Determinants of Acquisition, Persistence, and Clearance of Oncogenic Cervical Human Papillomavirus Infection in the Philippines Using a Multi-Omics Approach: DEFEAT HPV Study Protocol.

HPV infection is one of the most studied risk factors in cervical cancer-the second most common cancer site and cause of death due to cancer in the Philippines. However, there is a lack of population-based epidemiological data on cervical HPV infection in the Philippines. Local reports on co-infections with other lower genital tract pathogens, commonly reported globally, are also lacking, which emphasizes the need to increase efforts in targeting HPV prevalence, genotype, and distribution. Hence, we aim to determine the molecular epidemiology and natural history of HPV infection among reproductive-age Filipino women using a community-based prospective cohort design. Women from rural and urban centers will be screened until the target sample size of 110 HPV-positive women (55 from rural sites and 55 from urban sites) is reached. Cervical and vaginal swabs will be collected from all screened participants. For HPV-positive patients, HPV genotypes will be determined. One hundred ten healthy controls will be selected from previously screened volunteers. The cases and controls will comprise the multi-omics subset of participants and will be followed up after 6 and 12 months for repeat HPV screening. Metagenomic and metabolomic analyses of the vaginal swabs will also be performed at baseline, after 6 months, and after 12 months. The results of this study will update the prevalence and genotypic distribution of cervical HPV infection among Filipino women, determine whether the current vaccines used for HPV vaccination programs capture the most prevalent high-risk HPV genotypes in the country, and identify vaginal community state types and bacterial taxa associated with the natural history of cervical HPV infection. The results of this study will be used as the basis for developing a biomarker that can help predict the risk of developing persistent cervical HPV infection in Filipino women.

Improving diagnostic strategies for ovarian cancer in Filipino women using ultrasound imaging and a multivariate index assay.

OBJECTIVE: To evaluate the clinical performance and overall utility of imaging and biomarker assays in discriminating between benign and malignant ovarian masses in a Filipino population. METHODS: This is a prospective cohort study among Filipino women undergoing assessment for an ovarian mass in a tertiary center. All included patients underwent a physical examination before level III specialist ultrasonographic and Doppler evaluation, multivariate index assay (MIA2G), and surgery for an adnexal mass. Ovarian tumors were classified as high-risk for malignancy based on the International Ovarian Tumour Analysis (IOTA) - Logistic Regression 2 (LR2) score. The ovarian imaging and biomarker results were correlated with the reference standard: histological findings. RESULTS: Among the 379 women with adnexal masses enrolled in this study, 291 were evaluable with ultrasound imaging, biomarker assays, and histopathological results. The risk of malignancy was higher for women classified as high-risk based on IOTA-LR2 (≥10%). The sensitivity, specificity, and diagnostic accuracy for the prediction of malignancy were 81.2%, 81%, and 0.81 (95% CI: 0.77-0.86) for IOTA-LR2; 77.5%, 66.7%, and 0.72 (95% CI: 0.67-0.77) for CA-125; and 91.3%, 41.2%, and 0.66 (95% CI: 0.62-0.71) for MIA2G. A combination of IOTA-LR2 and MIA2G significantly influenced the diagnostic performance and the result. When MIA2G was combined with IOTA-LR2 in parallel, the sensitivity (94.2%) and NPV (87.7%) increased, but the specificity (37.3%) decreased. When combined with IOTA-LR2 in series, there were fewer false positives, which resulted in improved specificity (85%). CONCLUSION: This study determined the utility of ovarian imaging and a second-generation multivariate index assay in predicting the risk of ovarian malignancy. IOTA-LR2 and MIA2G were useful in classifying patients with a high risk for ovarian malignancy.

Clinical Performance of a Multivariate Index Assay in Detecting Early-Stage Ovarian Cancer in Filipino Women.

