Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G.

In brief: Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. Abstract: Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered 'fingerprints of parent cells', which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30-150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.

First intrauterine transfusion in Rh isoimmunised triplet pregnancy.

G3P2L1, 28+4 weeks of gestation rhesus (Rh) isoimmunised pregnant women, was referred with trichorionic triamniotic triplet pregnancy with Rh antibody titres of 1:32. Nuchal translucency and anomaly scan were within normal limits with no major malformation for any of the fetuses. Obstetric colour Doppler with middle cerebral artery peak systolic volume revealed foetal anaemia in all three fetuses having velocities corresponding to around 1.5 times the median. Decision of intrauterine transfusion of blood to all three fetuses was taken. Access to fetuses was challenging and expertise in interventional ultrasound was required for transfusion. The patient tolerated the procedure well and eventually went on to deliver uneventfully at 34 weeks of gestation for worsening pre-eclampsia. After birth, all three triplets received triple-surface intensive phototherapy and intravenous immunoglobulin at a dosage of 1 g/kg. Phototherapy was gradually reduced and discontinued within 72 hours, and the infants were discharged from the neonatal intensive care unit at 96 hours of age.

Double/Triple Intrauterine Blood Transfusion in Rh-isoimmunized Anemic Fetuses in Multiple Pregnancies with Favorable Outcome.

Background: Multiple pregnancies have increased with the use of assisted reproduction, and we expect more women reporting with Rh isoimmunization among multiple gestation in near future. Intrauterine transfusion in singleton itself is technically difficult and requires a lot of skill and precision. Performing double/triple transfusion in twins/triplets is expected to be more demanding. Aim: To create awareness on the technical difficulties encountered in intrauterine transfusion in twins and triplets. Methodology: We report a case series of four Rh-isoimmunized twins/triplets in 5 years who presented with severe anemia requiring intrauterine transfusion. Results: Each of the four sets of cases had their own intricacies that needed to be pondered before tackling them as not much was available in the literature. In Case 1, the first twin intrauterine transfusion in our 20-year-long experience, the difficulty in the approach to the first twin due to a posteriorly placed placenta has been highlighted. Case 2 was rare due to the concomitant presence of atypical antibodies in the mother in addition to Rh-D isoimmunization that made it difficult to cross match any donor blood for intrauterine transfusion. The third case was exclusive due to its monochorionic-diamniotic nature of the twins where the impact of inter-twin anastomosis on the transfusion was to be taken into consideration. Fourth case was a triplet gestation where the difficulty of which cord to be assigned to which fetus, the crowded space for intervention, as well as the risk of prolonged operative time and associated risk of preterm/premature rupture of membranes were our concern. Conclusion: Intrauterine transfusion (IUT) in twins/triplets is challenging. Difficulties encountered during IUT in multifetal gestation are due to different or uncertain chorionicity, intraplacental anastomosis between vessels, different degree of anemia in twins, difficult to ascertain cord-fetus relationship and difficulty to reach placental insertion site due to crowding by multiple fetal parts.

A Novel Neuroimaging Phenotype in the X-Linked Intellectual Disability with a Missense Mutation of CNKSR2 Gene.

Background: Mental retardation, X-linked, syndromic, Houge type (MRXSHG) is a form of mental retardation characterized by intellectual disability, speech and language impairments, and early-onset seizures. It has been recently recorded in Online Mendelian Inheritance in Man (OMIM), and only 10 cases have been reported in the literature so far. Objective: To highlight the novel neuroimaging findings in the pediatric X-linked intellectual disability with a missense mutation of connector enhancer of kinase suppressor of RAS2 (CNKSR2) gene. Material and Methods: We present a case of intellectual disability, refractory epilepsy, speech and language delay with subtle dysmorphism, and behavioral issues in an 11-year-old boy with novel neuroimaging findings in a CNKSR2 gene with missense mutation. Results: Brain MRI revealed involvement of the basal ganglia, predominantly the neostriatum, and along with the subependymal aspects with focal cavitations involving, especially the bilateral caudate heads. There was relative sparing of the globus pallidi and posterior putamina bilaterally. Whole-exome sequencing identified a hemizygous missense pathogenic variant in the CNKSR2 gene. The mother was found to be an asymptomatic carrier. Conclusion: This case report highlights the rare missense mutation in the CNKSR2 gene and abnormal neuroimaging findings, which further provide information about the phenotypic characteristics of X-linked syndromic intellectual disability.

