HPV infection and its correlation with p53 and Bcl-2 among pregnant mothers and their infants

doi:10.21203/rs.3.rs-3664859/v1

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HPV infection and its correlation with p53 and Bcl-2 among pregnant mothers and their infants

https://doi.org/10.21203/rs.3.rs-3664859/v1

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published 25 Apr, 2024

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Perinatal transmission of HPV leading to infection in infants due to swallowing of amniotic fluid or vaginal secretions is vital for early screening of cervical and oral cancers. Here, the perinatal transmission of HPV from the pregnant women to their infants was studied. p53 and Bcl-2 expressions in the mother-baby pairs were analyzed. The correlation of HPV infection with p53 and Bcl-2 in the mother-baby pairs were examined. The study included 135 mother-baby pairs. HPV infection was detected in the cervical and oral swabs employing both PCR and Hybrid Capture-II methods. Immunocytochemistry of p53 and Bcl-2 using the streptavidin-biotin peroxidase method with respective monoclonal antibodies was performed. The prevalence of HPV infection in the mothers was 28.14%, (38/135) and 30.37% (41/135) determined by the PCR and HC-II methods respectively. HPV 16 and/or 18 was identified in 81.57% (31/38) and 82.92% (34/41) of the HPV + women estimated by the PCR and HC-II methods respectively. The frequency of perinatal transmission was 21.05% (8/38) 23.68% (9/38) determined by the PCR and HC-II methods respectively. An abnormal nuclear expression of p53 and cytoplasmic expression of Bcl-2 were observed in the HPV + mother-baby pairs. A significant positive correlation was found between p53 and Bcl-2 expressions and HPV + mother-baby pairs. Cesarean section was not found protective for infants against perinatal HPV transmission. The detection of p53 and Bcl-2 proteins in the HPV-positive mother-baby pairs suggests that these biomarkers may be important in the early screening of these cancers in positive cases.

Bcl-2

HC-II

HPV

p53

PCR

Perinatal transmission

The Human Papillomavirus (HPV) belongs to Papillomaviridae family causing infection of epithelial cells of the skin, and oral and genital mucosa [1]. HPV causes 610,000 cancer cases [2] and 250,000 deaths every year [3]. The High-Risk-HPVs (HR-HPV) − 16, 18, 31, 33, 45 and the low-risk (LR)-HPVs- 6 and 11 comprise the most prevalent virus types [4–6]. The HR-HPVs-16, 18, 31, 33, and 45 are responsible for 75% of all HPV-associated squamous cell carcinoma and 94% of all adenocarcinomas [5–7]. Various groups reported about the presence of HPV DNA in blood, reproductive and placental cells, infants and children [8–10]. The horizontal transmission of HPV through saliva or other contact has been reported to explain cases of oral infection in infants whose mothers are HPV-negative, and investigations of vertical transmission have led to conflicting results and transient HPV detection in children [11]. Perinatal transmission of HPV leading to subclinical infection in infants due to swallowing of the maternal blood, amniotic fluid, or vaginal secretions as the fetus passes through an infected birth canal is reported [12]. The frequency of HPV infection among pregnant women [13] and recurrent laryngeal papillomatosis [14] and condyloma acuminata[15] in newborn infants are in agreement with the perinatal transmission of HPVs. We also reported on the vertical transmission of HPV from pregnant mothers to their infants (p = 0.0548) [16].

Cell cycle and apoptosis are critical events that occur during cell transformation and carcinogenesis [17]. Although several studies have demonstrated the relationship between HPV, p53, and Bcl-2 in malignant and premalignant lesions of the uterine cervix [18], vulvar carcinoma [19], and oral carcinoma [20], little is known about their effects on newborns of the HPV + mothers.

The present study investigated the perinatal transmission of HPVs using the conventional PCR and Hybrid capture-II methods. Additionally, the expressions of p53, and Bcl-2 associated with HPV infection in pregnant women and their infants were examined.

2.1. Subjects:

The study population comprised of 135 mother-infant pair. This study was initiated after obtaining approval from the institutional ethical board. Pregnant women (20-39 years) without any previous history of cervical abnormality or visible genital warts, complicated pregnancies and HIV positivity were included in this study. Informed consent for sample collection was collected from the women for themselves and their infants. Necessary approval for conducting this study was obtained from the internal ethics committee. Cervical smears were collected from the women at their third-trimester, 10-15 days prior to delivery. Neonatal buccal swabs were collected between 1- 4 days after birth before hospital discharge. Women were asked to return with their infants for further evaluation if either of them were HPV positive at first examination. Interval between enrollment and first return visit was 6 months postpartum for the mothers and 6 weeks for the babies. HPV positive cases were attempted to follow-up to 1 year with an interim visit at 3 months for infants and 6 months for mothers.