This study evaluated the clinical performance and overall utility of a multivariate index assay in detecting early-stage ovarian cancer in a Filipino population. This is a prospective cohort study among Filipino women undergoing assessment for an ovarian mass in a tertiary center. Patients diagnosed with early-stage ovarian cancer and who underwent a physical examination before level III specialist ultrasonographic and Doppler evaluation, multivariate index assay (MIA2G), and surgery for an adnexal mass were included in this study. Ovarian tumors were classified as high-risk for malignancy based on the IOTA-LR2 score. The ovarian imaging and biomarker results were correlated with the reference standard: surgico-pathologic findings. The MIA2G exhibited the best overall performance among individual classifiers with a sensitivity of 91.7% and NPV of 84.7%, with a concomitant higher sensitivity in early-stage disease, whether as an individual classifier (93.5%) or in serial combination with ultrasound (85.5%). The performance of biomarkers (specificity, positive predictive values, and AUROC) such as MIA2G and CA-125 significantly improved when combined with an ultrasound risk scoring approach (p < 0.01). MIA2G showed a higher sensitivity for detecting lesions among EOC and late-stage ovarian cancers than otherwise. The application of biomarkers for evaluating ovarian masses in our local setting is secondary to ultrasound but adopting multivariate index assays rather than CA-125 would increase the detection of early-stage ovarian cancers regardless of menopausal status. This is most relevant in areas where level III sonographers or gynecologic oncologists are limited and preoperative referrals to these specialists can improve the survival of our patients.

Current Status of Human Papillomavirus Infection and Cervical Cancer in the Philippines.

Cervical cancer is estimated to cause 341,831 deaths each year, with 9 of 10 deaths occurring in developing countries. Over the past decade, there has been a significant increase in cervical cancer incidence among women in the Philippines. Persistent infection with high-risk human papillomavirus (HPV) is the well-established necessary cause of cervical cancer. Based on limited studies conducted in the Philippines, the prevalence of infection with any HPV genotype was 93.8% for cervical squamous cell carcinoma and 90.9% for cervical adenocarcinomas. HPV types 16 and 18 were the most common HPV genotypes among Filipino patients with cervical cancer. On the other hand, the incidence of HPV infection among Filipino women with normal cervices was 9.2%. The World Health Organization has launched a global agenda of eliminating HPV infection by 2030. One of its key milestones is to vaccinate 90% of girls with the HPV vaccine by 15 years. However, the HPV vaccination rate among Filipino women remains to be unsatisfactory. HPV vaccination has only been included in the Philippine Department of Health's community-based National Immunization Program in 2015. Despite these efforts, the Philippines currently ranks last on HPV program coverage among low-middle income countries, with coverage of only 23% of the target female population for the first dose and 5% for the final dose. The principal reason for the non-acceptance of HPV vaccines was the perceived high cost of vaccination. The low utilization of available cervical cancer screening tests such as Pap smear and visual inspection with acetic acid hampered the Philippines' control and prevention of HPV infection and cervical cancer. Among those diagnosed with cervical cancer in the Philippines, only an estimated 50% to 60% receive some form of treatment. To this end, we summarize the burden of HPV infection and cervical cancer on Filipinos and the risk factors associated with the disease. We present the current screening, diagnostics, treatment, and prevention of HPV-related diseases in the Philippines. Lastly, we also propose solutions on how each building block in health systems can be improved to eliminate HPV infection and reduce the burden of cervical cancer in the Philippines.

Diagnostic Performances of Ultrasound-Based Models for Predicting Malignancy in Patients with Adnexal Masses.

