GENType: all-in-one preimplantation genetic testing by pedigree haplotyping and copy number profiling suitable for third-party reproduction.

STUDY QUESTION: Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction? SUMMARY ANSWER: Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping. WHAT IS KNOWN ALREADY: Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting. STUDY DESIGN, SIZE, DURATION: This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods. MAIN RESULTS AND THE ROLE OF CHANCE: Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla. LIMITATIONS, REASONS FOR CAUTION: Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation. WIDER IMPLICATIONS OF THE FINDINGS: We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting. STUDY FUNDING/COMPETING INTEREST(S): L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Evaluation of weight-based prescription of antiretroviral therapy in children.

OBJECTIVES: The aim of the study was to investigate the extent of and factors associated with incorrect dosing of antiretroviral therapy (ART) in HIV-infected children in Harare, Zimbabwe. METHODS: All children aged 0-10 years and children aged 11-17 years who weighed < 35 kg and taking ART were recruited from the paediatric HIV clinic at Harare Hospital. Their current doses of ART drugs were compared against doses recommended by the national guidelines. RESULTS: Among 309 children recruited [55% male; median age 7 years (interquartile range (IQR) 5-10 years)], the median CD4 count was 899 cells/µL and the median duration of their current ART regimen was 11.2 months (IQR 4.9-17.1 months). Overall, 110 (35.6%) children were prescribed incorrect doses of at least one drug component within their ART regimen; 64 (20.7%) under-dosed and 49 (15.9%) over-dosed on at least one drug. Children receiving a higher than recommended dose of at least one drug were younger compared with correctly dosed children (median 6 versus 7 years, respectively; P = 0.001), had been on their current ART regimen for a shorter time (median 7.2 versus 13 months, respectively; P = 0.003) and were less likely to be receiving a three-drug fixed-dose combination (FDC; 42.9 versus 63.3%, respectively; P = 0.009). Those who were under-dosed were also less likely to be on a three-drug FDC (25 versus 63.3%, respectively; P < 0.001). CONCLUSIONS: Over a third of children were prescribed incorrect doses of ART. Children taking triple-drug FDCs were likely to be correctly dosed. Our study highlights the importance of weight monitoring at each clinical contact, training of health care providers on paediatric drug dosing and the need for wider availability of FDCs for children.

Renal Artery Diameter Is a Surrogate Marker for Kidney Volume in Living Kidney Donors.

Donor kidney volume (KV) is an increasingly important parameter evaluated before living kidney donation; however, KV measurements on computed tomographic (CT) scanning requires a manually intensive process of manual or semiautomatic segmentation of kidneys with interobserver variation. Renal artery diameter (RAD) is an easier marker to measure, and this study aims to investigate the relationship between donor RAD and KV. METHODS: A retrospective review of 77 patients who underwent living donor nephrectomy was conducted. Bilateral KVs were measured based on contrast-enhanced CT scan imaging, and renal artery maximum diameter was measured by direct visualization on the arterial phase of transverse CT sections. RESULTS: On regression analysis, there was a significant association between the right and left RADs and their ipsilateral KVs with a regression coefficient of 7.9 (95% CI, 1.3-14.5; P = .02) and 9.8 (95% CI, 3.3-16.3; P = .004), respectively. Mean total RAD correlated with total KV with a regression coefficient of 9.3 (95% CI, 3.8-14.7; P = .001) and weakly correlated with estimated glomerular filtration rate with a Pearson coefficient of .10. CONCLUSIONS: This study demonstrates that renal artery size is positively associated with KV and may be used as an easily measured surrogate marker for kidney size with its attended implications in living donor transplantation.

Outcome of Living Donor Transplant Kidneys With Multiple Arteries.