2.2. Sample collection:

The cell scrapes were collected with a small endocervical brush. Simultaneously, cervical and oral smear for each mother-baby pair was also collected for cytological analysis and immunostaining of p53 and Bcl-2 proteins. All samples were taken in sterile phosphate-buffered saline (pH 7.4) and stored at –20⁰c until processed.

2.3. Detection of HPV DNA and genotyping:

a) PCR:

Cells were lysed by Proteinase K digestion and DNA was extracted by phenol–chloroform. A 268 bp fragment of the beta-globin gene was amplified to assess the adequacy of the DNA in each sample [21]. HPV detection was performed by the conventional PCR using the degenerate consensus primers MY09/MY1 [22] targeting a 450 bp region in the highly conserved L1 ORF [16]. The PCR protocol was followed as mentioned previously [16]. Positive cases were amplified further with type-specific primers for HPVs 6/11 [23], 16,18 [24], 31, and 33 [25]. The length of the final amplified HPV types are 113, 109, 334, 514, and 450 base-pair respectively. The PCR protocol was followed as mentioned earlier [16]. All PCRs included negative controls (distilled water and DNA from HPV-negative cell line C33A) and precautions were taken to avoid contamination. Plasmid DNA containing type-specific HPV genome and HPV-positive cell lines (SiHa/HeLa) were used as positive controls. The primer sequences of HPV L1 ORF and 6, 11,16, 18, 31, 33, and β-Globin are presented in Table 1. The genotyping was restricted to the aformentioned HPV subtypes depending on the availability of the primers.

b) HC-II assay:

HPV DNAs were detected in cervical and oral smears using the second-generation HC-II assay kit (Digene) following the manufacturer’s instructions. The specimens were first denatured followed by hybridization with probe B which included a pool of RNA probes for HPV genotypes 6, 11, 16, 18, 31 and 33. The HPV DNAs hybridized with the RNA probe and the RNA-DNA hybrids were captured on antibody-coated microplate wells. The immobilized hybrids were reacted with alkaline phosphatase-conjugated antibody and detected by cleavage of the chemiluminescent substrate. The light emitted was measured as a relative light unit (RLU) in a luminometer (DML 2000, Digene). The intensity of the light was proportional to the amount of target DNA in the sample. Specimens with RLU greater than or equal to the mean of the three positive control values were considered HPV positive.

c) HPV Genotyping:

RNA probes specific for each of the HPV types 6/11, 16, 18, 31 and 33 (Digene Corp.) were used for the HC-II test. The manufacturer’s instructions were followed to perform the tests. The method of type-specific HC-II test was almost similar to that of high-risk group HPV DNA detection using probe B but was different in three ways: i) instead of one, three positive controls - low positive control (LPC of concentration 10pg/ml), high positive control (HPC of concentration 2ng/ml) and cross-reactivity control (CRC of concentration 4ng/ml) were includedt, ii) the highly concentrated individual RNA probes were diluted many folds (1:50) and iii) after capture of the hybrids (DNA-RNA) each microplate well was treated with diluted RNase reagent. The samples were classified as positive for the relevant HPV type if the ratio of the RLUs of the specimens to the mean RLU of the LPC was greater than or equal to 1.0.

2.4. Immunocytochemistry of p53 and Bcl-2 proteins:

The cervical and oral smears were fixed on poly-L-lysine-coated glass slides. One slide from the study population was Pap stained for the routine and conventional examination by light microscopy and duplicate slides were used for immunocytochemistry of p53 and Bcl-2 proteins. Briefly, after fixing the cells, they were rinsed with 1X PBS, and then permeabilized and blocked with 10% horse serum, 2% BSA, and 0.5% Triton-X-100 in PBS for 1 h. The cells were then incubated overnight with primary antibodies namely anti-p53 (sc-6243 FL-393, Santa Cruz Biotech) and anti-Bcl-2 (mouse monoclonal, sc-7382, Santa Cruz Biotech) in a blocking buffer. After primary antibody treatment, the cells were washed and incubated with the respective Fluorescein isothiocyanate (FITC) conjugated secondary antibodies for 3 h followed by three washes with 1X PBS. The slides were then mounted with coverslips and cell images were acquired using a Zeiss Axio Observer Z1 microscope. Images of different, randomly chosen fields were acquired with identical exposure times for quantification.