This study compared the diagnostic performance of different ultrasound-based models in discriminating between benign and malignant ovarian masses in a Filipino population. This was a prospective cohort study in women with findings of an ovarian mass on ultrasound. All included patients underwent a physical examination before level III specialist ultrasonographic and Doppler evaluation using the different International Ovarian Tumor Analysis (IOTA) Group's risk models. Serum CA-125 and a second-generation multivariate index assay (MIA2G) were also determined for all patients. The ovarian imaging and biomarker results were correlated with the histological findings. A total of 260 patients with completed ultrasound, CA-125, MIA2G, and histopathologic results was included in the study. The presence of papillae with blood flow and irregular cyst walls during the ultrasound were significantly associated with a 20-fold (OR: 20.13, CI: 8.69−46.67, p < 0.01) and 10-fold (OR: 10.11, CI: 5.30−19.28, p < 0.01) increase in the likelihood of a malignant lesion, respectively. All individual sonologic procedures performed well in discerning malignant and benign ovarian lesions. IOTA-LR1 showed the highest accuracy (82.6%, 95% CI: 77.5−87%) for identifying ovarian cancer. IOTA-ADNEX showed the highest sensitivity (93.3%, 95% CI: 87.2−97.1%) while IOTA-LR2 exhibited the highest specificity (84.4%, 95% CI: 77.3−90%). Among the different serial test combinations, IOTA-LR1 with MIA2G and IOTA-LR2 with MIA2G showed the highest diagnostic accuracy (AUROC = 0.82). This study showed that all individual ultrasound-based models performed well in discerning malignant and benign ovarian lesions, with IOTA-LR1 exhibiting the highest accuracy.

The role of genital mycoplasma infection in female infertility: A systematic review and meta-analysis.

PROBLEM: Recent studies show that lower genital tract infection with genital mycoplasma may be associated with the pathology of female infertility. However, this association remains controversial due to the variable prevalence, sample sizes, and different methods used to diagnose genital mycoplasma infection. The aim of the present meta-analysis was to gain better understanding of the specific impact of genital mycoplasma on female infertility. METHOD OF STUDY: A systematic review of literature on the association of genital mycoplasma (Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum) infection and female infertility was performed using three electronic databases: PubMed, Scopus, and CINAHL, from January 2000 to January 2020. Pooled odds ratio (OR) and 95% confidence intervals for genital mycoplasma infection and female infertility were derived from a fixed effects model. RESULTS: This meta-analysis included eight studies conducted in six countries. Based on the results, women with infertility had a statistically higher odds of having any genital mycoplasma infection (p < .0001) compared to the control group. The pooled OR of all the included studies was 3.82 (95% CI: 2.55, 5.72). There was an unremarkable heterogeneity in all the studies included in this meta-analysis (I2  = 0%, p = .48). A subgroup analysis also showed that M. genitalium, M. hominis, and U. urealyticum infections are significantly associated with female infertility. CONCLUSION: Our meta-analysis showed a significant association between M. genitalium, M. hominis, and U. urealyticum infections and female infertility. This evidence supports the development of guidelines for the diagnosis and treatment of genital mycoplasma infections to prevent female infertility.

Evaluation of Abdominal Fetal Electrocardiography in Early Intrauterine Growth Restriction.

Objectives: This descriptive study was performed to evaluate the capability of a non-invasive transabdominal electrocardiographic system to extract clear fetal electrocardiographic (FECG) measurements from intrauterine growth restricted (IUGR) fetuses and to assess whether abdominal FECG parameters can be developed as markers for evaluating the fetal cardiac status in IUGR. Methods: Transabdominal FECG was attempted in 20 controls and 15 IUGR singleton pregnancies at 20+0-33+6 weeks gestation. Standard ECG parameters were compared between the study groups and evaluated for their correlation. Accuracy for the prediction of IUGR by cut off values of the different FECG parameters was also determined. Results: Clear P-QRST complexes were recognized in all cases. In the IUGR fetuses, the QT and QTc intervals were significantly prolonged (p = 0.017 and p = 0.002, respectively). There was no correlation between ECG parameters and Doppler or other indices to predict IUGR. The generation of cut off values for detecting IUGR showed increasing sensitivities but decreasing specificities with the prolongation of ECG parameters. Conclusion: The study of fetal electrocardiophysiology is now feasible through a non-invasive transabdominal route. This study confirms the potential of FECG as a clinical screening tool to aid diagnosis and management of fetuses after key limitations are addressed. In the case of IUGR, both QT and QTc intervals were significantly prolonged and thus validate earlier study findings where both these parameters were found to be markers of diastolic dysfunction. This research is a useful prelude to a test of accuracy and Receiver Operating Characteristics (ROC) study.