Assessment of knowledge, attitude and practice (KAP) of parents/caregivers towards epilepsy in children - A cross- sectional observational study.

Objectives: The study aimed to assess the knowledge, attitude, and practice (KAP) of parents/caregivers toward epilepsy in paediatric patients at a tertiary care centre of North India. Methods: A cross sectional study was carried out among 418 parents or caregivers using convenience sampling technique with 16-item questionnaire in English language and also translated to local language that is Hindi. Children with epilepsy who visited the paediatric outpatient department within a year were included in the study (January 2021-22). A total of 450 children visited the clinic, 32 of whom were excluded for various reasons, and the final analysis was conducted among the 418 parents or caregivers who completed the questionnaire. Results: The male and female patients were 56.7% (n = 237) and 43.3% (n = 181) respectively. The age distribution of patients with less than 5 years, 6-10 years and more than 10 years were 35.6% (n = 149), 54.5% (n = 228), 9.8% (n = 41) respectively. Only one third of parents and caregivers did not consider epilepsy as psychiatric illness. Most of the parents and caregivers think that epilepsy affects school performance (77.2%) and hinders family life (71.0%). More than half of the parents or caregivers believes that the society discriminates against person with epilepsy and around 46.6% consider that alternative medicine can cure epilepsy. The parents or caregivers felt financial burden due to epilepsy was in 72.5% and approximately 78.5% perceived that their work is affected because of their child's epilepsy. Perception of epilepsy as a psychiatric illness was found to be significantly higher in parents with primary and secondary level education, when compared to parents who were graduates. The practice of the parents or caregivers towards administration of drugs to their child was good, however around 36.6% (n = 153) missed the dose of anti-seizure medications. Conclusion: The study highlights the substantial knowledge, attitude and practice gap amongst parents and caregivers for children with epilepsy which indirectly has huge impact on the management of epilepsy. Thus it becomes utmost important to educate the family as well as the community regarding epilepsy which will help in improving the therapeutic outcomes, overall quality of life and interpersonal and social relationships of these children.

DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss.

Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

Whole genome bisulfite sequencing of sperm reveals differentially methylated regions in male partners of idiopathic recurrent pregnancy loss cases.

OBJECTIVE: To study the genome wide alterations in sperm DNA methylation in male partners of idiopathic recurrent pregnancy loss (iRPL) cases and note regions as potential diagnostic markers. DESIGN: Case-control study and methylome analysis of human sperm. SETTING: Obstetrics and Gynaecology clinics. PATIENT(S): Control group consists of apparently healthy fertile men having fathered a child within the last 2 years (n = 39); and case group consists of male partners of iRPL cases having ≥2 consecutive 1st trimester pregnancy losses (n = 47). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm DNA samples of controls and cases were selected for whole genome bisulfite sequencing analysis based on the previously set thresholds of global methylation levels and methylation levels of imprinted genes (KvDMR and ZAC). Whole genome bisulfite sequencing of selected sperm genomic DNA was performed to identify differentially methylated CpG sites of iRPL cases compared with fertile controls. Pathway analysis of all the differentially methylated genes was done by Database for Annotation, Visualization, and Integrated Discovery annotation tool and Kyoto Encyclopedia of Genes and Genomes tool. Differentially methylated CpGs within genes relevant to embryo and placenta development were selected to further validate their methylation levels in study population by pyrosequencing. RESULT(S): A total of 9497 differentially methylated CpGs with highest enrichment in intronic regions were obtained. In addition, 5352 differentially methylated regions and 2087 differentially methylated genes were noted. Signaling pathways involved in development were enriched on pathway analysis. Select CpGs within genes PPARG, KCNQ1, SETD2, and MAP3K4 showed distinct hypomethylated subpopulations within iRPL study population. CONCLUSION(S): Our study highlights the altered methylation landscape of iRPL sperm, and their possible implications in pathways of embryo and placental development. The CpG sites that are hypomethylated specifically in sperm of iRPL subpopulation can be further assessed as predictive biomarkers.

