Prenatal diagnosis (PND) is an effective measure to prevent births with transfusion-dependent β-thalassemia (TDT) and other severe hemoglobinopathies. However, multicentric consolidated data from high-volume Indian referral laboratories is limited. The authors collated a multi-center experience of invasive PND for β-thalassemia and the major hemoglobinopathies, including center-wise volumes, affected-fetus rates, trends in primigravida referrals, and diagnostic challenges from seven Indian tertiary-care referral centers with a minimum of 10 y PND services. Retrospective data were contributed by SGRH-New Delhi, ICMR-NIIH-Mumbai, PGIMER-Chandigarh, AIIMS-New Delhi, NIMS-CDFD-Hyderabad, CMC-Vellore and SGPGI-Lucknow. Carrier evaluation was based on blood counts and Hb-HPLC. Fetal diagnosis utilized chorionic villus sampling or amniocentesis with molecular testing by ARMS-PCR, reverse dot-blot and/or Sanger sequencing as per local practices. Descriptive analyses summarized volumes, proportions of fetuses with biallelic HBB variants, and primigravida referral trends. Across centers, 6,780 PND procedures were performed over 2015–2024, with ICMR-NIIH and SGRH contributing 58.2

Abstract not available Bangladesh J Obstet Gynaecol, 2019; Vol. 34(1): 43-47

Small supernumerary marker chromosomes (sSMCs) are defined as structurally abnormal chromosomes that cannot be identified or characterized by conventional karyotype analysis and are generally equal in size or smaller than chromosome 20. Here, we present the molecular characterization of an sSMCs derived from chromosome 15 in prenatal diagnosis in a 38-years-old female

Background & objectives: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder with a wide range of clinical manifestations. The disease is attributed to mutations in CYP21A2 gene encoding 21-hydroxylase enzyme. In view of severe phenotype in salt-losing cases, issues related to genital ambiguity in girls and precocity in boys, most families opt for prenatal testing and termination of affected foetus. CAH can be diagnosed in utero through direct molecular analysis of CYP21A2 gene, using DNA extracted from foetal tissues or cells obtained from chorionic villus sampling or amniocentesis. The objective of this study was to evaluate the feasibility and accuracy of prenatal diagnosis (PND) using sequencing and multiplex ligation probe amplification (MLPA) methods in families at risk for CAH. Methods: Fifteen pregnant women at risk of having an affected offspring with CAH were included in this study. Ten families had previous affected children with salt-wasting/simple virilising form of CAH and five families did not have live children but had a high index of suspicion for CAH in previous children based on history or records. Mutation analysis was carried out by Sanger sequencing and MLPA method. Results: Seven different mutations were identified in 15 families. Deletions and I2g mutation were the most common. Of the 15 foetuses analyzed, nine were unaffected while six were affected. Unaffected foetuses were delivered, they were clinically normal and their genotype was found to be concordant to the prenatal report. All except two families reported in the second trimester. None of the couples opted for prenatal treatment. Interpretation & conclusions: Our preliminary findings show that PND by direct mutation analysis along with MLPA is a feasible strategy that can be offered to families at risk.

Background & objectives: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder with a wide range of clinical manifestations. The disease is attributed to mutations in CYP21A2 gene encoding 21-hydroxylase enzyme. In view of severe phenotype in salt-losing cases, issues related to genital ambiguity in girls and precocity in boys, most families opt for prenatal testing and termination of affected foetus. CAH can be diagnosed in utero through direct molecular analysis of CYP21A2 gene, using DNA extracted from foetal tissues or cells obtained from chorionic villus sampling or amniocentesis. The objective of this study was to evaluate the feasibility and accuracy of prenatal diagnosis (PND) using sequencing and multiplex ligation probe amplification (MLPA) methods in families at risk for CAH.Methods: Fifteen pregnant women at risk of having an affected offspring with CAH were included in this study. Ten families had previous affected children with salt-wasting/simple virilising form of CAH and five families did not have live children but had a high index of suspicion for CAH in previous children based on history or records. Mutation analysis was carried out by Sanger sequencing and MLPA method.Results: Seven different mutations were identified in 15 families. Deletions and I2g mutation were the most common. Of the 15 foetuses analyzed, nine were unaffected while six were affected. Unaffected foetuses were delivered, they were clinically normal and their genotype was found to be concordant to the prenatal report. All except two families reported in the second trimester. None of the couples opted for prenatal treatment.Interpretation & conclusions: Our preliminary findings show that PND by direct mutation analysis along with MLPA is a feasible strategy that can be offered to families at risk.

