 10-fold dilutions, from 10 8 to 10 copies, of specific recombinant plasmid to calculate the viral DNA load [50]. Human β-globin gene was used to determine the human cell equivalents of each sample under qPCR analysis.…”

Section: Viral Dna Load Quantificationmentioning

confidence: 99%

High Human Papillomavirus DNA loads in Inflammatory Middle Ear Diseases

et al. 2020

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Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. We investigated HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n = 52), including chronic suppurative otitis media (CSOM) (n =38) and cholesteatoma (COMC) (n = 14), as well as normal middle ear (NME) specimens (n = 56) were analyzed. HPV sequences and DNA loads were analyzed by quantitative-PCR. HPV genotyping was performed by direct sequencing. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) of NME (p > 0.05). Specifically, HPV DNA sequences were found in 26.3% (10/38) of CSOM and in 14.3% (2/14) of COMC (p > 0.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell) and NME (mean 1.18 copy/cell) (P = 0.03 and P = 0.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. These data suggest that HPV may infect the middle ear mucosa, whereas HPV-positive COMCs are associated with higher viral DNA loads as compared to NME.Pathogens 2020, 9, 224 2 of 10 mucosa sampled from CSOM patients [11]. However, the etiology of CSOM remains to be determined. The relationship between HPV infection and inflammation has been previously reported [12]. It has been shown that persistent infection with high-risk HPVs leads to an increase in pro-inflammatory cytokines, including IL-6, TNF-α and MIP-1α [13]. In addition, high-risk HPV type 16 (HPV16) is able to increase the expression of cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins, which are important mediators of inflammation [14,15]. Until now, only a single study has reported HPV DNA sequences in CSOM, whereby different HPV genotypes, including HPV16, HPV18 and HPV6, have been detected in 30.7% of CSOM [4].COMC is a form of expanding growth consisting of keratinizing squamous cell epithelium [16]. There is great interest in the etiopathogenesis of HPV-associated cholesteatoma because HPV commonly infects the stratified epithelium [17,18]. However, conflicting data have been reported for HPV in COMC [10,[19][20][21]. HPV sequences have been detected in COMC with different prevalence, ranging from 3% to 70% [10,[19][20][21]. Moreover, no specific HPV genotypes have been associated with COMC, as high-and low-risk HPVs, such as HPV16, HPV18 and HPV6 and HPV11, have been detected [10,[19][20][21].There is emerging evidence that HPV infection can occur in different anatomical sites. Since HPV infects epithelia [22], all anatomical sites covered with epithelial tissue are potentially exposed to HPV infection. Apart from pluristratified tissues of the cervix [23], vulva [24] and oral pharynx [25], HPV sequences have been detected in simple epithelia from several anatomical sites such as lung [26], upper respiratory tract [27], larynx [28] and nose [29]. Since the middle ear muc...

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Droplet‐digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females

Cited by 12 publications

References 38 publications

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women

Investigation on Spontaneous Abortion and Human Papillomavirus Infection

High Human Papillomavirus DNA loads in Inflammatory Middle Ear Diseases

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