2.5. Cytological analysis:

Cytological preparations were assessed by the standard Pap staining. HPV diagnosis was performed based on the parameters such as parakeratosis, and /or dyskeratosis, keratinization, nuclear enlargement, nuclear membrane irregularities, and sharply demarcated paranuclear halo.

2.6. Statistical analysis:

The results were analyzed using the EPI INFO statistical software, version 6.0. The association between HPV prevalence in the mothers and their infants was tested by using the test of proportion and Pearson χ2. P<0.05 was considered significant. The correlation between p53 and Bcl-2 expression was analyzed by Spearman’s correlation test. The association between HPV infection and p53 and Bcl-2 were analyzed by univariate statistical correlation analysis.

3.1. Prevalence of cervical HPV infection among pregnant women with respect to age, gravida, and cytology, comparison of PCR with HC-II:

The overall prevalence of cervical HPV infection in pregnant women determined by the PCR method was 28.14% (38/135) at the time of enrolment presented in Table 2. On the other hand, the overall HPV prevalence in the cervical swabs of pregnant women determined by the HC-II method was 30.37% (41/135) (Table 2). The mean age of the pregnant women was 26.55 years (range: 20–39 years). The average age of the HPV-positive and HPV-negative women was 25 and 21 years respectively. Thus, the overall cervical HPV prevalence in the pregnant women determined by the HC-II method was found to be slightly more compared to the PCR method using the consensus primers (Table 2). With respect to gravida, the overall prevalence of HPV in pregnant women with gravida 1 was 32.85% (23/70) determined by the PCR method (Table 2). HC-II assay of these samples revealed 35.71% (25/70) positivity shown in Table 2. With respect to gravida 2, the overall HPV prevalence in the pregnant mothers estimated by the PCR method was found to be 31.25% (10/32) and by HC-II method it was 34.37% (11/32). With respect to gravida 3, the overall HPV prevalence in the pregnant mothers was found to be equal (15.62%, 5/32) following both the methods.

3.2. Comparison of genotyping of HPV of the infected mothers by PCR and HC-II methods:

HPV 16 and/or 18 was identified in 81.57% (31/38) of the HPV + women estimated by the PCR method. HPV 16 was detected in 63.15% (24/38) of the total positive cases. Out of the 31 HPV-positive cases 13 had HPV type 16, 7 had HPV 18, and 11 had both HPV 16 and 18. Out of 13 HPV 16 positive cases, 3 had HPV 6/11 DNA, and one had additional HPV 31 infections. Among the 7 HPV 18 positive cases, 2 had, in addition, HPV 31, and one had both HPV 31 and 33. Out of the 11 HPV 16 and 18 positive cases, 4 were also positive for HPV 6/11, 1 for HPV 31, and one for both HPV 31 and 33. Thus, out of 38 overall HPV-positive mothers, 31 had HPV 16 and/or 18, and one was infected with both HPV 6/11 and 31 and 6 had HPVs of other types (Table 3). HPV genotyping in the infected pregnant women was also performed employing the HC-II method. HPV 16 and/or 18 was identified in 82.92% (34/41) of the HPV + women by the HC-II method. HPV 16 was detected in 65.85% (27/41) of the positive cases. Out of the 34 HPV-positive cases 15 had HPV type 16, 9 had HPV 18, and 10 had both HPV 16 and 18. Out of 15 HPV 16 + cases one had HPV 31 infection. Among the 9 HPV 18 positive cases, 3 had in addition HPV 31 infection. Out of the 10 HPV 16 and 18 positive cases, 2 were HPV 31 positive (Table 3 ).