INTRODUCTION:It is estimated that 3-10% of infants are growth restricted. Growth disturbances may have long-term issues. Doppler allows insight into the fetal response to intrauterine stress.OBJECTIVE:The aim of this study was to detect fetal compromise in intrauterine growth-restricted (IUGR) fetuses by means of biophysical profile (BPP) vis-à-vis Doppler velocimetry studies of the fetal umbilical artery, and to find out which of the two is a better and earlier predictor of fetal compromise.METHODS:A prospective study was conducted on a total of 50 singleton pregnancies with IUGR between 28 and 42 weeks of gestation. Study patients were managed expectantly with nonstress testing and amniotic fluid assessment, BPP and Doppler velocimetry studies of the fetal umbilical artery.RESULTS:Fetal outcome was poor in 5/50 (10%) of the fetuses, defined as presence of all of the following: poor Apgar test score, neonatal intensive care unit stay, necrotizing enterocolitis, and low birth weight. Of the four with abnormal BPP, 50% had poor fetal outcomes. Out of 46 with normal BPP, 6.5% had poor fetal outcomes.CONCLUSION:Inference drawn from the study is that the Doppler technology provides us the opportunity for repetitive noninvasive hemodynamic monitoring in IUGR pregnancies.

Aims: Study on prognostic significance of antenatal ultrasonography and renin angiotensin system activation in predicting disease severity in posterior urethral valves. Materials and Methods: Antenatally diagnosed hydronephrosis patients were included. Postnatally, they were divided into two groups, posterior urethral valve (PUV) and non-PUV. The studied parameters were: Gestational age at detection, surgical intervention, ultrasound findings, cord blood and follow up plasma renin activity (PRA) values, vesico-ureteric reflux (VUR), renal scars, and glomerular filtration rate (GFR). Results: A total of 25 patients were included, 10 PUV and 15 non-PUV. All infants with PUV underwent primary valve incision. GFR was less than 60 ml/min/1.73 m 2 body surface area in 4 patients at last follow-up. Keyhole sign, oligoamnios, absent bladder cycling, and cortical cysts were not consistent findings on antenatal ultrasound in PUV. Cord blood PRA was significantly higher (P < 0.0001) in PUV compared to non-PUV patients. Gestational age at detection of hydronephrosis, cortical cysts, bladder wall thickness, and amniotic fluid index were not significantly correlated with GFR while PRA could differentiate between poor and better prognosis cases with PUV. Conclusions: Ultrasound was neither uniformly useful in diagnosing PUV antenatally, nor differentiating it from cases with non-PUV hydronephrosis. In congenital hydronephrosis, cord blood PRA was significantly higher in cases with PUV compared to non-PUV cases and fell significantly after valve ablation. Cord blood PRA could distinguish between poor and better prognosis cases with PUV.

Background Chromosomal Microarray (CMA) and Multiplex Ligationdependent Probe Amplification (MLPA) are relatively newer techniques for detecting cryptic copy number variations (CNVs). Here we are presenting the data of eight families where CMA and MLPA were used for prenatal diagnosis (PND) in view of their previous child with Intellectual disability/ developmental delay (ID/DD) and normal karyotype.

OBJECTIVES:To study the usefulness of direct fetal intravenous immunoglobulin (IVIG) infusion along with intrauterine transfusion (IUT) in the management of severe fetal anemia in rhesus (Rh) alloimmunized pregnancies. METHODS:Thirty-four consecutive Rh-isoimmunized pregnant women who required serial IUTs received either blood alone (control group, n = 16) or IVIG and blood (study group, n = 18). Pregnancies were followed up to delivery, and fetal outcome was recorded. The rate of fall of hematocrit was measured and compared between the two groups. RESULTS:There was a slower rate of fall of hematocrit in the study group (IUT and IVIG) compared to the control group (only IUT). The mean rate of fall was 0.72 ± 0.54% per day in the study group while it was 1.29 ± 0.95% per day in the control group (p = 0.005). CONCLUSION:Fall of fetal hematocrit was reduced in the study group. The results of this pilot study can be used to time the next transfusion in patients receiving IVIG along with IUT (taking the rate of fall as 0.70%). This may eventually result in decreasing the number of transfusions per fetus.