Ultrasound Imaging of Mouse Fetal Intracranial Hemorrhage Due to Ischemia/Reperfusion.

Despite vast improvement in perinatal care during the 30 years, the incidence rate of neonatal encephalopathy remains unchanged without any further Progress towards preventive strategies for the clinical impasse. Antenatal brain injury including fetal intracranial hemorrhage caused by ischemia/reperfusion is known as one of the primary triggers of neonatal injury. However, the mechanisms of antenatal brain injury are poorly understood unless better predictive models of the disease are developed. Here we show a mouse model for fetal intracranial hemorrhage in vivo developed to investigate the actual timing of hypoxia-ischemic events and their related mechanisms of injury. Intrauterine growth restriction mouse fetuses were exposed to ischemia/reperfusion cycles by occluding and opening the uterine and ovarian arteries in the mother. The presence and timing of fetal intracranial hemorrhage caused by the ischemia/reperfusion were measured with histological observation and ultrasound imaging. Protein-restricted diet increased the risk of fetal intracranial hemorrhage. The monitoring of fetal brains by ultrasound B-mode imaging clarified that cerebral hemorrhage in the fetal brain occurred after the second ischemic period. Three-dimensional ultrasound power Doppler imaging visualized the disappearance of main blood flows in the fetal brain. These indicate a breakdown of cerebrovascular autoregulation which causes the fetal intracranial hemorrhage. This study supports the fact that the ischemia/reperfusion triggers cerebral hemorrhage in the fetal brain. The present method enables us to noninvasively create the cerebral hemorrhage in a fetus without directly touching the body but with repeated occlusion and opening of the uterine and ovarian arteries in the mother.

Vaginal LPS changed gene transcriptional regulation response to ischemic reperfusion and increased vulnerability of fetal brain hemorrhage.

During pregnancy, both ischemic reperfusion and bacterial agent LPS are known risk factors for fetal brain damage. However, there is a lack of evidence to explain whether vaginal LPS affects the fetus response to ischemic reperfusion. Here we reported that there was more than 2 folds higher vulnerability of fetal brain hemorrhage response to ischemic reperfusion when mother mouse was treated with vaginal LPS. As our previously reported, ischemic reperfusion induces P53-dependent fetal brain damage was based on a molecular mechanism: the transcriptional pattern was changed from HIF-1alpha-dependent to P53-dependent immediately. In the present work, only with vaginal LPS precondition, phosphorylation of activated transcriptional factor (ATF) 2 at Thr71 appeared in response to ischemic reperfusion. Moreover, this phosphorylation was completely blocked by pre-treatment with a P53 inhibitor, pifithrin-α. We concluded that vaginal LPS precondition trigged the p53-dependent phosphorylation of ATF2 in response to ischemic reperfusion, which played an important role of increasing vulnerability to hemorrhage in fetus.

Intrauterine ischemic reperfusion switches the fetal transcriptional pattern from HIF-1α- to P53-dependent regulation in the murine brain.

Ischemic reperfusion (IR) during the perinatal period is a known causative factor of fetal brain damage. So far, both morphologic and histologic evidence has shown that fetal brain damage can be observed only several hours to days after an IR insult has occurred. Therefore, to prevent fetal brain damage under these circumstances, a more detailed understanding of the underlying molecular mechanisms involved during an acute response to IR is necessary. In the present work, pregnant mice were exposed to IR on day 18 of gestation by clipping one side of the maternal uterine horn. Simultaneous fetal electrocardiography was performed during the procedure to verify that conditions resulting in fetal brain damage were met. Fetal brain sampling within 30 minutes after IR insult revealed molecular evidence that a fetal response was indeed triggered in the form of inhibition of the Akt-mTOR-S6 synthesis pathway. Interestingly, significant changes in mRNA levels for both HIF-1α and p53 were apparent and gene regulation patterns were observed to switch from a HIF-1α-dependent to a p53-dependent process. Moreover, pre-treatment with pifithrin-α, a p53 inhibitor, inhibited protein synthesis almost completely, revealing the possibility of preventing fetal brain damage by prophylactic pifithrin-α treatment.