Comparison of Punch Biopsy and Loop Biopsy for the Management of Severe Cervical Intraepithelial Neoplasia (CIN).

Objective: The study was undertaken for comparing the colposcopy directed punch versus loop excision biopsy technique in order to get the most précised sample for study as well as comparing the complications associated with them. Materials and Methods: The prospective clinical study was conducted on 50 women of age group 20-80 years. Women who were found to have a Reid colposcopic index of high grade disease, low grade disease or invasive disease recognized as the patients needed biopsy. They were randomly divided into either the punch biopsy forceps group (PB group) or the round loop electrode group (LE group). Result: The quality of tissue of the LE group was found significantly better than the tissue quality of PB group based on the size, site and tissue damaged measures. Both the groups were found to have the same cytology report as well as colposcopic diagnosis and histology reports. Mild degree of bleeding has been reported as a frequent complication in LEEP and punch biopsy. The VAS pain score associated in each procedure was found to be 2.4 and 2.8, respectively, in both groups with no significant difference. Conclusion: The present study has concluded that the quality of tissue from the LEEP biopsy electrode was better than the punch biopsy forceps with no difference in the pain score, and it could be suggested that use of the round loop electrode in CDB provides good quality tissue to evaluate the histology.

Evaluation of cytokine profile in cervicovaginal lavage specimens of women having asymptomatic reproductive tract infections.

Reproductive tract infections (RTIs) such as vaginal candidiasis and bacterial vaginosis (BV) are common among sexually active women and can be both symptomatic or asymptomatic. The microbiota of the reproductive tract triggers immune response at the cervicovaginal interface resulting in secretion of cytokines during the course of these RTIs. The objective of this study was to evaluate the cytokine profile in cervicovaginal lavage of women having asymptomatic vaginal infections. Measurement of vaginal cytokines was done for various interleukins including IL-1ß, IL-6, IL-8, IL-10, IL-12/IL23p40, IL-17A, tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) by ProcartaPlex™ Multiplex Immunoassay. Women having vaginal Candida infection had increased concentration of IL-1ß (p=.01), IL-6 (p=.007), IL-8 (p=.327), IL-12/IL23p40 (p=.049) and IFN-γ (p=.125). The results of our study suggest that evaluation of these cytokines could be explored as an additional measure to determine host inflammatory response in women having asymptomatic vaginal candidiasis.Impact StatementWhat is already known on this subject? Studies assessing the vaginal cytokine profile to assess the vaginal milieu in various cohorts such as post-menopausal women, pregnant women, women with history of preterm birth, CIN and scheduled IVF cycle are being undertaken. Variable cytokine response has been reported in literature in women with symptomatic bacterial vaginosis and Candida infection. However, much less is known about vaginal cytokine profile in asymptomatic infection.What do the results of this study add? The results of the study show increased concentration of the pro-inflammatory cytokines IL-1ß, IL-6 IL-8, IL-12/IL23p40 and interferon gamma (IFN-γ) in women having asymptomatic Candida, vaginal leucocytosis and raised vaginal pH.What are the implications of these findings for clinical practice and/or further research? Evaluation of vaginal cytokine profile (IL-1ß, IL-6 IL-8, IL-1ß, IL-12/IL23p40 and IFN-γ) could be explored as an additional measure to determine inflammation in asymptomatic women. Vaginal cytokines (IL-1ß, IL-6 IL-8, IL-1ß, IL-12/IL23p40 and IFN-γ) could be used further for development of a point of care test.

Prenatal Invasive Testing at a Tertiary Referral Center in India: A Report of 433 Cases Under a Single Operator.