Standard semen parameters are poor predictors of fertility potential. To date, apart from, paternal karyotyping sperm factors are not evaluated in recurrent pregnancy loss (RPL), only recent studies have emphasized the role of sperm factors in early embryonic development as sperm transcribes genes critical for early embryonic development. Sperm DNA integrity is useful diagnostic and prognostic marker and has clinical implications in idiopathic recurrent pregnancy loss (iRPL) following spontaneous conception. The aim of this study was to assess DNA integrity in cases experiencing iRPL following spontaneous conception.Semen samples from 45 patients and 20 controls were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was used to measure DNA fragmentation index (DFI).By applying receiver operating curve (ROC) analysis, sperm DFI of approximately 26 % was found in male partner of couples experiencing iRPL.Our data indicate that sperm from men with a history of iRPL have a higher percentage of DNA damage as compared to control group, and this can explain pregnancy loss in these patients. Men with higher DFI are infertile whereas men with lower DFI (26 %) are able to conceive but experience recurrent pregnancy loss. Thus it is important to evaluate sperm DFI in couples experiencing iRPL to understand exact aetiology of RPL and determine prognosis and management.

Fetal umbilical cord blood sampling is now being performed worldwide, using an ultrasound guided technique, for prenatal diagnosis in pregnancies at high risk for several congenital and genetic defects in the fetus. Awareness of feasibility of the procedure and indications for the same should be known to every obstetrician.

Norrie Disease (ND) is a rare X-linked recessive disorder characterised by congenital blindness due to severe retinal dysgenesis. Hearing loss and intellectual disability is present in 30-50 % cases. ND is caused by mutations in the NDP gene, located at Xp11.3. The authors describe mutation analysis of a proband with ND and subsequently prenatal diagnosis. Sequence analysis of the NDP gene revealed a hemizygous missense mutation arginine to serine in codon 41 (p.Arg41Ser) in the affected child. Mother was carrier for the mutation. In a subsequent di-chorionic di-amniotic pregnancy, the authors performed prenatal diagnosis by mutation analysis on chorionic villi sample at 11 wk of gestation. The fetuses were unaffected. This is a first mutation report and prenatal diagnosis of a familial case of Norrie disease from India. The importance of genetic testing of Norrie disease for confirmation, carrier testing, prenatal diagnosis and genetic counseling is emphasized.