3.3. HPV prevalence, genotyping and cytological screening in the infants:

The overall prevalence of HPV infection among the infants was 11.11% (15/135) at the time of birth estimated by the PCR method. Among the 15 HPV-positive infants, 8 (vaginal/cesarean delivery = 3/5) were born to HPV-positive mothers and 7 (vaginal/cesarean delivery = 2/5) to HPV-negative mothers. Therefore at birth, the overall frequency of HPV transmission from HPV-positive mothers to their infants was 21.05% (8/38) shown in Tables 3 and 4. Out of the 15 HPV-positive infants, 13 had HPV 16 DNA, 1 HPV 31, and 1 HPV 33. Out of the 13 HPV 16-positive babies HPV 31 was found in seven, HPV 33 in one, and HPV 18 and 31 in one. On the other hand, the overall HPV prevalence in infants was 23.68% (9/38) at the time of birth determined by the HC-II method (Tables 3 and 4). Among the 16 HPV-positive infants, 9 (vaginal/cesarean delivery = 3/6) were born to HPV-positive mothers and 7 (vaginal/cesarean delivery = 2/5) to HPV-negative mothers. Therefore, at birth, the overall frequency of HPV transmission from HPV-positive mothers to their infants was 21.95% (9/41). Out of the 16 HPV-positive infants, 14 had HPV 16 DNA, and 2 had HPV infection other than HPV 16 (Tables 3 and 4).

3.4. Immunocytochemistry of p53 protein and its association with HPV infection:

A distinct nuclear immunoreactivity for p53 was observed in the HPV-positive cervical cells of the mothers (Fig. 1B) and oral squamous epithelial cells of the of their infants (Fig. 1D) compared to those of HPV-negative cervical (Fig. 1A) and buccal cells (Fig. 1C). p53 accumulation was highly associated with the HPV positive mother-baby pairs with increasing gravida with more intense expression. HPV infection and p53 immunoreactivity revealed a highly significant positive correlation (p < 0.05).

3.5. Immunocytochemistry of Bcl-2 protein and its association with HPV infection:

Bcl-2 immunoreactivity was found throughout the cytoplasm with low concentrations in the perinuclear zone in cervical cells of the HPV-positive mothers (Fig. 2B) and squamous cells of their offspring (Fig. 2D) compared to the HPV-negative mothers (Fig. 2A) and their offspring (Fig. 2C). A highly significant positive correlation between Bcl-2 expression and HPV infection was observed in HPV-positive mothers and their offspring (p < 0.05).

3.6. Correlation between p53 expression and Bcl-2 expression:

p53 protein expression was analyzed against the Bcl-2 protein expression in the mother-baby pairs. All the cases with negative p53 expression were also negative for Bcl-2 expression (100%). Among the 41 HPV-positive mothers (HC-II data) with p53 expression 38 showed Bcl-2 expression in the cervix smears. Among the sixteen HPV-positive infants (HC-II data) fifteen showed Bcl-2 expression in the buccal swabs. Statistical correlation analysis also revealed a highly significant positive correlation between p53 and Bcl-2 expressions in both the HPV-positive mothers and their infants at the time of birth (p < 0.05).

3.7. Cytological analysis by Pap staining:

Cytological analysis by Pap staining revealed 28.88% (39/135) of the pregnant had HPV-related lesions (Table 2). Twenty-one HPV-positive samples (PCR and HC-II) whereas eighteen HPV-negative samples (PCR and HC-II) exhibited HPV-related lesions out of the total 39 Pap-positive cases (Table 2). On the other hand, fifteen samples didn’t show Pap positivity though they were HPV positive determined by PCR and HC-II methods out of a total of 94 Pap-negative smears (Table 2). The frequencies of cytological abnormalities related to the HPV-related lesions in the HPV-positive as well as negative mother-baby pairs are shown in Tables 2 and 3.

In this study cervical swabs were collected during the third trimester of pregnancy, a few days prior to delivery when a detectable level of HPV DNA was found [13]. Here, the prevalence of HPV infection in pregnant mothers was compared by using the conventional PCR and HC-II methods. Additionally, the vertical transmission of HPV infection from the mothers to their infants at birth was investigated.