PURPOSE OF THE STUDY: Chromosomal aneuploidies are major causes of perinatal death and childhood handicap. Awareness about screening and prenatal diagnosis for these disorders among obstetricians and primary care physicians is increasing. Since invasive tests like amniocentesis or chorionic villus sampling (CVS) are associated with a risk of miscarriage these tests should be carried out judiciously in pregnancies considered to be at high risk for aneuploidies and other genetic disorders. The purpose of our study was to examine the patterns, trends and outcomes of the various screening procedures and invasive tests results. METHODOLOGY: Retrospective observational study done over a period of 3 years and one month including 433 pregnant women with high risk for genetic disorders undergoing invasive prenatal testing like chorionic villus sampling, amniocentesis or cordocentesis. Data were collected from our department records regarding the maternal age, indication for invasive testing, past obstetric history, family history of genetic syndromes, ultrasound findings in the current sonographic examination and the results of the tests done. Any immediate or late complications of the procedure if any were telephonically addressed. RESULTS: A total of 436 procedures on 433 patients (418 singleton,12 single fetus of twin, 3 both fetuses of twins) were done out of which 281 were amniocentesis(64.4%), 153 were chorionic villus sampling (35.1%) and 2 were cordocentesis(< 1%). Of the 436 procedures, 373(85.5%) were done for positive screening tests for chromosomal aneuploidies and 63(14.4%) were done for previous history of genetic syndromes. The positive predictive value of biochemical marker alone was around 2.7% and higher around 13% for a combined first trimester or a second-trimester screen along with ultrasound abnormalities. The higher the biochemical risk does not translate into higher chance of chromosomal abnormality. Nineteen percentage of fetuses with NT above 95th centile had chromosomal abnormality. Twenty-one percentage of fetuses with absent nasal bone in our study had trisomy 21. CONCLUSION: Aneuploidy screening is the most common indication for prenatal invasive testing with dual marker combined with nuchal translucency, nasal bone, tricuspid regurgitation and ductus venosus flow providing the best detection rates. The chance of an affected fetus in a patient with aneuploidy screen positive overall is only 6.7%.

Efficacy of Antenatal Magnesium Sulfate for Neuroprotection in Extreme Prematurity: A Comparative Observational Study.

Background: Survival of preterm infants has improved drastically. In addition to significant contribution to neonatal mortality, impact of prematurity among survivors may continue through life impairing long-term physical life through neuro-disability and increased risk of cerebral palsy. Maternal administration of magnesium sulfate prior to impending preterm birth is an effective strategy to reduce neuromorbidity. Aim: To investigate the effectiveness of antenatal magnesium sulfate for neuroprotection in preterm infants between 26 and 34 weeks in preventing early neonatal morbidity and mortality. Secondary objective was to assess any adverse events with the use of magnesium sulfate on the mother and neonate. Method: This was a prospective observational comparative study for 2 years at our tertiary care hospital of 100 pregnant women who gave preterm births. Fifty infants each were born to mothers who were either not given MgSO4 (Group 1) or given 4gm intravenous loading dose MgSO4 (Group 2), preferably 4 h prior to preterm birth. Results: Among all the preterm in our study, 81% delivered between 30 and 34 weeks. There was no significant difference in terms of maternal mortality or serious morbidity including postpartum hemorrhage, caesarian section rates or length of hospital stay among women receiving MgSO4 versus no MgSO4. Mild maternal side effects secondary to magnesium sulfate were experienced in 8% cases. There were no significant differences between both groups for low 5 min APGAR, need for NICU admission, neonatal convulsions, hyperbilirubinemia, necrotizing enterocolitis, periventricular leukomalacia and septicemia. There was a trend toward reduced risk in the magnesium sulfate group for need for mechanical ventilation and ongoing respiratory support, intraventricular hemorrhage, neonatal hypotension, hypothermia, length of NICU stay. IVH was less frequent and less severe in babies exposed to antenatal MgSO4 (8%) as compared to non-MgSO4 group (16%). Neonatal morbidities were more when antenatal MgSO4 was given less than 4 h from delivery. Conclusion: MgSO4 is a safe drug to use in antenatal women at risk for impending preterm. Antenatal magnesium sulfate given to women in established preterm labor conferred significant neuroprotective advantage to the neonate. MgSO4 also has protective effect on the need of invasive ventilatory support in preterm infants. Given the breadth of evidence in its favor, it is time for us to start using MgSO4 in clinical practice for neuroprotective intent in all our extreme preterm births.

A Retrospective Analysis to Evaluate Role of the New UTD Classification System in Prenatal Prediction of Severity and Postnatal Outcome in Antenatally Diagnosed Urinary Tract Dilatation Abnormalities.