Molecular patterns of neurodevelopmental preconditioning: a study of the effects of antenatal steroid therapy in a protein-restriction mouse model.

Introduction. Prenatal programming secondary to maternal protein restriction renders an inherent susceptibility to neural compromise in neonates and any addition of glucocorticosteroids results in further damage. This is an investigation of consequent global gene activity due to effects of antenatal steroid therapy on a protein restriction mouse model. Methods. C57BL/6N pregnant mice were administered control or protein restricted diets and subjected to either 100 µ g/Kg of dexamethasone sodium phosphate with normosaline or normosaline alone during late gestation (E10-E17). Nontreatment groups were also included. Brain samples were collected on embryonic day 17 and analyzed by mRNA microarray analysis. Results. Microarray analyses presented 332 significantly regulated genes. Overall, neurodevelopmental genes were overrepresented and a subset of 8 genes allowed treatment segregation through the hierarchical clustering method. The addition of stress or steroids greatly affected gene regulation through glucocorticoid receptor and stress signaling pathways. Furthermore, differences between dexamethasone-administered treatments implied a harmful effect during conditions of high stress. Microarray analysis was validated using qPCR. Conclusion. The effects of antenatal steroid therapy vary in fetuses according to maternal-fetal factors and environmental stimuli. Defining the key regulatory networks that signal either beneficial or damaging corticosteroid action would result in valuable adjustments to current treatment protocols.

Automated Identification of fetal cardiac valve timings.

In this paper a new noninvasive method is proposed for automated estimation of opening and closure timings of fetal cardiac valves. These timings are obtained from Doppler Ultrasound (DUS) signal and fetal electrocardiogram (fECG) as a reference. Empirical Mode Decomposition (EMD) is first applied to the DUS signal to decompose it into different components called Intrinsic Mode Functions (IMFs). The envelope of the first IMF is then taken and its peaks are identified. The opening and closure of the valves are then automatically assigned to the IMF peaks by using Hidden Markov Model (HMM). It is shown that this new method can continuously evaluate fetal cardiac valves' (aortic and mitral) motion timings for 82.5~99.7% of cardiac cycles. The estimated timings are verified using the Pulsed Doppler images. These findings can be used as sensitive markers for evaluating the fetal cardiac performance.

Evaluation of cardiac performance by abdominal fetal ECG in twin-to-twin transfusion syndrome.

OBJECTIVE: To investigate fetal cardiac performance by abdominal fetal electrocardiography (ECG) in monochorionic diamniotic pregnancies with twin-to-twin transfusion syndrome (TTTS-MCDA). METHODS: Abdominal fetal ECG was attempted in 16 singleton, 21 non-TTTS-MCDA, and 14 TTTS-MCDA pregnancies at 16-27 weeks' gestation. Standard ECG parameters were compared across study groups and evaluated for their correlation with Doppler ultrasound indices. RESULTS: Clear P-QRST complexes were recognized in all cases. In the TTTS-MCDA pregnancies, the QT interval and QTc were significantly longer than in both singletons and the non-TTTS-MCDA pregnancies (p = 0.002 and p = 0.0002, respectively), whereas in the recipient fetus, both the PR interval and PR/RR were significantly longer when compared with all other study groups (p = 0.019 and p = 0.012, respectively). Further comparison with Doppler ultrasound indices yielded significant reciprocal correlations between ductus venosus pulsatility index and the QT interval (r = 0.552, p = 0.041) and between umbilical artery pulsatility index and PR/RR (r = 0.654, p = 0.011) both demonstrated in recipient fetuses alone. CONCLUSIONS: Abdominal fetal ECG is feasible in second-trimester twin pregnancies. In TTTS, there is evidence of a higher risk of cardiac dysfunction in the recipient twin.

Understanding congenital heart defects through abdominal fetal electrocardiography: case reports and clinical implications.