Sir, Recurrent miscarriage (RM, recurrent spontaneous abortion - RSA) is defined as the loss of three or more consecutive pregnancies. Classic factors associated with RM include parental chromosome translocations, uterine malformations, endocrine and autoimmune factors. Even after extensive investigation approximately 40 per cent of RM cases remain unexplained and are classified as unexplained RM (URM)1. Female factor is believed to play a major role in recurrent miscarriages, but the role of male factor has only recently been realised2–5. Sperm DNA integrity plays a vital role in embryogenesis and foetal well being and sperm DNA damage may lead to pre- and post-implantation losses6, early pregnancy loss and congenital malformations. This pilot study was planned to analyse sperm factor in unexplained RM and determine cut-off value of DNA damage in cases of URM. After approval of the study protocol from the ethics committee of the institute [All India Institute of Medical Sciences (AIIMS), New Delhi, India], a total of 25 idiopathic RM cases and 20 control men were enrolled for the study from February 2010 to May 2011. These cases were referred from department of Obstetrics and Gynaecology, AIIMS, a tertiary research and referral hospital after detailed work up for female factor by the clinician. Selection criteria included a history of at least three prior pregnancy losses at <20 wk of gestation, with all pregnancies fathered by the same partner. All known causes for RM were ruled out and idiopathic cases were recruited. In brief, female partners of the couples had normal haemogram (Hb, TLC, DLC, ESR, platelets, PT, CT) with normal ovarian function (normal ultrasonography of ovary for PCOS), a normal uterus confirmed by vaginal ultrasound and/or hysterosalpingography/hysteroscopy, and had normal endocrinological parameters (level of T3, T4, TSH, FSH, LH, PRL), with normal perspeculum and pervaginal examination. Other causes for RM like thrombophilia both inherited (Proglob C) and acquired (anticardiolipin-IgG and IgM) were also screened and male partners having normal semen parameters only were included in the study. Also the karyotype of both partners were analysed to rule out any chromosomal abnormality. Male partners of couples who have recently fathered a child (not older than 2 yr) were enrolled as fertile controls. Informed consent was obtained from both the cases and controls. Semen samples were analysed as per the WHO guidelines7. An aliquot of 100 μl of the raw semen was stored at -80°C till sperm chromatin structure assay (SCSA) analysis was done. The SCSA was performed as per the protocol described by Evenson & Jost8. Frozen aliquots of semen were placed in 37°C water bath until just thawed, after which samples were diluted with TNE buffer (pH 7.4, working solution, 0.01 M Tris-HCl, 0.15 M NaCl, 1 mM EDTA) to 1-2×106 sperm cells per ml; 0.20 ml aliquots of diluted samples were mixed with 0.40 ml of acid-detergent solution (0.08 M HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2). After 30 seconds, the cells were stained by adding 1.2 ml acridine orange (AO) solution containing 6 μg AO (chromatographically purified; Polysciences Inc, USA) per ml buffer (0.037 M citric acid, 0.126 M Na2HPO4, 0.0011 M EDTA disodium, 0.15 M NaCl, pH 6.0). After complete analysis of the sample, X-mean (red fluorescence) and Y-mean (green fluorescence) values were recorded manually after selecting gate for sperm cells using FlowJo software (Oregon, USA). Extent of DNA denaturation (damage) was expressed in terms of the DNA fragmentation index (DFI), which is the ratio of red to total (red plus green) fluorescence, i.e. the level of denatured DNA over the total DNA. The percentage of high DNA stainability cells (HDS) was also recorded in each sample manually from the graph plot. HDS represents another distinct population in semen that characterizes immature spermatozoa with incomplete chromatin condensation. The DFI values of the same patient and controls were analysed in duplicate by a single operator and mean value was recorded. Statistical analyses were performed using MedCalc trial version for Windows (MedCalc Software, Mariakerke, Belgium). Data are represented as mean ± standard deviation. The data was analysed by using Student's t-test to compare DFI of cases and controls (29.35±4.93 vs 21.0±4.1, P<0.001). Receiver operating characteristic (ROC) curve analysis was applied to find the cut-off value of DFI to discriminate patient from control. The mean DFI of cases was 29.35 ± 4.93 with a range of 19.45 to 35.75. The median DFI of cases was significantly (P<0.05) higher when compared to controls. However, in controls, the lowest DFI was 18.34 and the highest was 28.67. Using ROC curve analysis, a threshold value of 23.54 was obtained to discriminate from control group. The area under the curve was 0.975 (P<0.0001; 95% CI, 0.845-1.000), with 97 per cent sensitivity (95% CI, 84.7 - 99.9) and 100 per cent specificity (95% CI, 79.4 - 100.0). Sperm DNA is highly compact, organized crystalline structure, the size of which is 1/6 to 1/20 that of somatic cells. This genome is organized into a central protamine bound fraction (toroid) while the peripheral histone bound fraction retains the nucleosomal structure. It is this peripheral loosely bound chromatin which has genes of critical developmental importance and maintains genomic imprints. This 15 per cent peripherally located histone bound chromatin is highly susceptible to environmental insult especially oxidative stress. We have shown in a previous study that sperm reactive oxygen species (ROS) levels are elevated in RM cases2. We have also reported that DFI in infertile cases is approximately 30 per cent3. Thus it is possible that cases with 30 per cent DFI and above are infertile where as cases with lower DFI (24%) are able to initiate pregnancy but have sufficient DNA damage which adversely affects embryogenesis and results in recurrent early pregnancy loss. Oxidative stress results in generation of ethenonucleoside9, an oxidative product which inhibits oocyte nucleotide excision repair. Thus sperm DNA damage results in impaired embryonic development and recurrent miscarriages. The role of sperm factor in recurrent assisted and spontaneous conception loss is now being realised. Earlier studies have suggested that pregnancy is unlikely to occur when sperm nuclear DNA fragmentation index values are above certain threshold value3,10,11. However, a cut-off value for DNA damage in unexplained recurrent miscarriages cases is still not documented. In our earlier study, we have established a cut-off value in infertile men unable to conceive even after 6 years of marriage3. This preliminary study on URM following spontaneous conception has established cut-off value of sperm DFI (24%) in URM cases. This will help to understand aetiology in a large numbers of couples experiencing URM. High level of DNA damage in cases of RM in our study is in accordance with previous studies11,12. In the study by Liu et al12 DFI of 37.5 per cent of the subjects was over 30 per cent as compared to the control group, where only 25.8 per cent of the subjects had DFI over 30 per cent. The chief cause of loss of DNA integrity was oxidative stress. Smoking has been shown to increase seminal leukocyte concentration by 48 per cent and cause a 107 per cent increase in seminal ROS levels. Peake and his associates14 documented that extremes of exercise (too little and rigorous exercise) are linked with oxidative stress. Another group reported that obesity produces oxidative stress as adipose tissue releases proinflammatiry cytokines that increases leukocyte production of ROS15. Psychological stress results in decline in semen quality16. Psychological stress has also been reported to result in poor sperm quality which was mediated by supraphysiological ROS levels and low antioxidant levels17,18. Thus minimizing lifestyle triggers of oxidative stress can prevent oxidative stress induced DNA damage19,20. The results of this study show that sperm DNA damage may be a cause of URM, and paternal factors may play a critical role in embryonic development.