BACKGROUND/PURPOSE OF THE STUDY: Foetal urinary tract dilation (UTD) abnormalities affect 1-5% of all pregnancies. However, exact incidence is difficult to estimate because of different terminologies used to define the condition and different grading systems to define its severity antenatally as well as postnatally worldwide. In order to overcome this problem, the new UTD classification system has been introduced in the year 2014 so as to have universal approach for diagnosis and management of UTD globally. Indian data about clinical utility of the UTD classification system and its role in prenatal prediction of severity of renal disease are lacking. The present study aims to investigate clinical utility of new UTD classification system in foetal UTD abnormalities and to evaluate the role of UTD classification system in antenatal prediction/prognostication of severity of UTD abnormalities. METHODS: We conducted a single-centre retrospective study between April 2014 and January 2017, which included 70 infants with antenatally diagnosed UTD delivered in our hospital and managed in our paediatric unit postnatally. Pre- and postnatal ultrasound findings were noted, and UTD-A and UTD-P classification were applied retrospectively in all cases as per criteria defined in the new UTD classification. Postnatal outcome in all cases was evaluated in terms of need for immediate postnatal urosurgical intervention, presence of persistent UTD pathology and severity of renal impairment in relation to their pre- and postnatal UTD A and P risk categories. RESULTS: None from UTD A1 risk group in the last prenatal scan showed significant postnatal UTD abnormality. In contrast to this, UTD A2-3 risk group in the last prenatal scan had persistent postnatal UTD pathology in 70% cases. All infants with abnormal postnatal UTD diagnosis were identified prenatally as UTD A2-3 (high risk). Nine infants (12.8%, n = 70) who needed urosurgical intervention postnatally were categorised as UTD A2-3 prenatally and UTD P3 postnatally. CONCLUSION: We found increased frequency of complications and urosurgical interventions in all infants with antenatal UTD A2-3 grades in the last prenatal scan in comparison with those with UTD A1 grades who showed complete resolution (100%) postnatally. Antenatal UTD classification may be useful in antenatal prediction and prognostication of postnatal severity, especially in high-risk cases (i.e. UTD A2-3).

DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

STUDY QUESTION: What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY: RPL is defined as loss of two or more pregnancies, affecting 1-2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION: In this case-control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION: In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS: Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Prenatal Diagnosis for Primary Immunodeficiency Disorders-An Overview of the Indian Scenario.

Prenatal Diagnosis (PND) forms an important part of primary preventive management for families having a child affected with primary immunodeficiency. Although individually sparse, collectively this group of genetic disorders represents a significant burden of disease. This paper discusses the prenatal services available for affected families at various centers across the country and the challenges and ethical considerations associated with genetic counseling. Mutation detection in the index case and analysis of chorionic villous sampling or amniocentesis remain the preferred procedures for PND and phenotypic analysis of cordocentesis sample is reserved for families with well-characterized index case seeking PND in the latter part of the second trimester of pregnancy. A total of 112 families were provided PND services in the last decade and the presence of an affected fetus was confirmed in 32 families. Post-test genetic counseling enabled the affected families to make an informed decision about the current pregnancy.

Prediction of preeclampsia using combination of biomarkers at 18-23 weeks of gestation: A nested case-control study.