BACKGROUND: Living donor transplantation (LDT) using kidneys with multiple arteries (MA) has previously been reported to be associated with increased complications and poorer outcomes in recipients. The objective of this study was to investigate outcomes of LDT with MA at the National University Hospital of Singapore, an institution with modest kidney transplant volumes. METHODS: From 2007 to 2014, a total of 109 consecutive living donor kidney transplantations were performed. Of the nephrectomies, 91% were left sided. A total of 19 cases involved MA, of which 7 with small polar vessels (<2 mm) were ligated and 12 were revascularized. Baseline characteristics and outcomes were comparable between donor-recipient pairs with MA and single artery (SA). Both groups had equivalent induction and maintenance immunosuppressive regimens. RESULTS: Mean warm ischemia time (minutes) was similar for kidneys with MA and SA (4.3 ± 3.2 vs 3.9 ± 3.2, P = .38). Operative time (minutes) in the recipients was also equivalent (P > .05) for MA and SA (158 ± 39.2 and 145 ± 57.2, respectively). The MA kidney recipients had a lower estimated glomerular filtration rate (eGFR) on postoperative day 5 compared to SA (56.6 ± 24.2 vs 74.1 ± 35.9 mL/min/1.73 m(2), P = .058). However, eGFR at 1 year was the similar for both groups (64.9 ± 16.2 vs 66.4 ± 18.1 mL/min/1.73 m(2), respectively, P = .76). Delayed graft function rates were 5.6% and 6.6% for MA and SA, respectively (P = .9). There were no surgical complications for LDT recipients within the MA group. Patient and graft survival was 100% in the MA group compared with 98% in the SA group (P > .05). CONCLUSIONS: With current surgical techniques, LDT with MA can achieve equally good functional outcomes at 1 year as SA kidneys, with minimal surgical complications.

Incidence of complications after transrectal ultrasonography-guided biopsy of the prostate in a local tertiary institution.

INTRODUCTION: This study aimed to evaluate the risk of complications for patients who received periprosthetic nerve block (PPNB) with one percent lignocaine before transrectal ultrasonography (TRUS) biopsy of the prostate. METHODS: From 2008 to 2009, data on 526 consecutive patients who underwent prostate biopsy was prospectively recorded and analysed. 475 (90.3 percent) patients received PPNB with 10 ml of one percent lignocaine (Group 1), which was carried out under TRUS-guidance and prior to biopsy. 51 (9.7 percent) patients received diclofenac (100 mg) intramuscular injections or no analgesia (Group 2). Complications were defined as any adverse effects after biopsy. Serious complications were defined as those requiring hospitalisation or invasive/operative procedures for treatment. RESULTS: At baseline, both groups were comparable. The mean prostate-specific antigen level in Group 1 was higher than that in Group 2 (48.6 +/- 13.8 versus 19.0 +/- 4.3 ng/ml; p-value is 0.04). There was no perioperative mortality. Post-procedural complications were reported in 23.4 percent (n is 111) of patients in Group 1 and 25.5 percent (n is 13) in Group 2 (p-value is 0.27). Serious complications were reported in 2.5 percent (n is 12) and 7.1 percent (n is 3) of Group 1 and 2 patients (p-value is 0.10), respectively. Both univariable and logistic regression revealed age below 65 years and pre-procedure complaints of lower urinary tract symptoms as independent predictors for complications (p-values are 0.02 and 0.006, respectively). CONCLUSION: PPNB with one percent lignocaine is a safe analgesic procedure to perform in patients undergoing TRUS biopsy.

Prevalence of specific IGM due to toxoplasma, rubella, CMV and c.trachomatis infections during pregnancy.

One hundred and seventy five apparently normal asymptomatic pregnant women were studied prospectively and 247 women with different complications of pregnancy were screened at the time of delivery for infections like Chlamydia trachomatis, Toxoplasma, Rubella and cytomegalovirus (CMV). One hundred and forty two women with normal outcome of pregnancy served as controls. Specific IgM due to these agents were determined in the sera using commercial diagnostic kits. Results of the study showed that chlamydial infection was the most prevalent (29.8%) followed by Toxoplasmosis (13.1), Rubella (6.5%) and CMV (5.8%). Adverse outcome was seen among those seropositive for Chlamydia, Toxoplasmosis and Rubella. CMV showed no association with adverse outcome of pregnancy. Since Chlamydia and Toxoplasmosis are amenable to treatment with antibiotics, screening for these infections and appropriate treatment would improve outcome of pregnancy.