At birth, the prevalence of HPV infection among infants was 11.11% and the transmission rate was 21.05% as determined by the PCR method. On the other hand, the overall HPV prevalence in infants was 23.68% (9/38) at the time of birth determined by the HC-II method. The concept of maternal–fetal transmission of HPV infection at birth was first proposed by Sedlacek et al 1989 [26]. This concept was also supported by the detection of HPV infection in nasopharyngeal aspirates (48%) [27]. Additionally, several other studies demonstrated the concept of perinatal transmission of HPVs [28,26,8]. Smith et al 1995 [29] and Puranen et al 1996 [30] using PCR reported about the transmission rates of HPVs of 4% and up to 80% respectively. Pakarian et al 1994 demonstrated a 55% vertical transmission rate, from HPV-positive mothers to their new-born [31]. These findings suggest transient prenatal contamination of infants’ specimens by maternal HPV did not occur. Tenti et al detected transmission of 30% in a population with a low prevalence (5.2%) of HPV infection among mothers [32]. Previously, our laboratory reported about 41.6% perinatal transmission of HPV in a small population (N = 30) estimated by in situ hybridization [33]. We also reported on the vertical transmission of HPV from pregnant mothers to their infants (p=0.0548) [16]. Recently the HERITAGE study reported about the presence and persistence of HPV infection in the conjunctiva of the children of infected mothers [34]. These findings demonstrated a widely varying rate of vertical transmission. Such variation may be due to the varied risk factors for cervical neoplasia in the population screened with different sample collection techniques and sensitivities of the HPV detection methods. In the present study, the prevalence of HPV infection among pregnant mothers and the frequency of transmission to their infants investigated by the conventional PCR and HC-II methods are comparable though the latter was found to be more reliable. Identification of HPV infection using the HC-II test is a much more sensitive method for the detection of high-grade cervical lesions than the classic cytology [35]. The analytical sensitivity of HC-II is on the order of about 100,000 genome copies [36]. It has been suggested that HC-II could be able to detect high viral loads that are clinically relevant or most likely to [37].

Here the high rate of HPV infection among neonates after a cesarean delivery suggests that cesarean delivery did not effectively prevent perinatal transmission of HPVs. Various groups reported about the presence of HPV DNA in the amniotic fluid,⁹peripheral blood mononuclear cells of pregnant women and in the cord blood specimens of neonates born to HPV+ mothers [12]. Thus, it can be suggested that HPV can cross the placental barrier and infection might occur in the uterus. Here, multiple HPV infection was found among mothers and their infants. A previous study found HPV 16 infection to be usually associated with a greater risk of acquisition of other HPV types [38].In the present study, all HPV-positive infants born to HPV-positive mothers did not carry the maternal HPV subttypes. Additionally, HPV infection was found among babies of HPV-negative mothers. These findings are consistent with other reports of discordant mother-infant pairs and HPV-positive babies from HPV-negative mothers [8]. This may occur due to HPV transmission by transplacental route in advance of delivery or by some other route (horizontal spread, from relatives/friends, infected fomites/clothing) in the intervening period between delivery and testing [39]. Presence of HPV in the human sperm cells and active transcription of HPV-specific genes in infected cells have been reported [40,41]. These virus-infected sperm cells behaving as vectors or carriers cause transmission of HPV to fetuses through fertilized eggs [40].

Here we report a higher p53 expression in the HPV-positive mothers and their neonates. A highly significant correlation was also observed between the abnormal p53 expression and the status of gravida. It is suggested from these results that the immunodetection of p53 in cervical smears can be used as a useful diagnostic marker for HPV infection and its vertical transmission. Here a significant association was observed between p53 expression and HPV status in the study population. Higher numbers of p53-positive cases were found in HPV-positive mother-baby pairs compared to the HPV-negative mother-baby pairs. In accordance with the findings of Paquette et al 1993 [42] and Nair et al 1999 [43], these results also suggest that the p53 protein inactivation by complex formation with high-risk HPV 16/18 subtypes may be responsible for the overexpression of p53 observed in the HPV positive mother-baby pairs. Crook et al 1994 suggested that some degree of modulation of p53 function is necessary for the normal life cycle of the virus [44]. The same group demonstrated a correlation between the efficiency of this activity and the oncogenic potential of the virus [44]. The loss of function of the p53 check-point regulation due to its interaction with high-risk HPV E6 may thus impair the apoptotic response.

Here, Bcl-2 protein expression was also analyzed during the vertical transmission of HPVs from pregnant mothers to their infants. A significant correlation was found between Bcl-2 expression and perinatal transmission of HPVs that increased with the gravida status. It is therefore suggested that the immunodetection of Bcl-2 in cervical lesions may be used as a diagnostic marker for vertical transmission of HPVs. Increased Bcl-2 protein expression has been reported in cervical carcinoma cell lines containing mutated or HPV E6-inactivated p53 [45]. Another study also has demonstrated a strong association between the presence of HPV E6 protein and Bcl-2 [46]. They suggested that the detection of HPV E6 protein and Bcl-2 may be helpful for the identification of women who are at increased risk for developing cervical cancer in future [46]. The present study also focused on the association of Bcl-2 protein expression with HPV-positive mother-baby pairs. A highly significant positive correlation was observed between HPV infection and Bcl-2 expression. The present findings on Bcl-2 protein expression in the HPV positive mother-baby pairs suggest a highly interlinked association between HPV infection especially high-risk HPVs and Bcl-2. It has been also reported that viruses interact with the cell death pathway at the checkpoints of the Bcl-2 family proteins for their site of intervention. It has been reported that DNA viruses parasitize the host cellular machinery to drive their own replication [47]. This suggests a strong effect of HPV on Bcl-2 at the gene level itself. It has been also suggested that the expression of bcl-2 is lost during tumor progression and is a strong prognostic parameter, suggesting that the regulation of apoptosis plays an important role in the behavior of cervical carcinomas [48] bcl-2 can probably not only mediate cell death but also regulate cell growth [49].