OBJECTIVE: To evaluate the combination of plasma activated endothelial microparticles (CD62e), serum Copeptin (CPP) and placental growth factor (PlGF) levels at 18-23 weeks of gestation for prediction of preeclampsia (PE) in primigravid women. METHODS: This was a nested case-control study from a prospective cohort of 1115 primigravid women attending antenatal care clinic. Plasma levels of CD62e and serum Copeptin, PlGF levels were measured by flow cytometry and ELISA, respectively. Data were presented as median (Interquartile range) and biomarker levels were compared between patients and controls using Mann-Whitney Test. Using binary logistic regression, predictive potential of a combination of biomarkers for PE prediction was determined. RESULTS: Women who developed PE 41 (3.97%) showed significantly increased levels of plasma CD62e [799.33 (546.86-1249.29) versus 384.08 (245.03-576.00), p < 0.0001], serum Copeptin [303.42 (226.01-484.18) versus 207.24 (169.73-276.46), p < 0.0001] and reduced level of PlGF [238.38 (161.36-312.62) versus 947.21 (466.7-1428.56), p < 0.0001] compared to controls at 18-23 weeks of gestation. None of the marker showed statistically significant alteration in levels in fetal growth restriction (FGR) group 68 (6.58%) compared to controls. Using binary logistic regression analysis, AUC, Sensitivity, specificity, PLR, NLR, PPV, and NPV of combination of CD62e, Copeptin and PlGF for prediction of PE at 18-23 weeks of gestation was 0.969, 92.3%, 90.3%, 9.73, 0.08, 79.17%, and 96.94%, respectively. CONCLUSION: At 18-23 weeks, Combination of CD62e microparticles, copeptin, and PlGF levels can effectively identify women at risk of developing PE later in gestation.

Combination of copeptin, placental growth factor and total annexin V microparticles for prediction of preeclampsia at 10-14 weeks of gestation.

INTRODUCTION: Preeclampsia (PE) remains to be an enigmatic puzzle for clinicians and researchers perplexing them for decades. As delivery remains only choice of treatment, early prediction of PE will offer timely therapeutic invention and hence extensive research efforts have been put in identification of biomarkers which will facilitate early prediction of PE. METHODS: Serum levels of CPP, PlGF and plasma total annexin V MPs were assessed in women who subsequently developed PE (n = 33), IUGR (n = 81) and normal pregnancy outcome (n = 112) at 10-14 weeks of gestation. Comparison of biomarker levels between patients and control group was done using Mann Whitney test. Receiver operating curve (ROC) analysis and binary logistic regression analysis were used to evaluate predictive utility of combination of CPP, PlGF and total annexin V MPs for prediction of PE. RESULTS: Women who subsequently developed PE showed significantly elevated levels of total annexin V MPs [2766.04 (2086.88-3794) versus 1090.74 (631.91-2197.16)] and CPP [440.98 (365.12-488.92) versus 217.8 (171.13-308.98)] compared to controls. Serum PlGF levels were significantly reduced in women with PE 17.68 (12.66-22.32) compared to controls 105.22 (35.02-255.1). Using logistic regression analysis, the combination of CPP, PlGF and total annexin V MPs gave high predictive value with AUC of 0.970, 93.1% sensitivity, 90.7% specificity, 77.50% Positive predictive value, 98.10% Negative predictive value, 11.69 Positive likelihood ratio and 0.07 Negative likelihood ratio for PE prediction at 10-14 weeks. CONCLUSION: The combination of serum markers and plasma microparticles can be used for 10-14 weeks prediction and discrimination of PE from other pregnancy complications.

Lenz-Majewski syndrome: Report of a case with novel mutation in PTDSS1 gene.

Lenz-Majewski syndrome (LMS) is an extremely rare syndrome characterized by osteosclerosis, intellectual disability, characteristic facies and distinct craniofacial, dental, cutaneous and distal - limb anomalies. Recently, mutations in PTDSS1 gene have been identified as causative in six unrelated individuals. We report the seventh mutation proven case of LMS and provide a concise review of all known patients till date.

Promising prognostic markers of preeclampsia: new avenues in waiting.

Preeclampsia is a pregnancy related condition identified by hypertension and either proteinuria or end-organ dysfunction after 20(th) week of gestation and complicates 2-8% pregnancies worldwide. Enigmatic pathophysiology and multi-system involvement hinder accurate identification and clinical management of patients. Inadequate trophoblast invasion and subsequent inflammatory response have been implicated in the onset of PE. In absence of effective treatment of preeclampsia except delivery, recent research has been focused on identification of specific and sensitive biomarkers for early prediction of PE. Several angiogenic, anti-angiogenic, inflammatory, biophysical (mean arterial pressure and uterine artery Doppler) biomarkers, alone and in combination, have been proposed for prediction but limited predictive values have hindered their use in clinical settings. Current review summarizes some of relatively new biomarkers such as corin, copeptin, microparticles and miRNA, the prognostic efficiency of which are either analyzed in associated disorders or recently discovered.