Objective: To study patient perception of pain and anxiety before and after amniocentesis (AC) and transabdominal chorionic villus sampling (CVS) and the clinical correlates of pain and anxiety. Methods: 92 women underwent AC and 78 CVS. Visual analog scale was used to quantify pain and anxiety, immediately before and after the procedure. Factors which could affect pain and anxiety were noted. Results: The pain and anxiety anticipated before the procedures were significantly less than actually perceived. The pre-procedure anxiety did not correlate with post-procedure pain. There was no correlation between anticipated pain or anxiety and age, parity, education, socioeconomic status, and history of procedure in previous pregnancy in both the AC and CVS groups. Post-procedure pain did not correlate with age, parity, education, socioeconomic status, abdominal scar, placental location, number of needle insertion, repeat procedure or abdominal wall thickness in either group. However, on multiple linear regression the overall post-procedure pain was associated with the number of needle insertions. Conclusion: Though pre-procedure pain and anxiety levels are high, most patient experience less pain and anxiety after the procedure. Copyright (C) 2012 S. Karger AG, Basel

Heterotopic pregnancy, the presence of two gestational sacs simultaneously, is a rare event but with the advent of Assisted Reproductive Technology, it is now an increasingly common complication. The reported incidence of a heterotopic pregnancy in a spontaneous cycle is quoted as 1 in 30,000. We report the case of a 38-year-old primigravida who was referred to our center at 11 + 2 weeks gestation with a diagnosis of heterotopic pregnancy for further management. A non-surgical intervention comprising of transvaginal ultrasound-guided potassium chloride and methotrexate into the cervical pregnancy resulted in a successful outcome. As an obstetrician, a high index of clinical suspicion and an early scan is mandatory to make a diagnosis of a heterotopic pregnancy and manage accordingly.

Sperm nuclear proteins, the protamines (PRM) and transition nuclear proteins (TNP) play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in PRM and TNP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyze PRM and TNP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. Protamine and TNP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry-sperm chromatin structure assay (FC-SCSA) was applied to measure the DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO [1999] guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of PRM2 gene (c.443C > A) and a novel insertion of T (c.396_397InsT) at the 3′ UTR region of TNP1 were detected. None of the nucleotide changes were observed with increased risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p < 0.0001) higher in infertile men compared to controls (36.31 ± 7.25 vs. 26.49 ± 2.78) irrespective of nucleotide changes, no such difference was observed between 100 infertile men or pooled population of 200 with and without mutations. However it was observed that two cases with novel nucleotide changes PRM2 c.443C > A and TNP1 c.396_397InsT had higher DFI value of 34.82% and 43.85%, respectively. In conclusion, our pilot study for the first time in the Indian population revealed two rare novel mutations in sperm nuclear protein genes that are perhaps associated with higher sperm DNA fragmentation.

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