Since p53 and Bcl-2 are antagonistic in their function, an inverse correlation between their protein expression level as reported in many of cancers is expected [46,50]. But, we report a positive correlation between the expression level of p53 and Bcl-2 proteins. This suggests the role of HPV infection by disturbing the apoptosis regulatory pathway. Moreover, the correlation between p53 and Bcl-2 and HPV infection is more or less similar, thus suggesting the aberrant expression of these proteins may be interlinked with high-risk HPV infection. The present study is therefore in good agreement with the hypothesis as suggested by Sidransky and Hollstein 1996 [51] that the p53 protein inactivation by the oncoprotein HPV E6 releases the repression of the Bcl-2 gene thus leading to the over-expression of Bcl-2 and nonfunctional p53 proteins in the HPV positive cases. Conversely, it has been reported that no significant association was found between HPV 16/18 infection and p53 and Bcl-2 expression in premalignant and malignant lesions of uterine cervix [52].

Though this study could not conclusively support the concept of perinatal transmission of HPV even when the infection is sub-clinical, there is a clear possibility of transplacental infection. Moreover, this study strongly suggests that the co-overexpression of p53 and Bcl-2 proteins in the HPV positive mother-baby pair is due to the high-risk HPV infection.

Acknowledgements:

The author is thankful to Dr. S. Lahiri, Dr. S. Chatterjee and all the personnel of the Department of Gynecology and Obstetrics, South Eastern Railway Hospital, Garden Reach, Kolkata, India for collection of the cervical swabs and buccal swabs of the mother-baby pairs. The author acknowledges the technical help provided by Drs. Sarmistha Bhattacharya (Avitide.Inc, USA), Debjani Guha (Dana-Farber Cancer Institute, USA) and Biplab Mandal (Dept. of Zoology, Vidyasagar University, WB, India) during this study. The author is thankful to Dr. Shyamsundar Mandal, Statistician, Chittaranjan National Cancer Hospital, Kolkata, India for the help in data analysis. The author also acknowledges Drs. Madhumita Roy and Sutapa Mukherjee (Dept. of Environmental Cancinogenesis, Chittaranjan National Cancer Institute, Kolkata, India) for the immunocytochemistry-related experiments. The author is indebted to Dr. Lakshmi Majumdar (Dept. of Pathology, Ramakrishna Mission Seva Pratishthan, Kolkata, India) for the Pap staining and cytological analysis. S.S received a Research Assistant fellowship from the Indian Council of Medical Research (ICMR), New Delhi, India. This work was supported by a research grant (IRIS section. 9502050) from the ICMR, New Delhi, India.

Ethical Approval:

This study was performed in line with the principles of the 1964, Declaration of Helsinki and its later amendments or comparable ethical standards. This study was commenced after obtaining approval from the institutional ethical board. Informed consent for sample collection were received from the women for themselves and their infants.

Competing interests:

The author declare no competing interests.

Authors' contributions:

The corresponding author is the only author of this manuscript.

Funding:

This work was supported by a research grant (IRIS section. 9502050) from the ICMR, New Delhi, India.

Availability of data and materials:

Data cannot be shared openly but are available on request from author.

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Tables 1 to 4 are available in the Supplementary Files section.

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HPV infection and its correlation with p53 and Bcl-2 among pregnant mothers and their infants

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Abstract

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1. Introduction

2. Materials and methods

3. Results

3.2. Comparison of genotyping of HPV of the infected mothers by PCR and HC-II methods:

3.3. HPV prevalence, genotyping and cytological screening in the infants:

3.4. Immunocytochemistry of p53 protein and its association with HPV infection:

3.5. Immunocytochemistry of Bcl-2 protein and its association with HPV infection:

3.6. Correlation between p53 expression and Bcl-2 expression:

3.7. Cytological analysis by Pap staining:

4. Discussion

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References

Tables

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Supplementary Files

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