AIMS: Congenital heart defects are the most common fetal structural anomalies of which a significant number remain unrecognized during postnatal life. Fetal electrocardiography (FECG) is an ideal clinical tool to complement ultrasonography for the screening and management of these cases where early and accurate diagnoses would allow definite rather than palliative treatment. The objective of this report was to correlate the particular FECG results found with the different types of congenital heart defects involved and to further demonstrate the usefulness of FECG in clinical settings. MATERIAL & METHODS: This is a report of four cases of prenatally diagnosed congenital heart defects seen at a university hospital in Sendai, Japan. Their complete and thorough evaluation included, among other tests, abdominal FECG analysis. RESULTS: The presence of premature ventricular contractions, a prolonged pre-ejection period (PEP > 75 msec), and prolonged QTc intervals (QTc > 440 msec) served as markers of hemodynamic alteration but were unlikely determinants of disease severity precluding further investigation. CONCLUSIONS: In practice, similar findings found on FECG should raise the index of suspicion for the presence of congenital heart disease and prompt a targeted ultrasound scan.

Effects of antenatal steroid therapy on neurodevelopment in an IUGR mouse model.

BACKGROUND/OBJECTIVE: To investigate the neurodevelopmental response in postnatal mice secondary to antenatal steroid treatment in association with maternal protein restriction. METHODS: C57BL/6N pregnant mice (n = 24; 4 per treatment group) were administered control (C) or protein-restricted (PR) diets and subjected to daily subcutaneous injection stress during late gestation (E10-E17) with either 100 microl/kg of dexamethasone sodium phosphate in normosaline (C-D/S, PR-D/S) or normosaline alone (C-S, PR-S). Non-treatment groups were also included (C, PR). Brain samples of pups were collected on postnatal day 7 and analyzed by immunohistochemistry and qRT-PCR. RESULTS: Neonatal weights in the treatment groups were smaller than their counterparts in the C group, but there were no significant differences in brain size. Immunohistochemical evaluation of neuroglial cells revealed a pronounced effect of protein restriction on oligodendrocytes and oligodendrocyte precursor cells with distinct fetal responses to stress and dexamethasone. Further evaluation using quantitative RNA analysis showed significant activation of Galr1, Galr2, Igfbp-1, Igfbp-3, Igfbp-6, and Fgf2 by 1- to 2.5-fold in the PR-D/S group and by much higher increments, 1- to 10.5-fold, in the PR-S group. CONCLUSION: This preliminary investigation revealed the possible role of dexamethasone in further increasing vulnerability to cell damage in injury-prone neuroglial cells. The distribution of key glial markers and the overexpression of several neurotrophic factors depicted ongoing cellular adaptation.

Aberrant behavior of mouse embryo development after blastomere biopsy as observed through time-lapse cinematography.

OBJECTIVE: To analyze whether blastomere biopsy affects early embryonal growth as observed through time-lapse cinematography. DESIGN: Comparative prospective study between embryos in which a blastomere was removed and embryos in which a blastomere was not removed. SETTING: An experimental laboratory of the university. MAIN OUTCOME MEASURE(S): We calculated the time between blastocele formation and the end of hatching, the time between the start and end of hatching, the number of contractions and expansions between blastocyst formation and the end of hatching, and the maximum diameter of the expanded blastocyst. RESULT(S): In blastomere removal embryos, compaction began at the six-cell stage instead of at the eight-cell stage. We also found that hatching was delayed in these embryos as compared with matched controls. Moreover, the frequency of contraction and expansion movements after blastocyst formation was significantly higher in the blastomere removal group as compared with the control group. Finally, the maximum diameter of the expanded blastocyst just before hatching was not significantly different between both groups. CONCLUSION(S): These findings suggested that blastomere removal has an adverse effect on embryonic development around the time of hatching. Thus, future developments in preimplantation genetic diagnosis and screening should involve further consideration and caution in light of the influence of blastomere biopsy on embryonal growth.

Rudimentary horn pregnancy

This is a case report and review of the diagnosis and management of rudimentary horn pregnancy in a unicornuate uterus.