Effect of dose and formulation on iron tolerance in pregnancy.

BACKGROUND: The National Nutritional Anaemia Prophylaxis Programme (NNAPP) in India was launched in 1971. However, anaemia continues to be a major public health problem. Partial coverage of the population, inadequate dose of the iron supplement, defective absorption due to intestinal infestations and problems with formulation have been recognized as factors responsible for its failure. Therefore, the bioavailability of iron from different formulations containing 60 mg of elemental iron and of tablets with varying doses of elemental iron was undertaken. METHODS: One hundred and fifteen women were randomly allotted to receive different formulations and doses of iron and then undergo iron tolerance tests. They received ferrous sulphate tablets containing 60 mg, 120 mg and 180 mg of elemental iron; formulations containing 60 mg of elemental iron as pure ferrous sulphate salt, ferrous fumarate tablets, ferrous fumarate syrup, excipients added to pure ferrous sulphate salts, powdered ferrous sulphate tablets, iron tablets distributed by the NNAPP and pure ferrous salt in gelatin capsules. RESULTS: The data obtained from 32 subjects were excluded because of non-compliance, intolerance of the medication and lack of results of blood tests. Data from the remaining 83 subjects indicated that increasing the dose of elemental iron from 60 mg to 180 mg improved the bioavailability of iron, but was associated with unacceptable side-effects. Also, liquid formulations of iron had a better bioavailability, with ferrous fumarate syrup and gelatin capsules being the most superior. CONCLUSION: Providing the iron formulation with a high bioavailability should enable the NNAPP to be more successful in decreasing the prevalence of anaemia.

Interaction between vitamin A and iron: effects of supplements in pregnancy.

Very few reports are available on the vitamin A status of Indian pregnant women in the rural population. The present study was carried out to assess the vitamin A status and the effect of vitamin A and iron supplementation in pregnancy on serum vitamin A levels in the rural women. It was observed that serum vitamin A levels showed a marginal decrease with advancing gestation. Though satisfactory as per WHO guidelines, supplementation with 60 mg of iron prevented this decrease. A higher dose of iron (120 mg) actually resulted in a similar vitamin A status as seen in vitamin A supplemented women. If this preliminary study can be confirmed it would appear that interactions between vitamin A and iron in pregnancy have a positive effect on the vitamin A nutriture.

Vitamin A requirements assessed by plasma response to supplementation during pregnancy.

Plasma vitamin A responses were examined in 30 low income pregnant women before and after supplementation with vitamin A. In the groups of women who had an initial (at < 20 weeks) plasma vitamin A of less than 30 micrograms/dl retinol, typical changes representing a peak at mid gestation and a significant decline at term were observed. Supplementation with vitamin A tablets equivalent to 1800 micrograms of retinol daily for 12 weeks was able to raise the plasma vitamin A content at term. On the other hand, those women who had an initial plasma vitamin A concentration of more than 30 micrograms/dl did not show any appreciable changes even with vitamin A supplementation. Consistent beneficial effects of supplementation on maternal and cord vitamin A and maternal hemoglobin and progesterone were noted in our previous studies only when the supplementation lasted for 12 weeks and not after 10 weeks. Using these data with dietary intake obtained from other studies, an attempt was made to estimate the requirements of vitamin A during pregnancy. The approximate requirement of 780 retinol equivalents (RE) obtained, is in agreement with earlier recommendations of FAO and Indian Council of Medical Research (ICMR) Expert Committees, but is higher than the recent ICMR recommendations.

The relationship between maternal nutritional status and spontaneous abortion.

BACKGROUND: The incidence of pregnancy wastage is high among poor women. Though the aetiology of spontaneous abortion is multifactorial, nutritional deficiency is considered to be an important contributory factor. METHODS: A case-control study was conducted to assess the relationship between maternal vitamin status and spontaneous abortion. RESULTS: The incidence of anaemia (15%), and riboflavin (84%) and folate deficiency (24.5%) were similar among the cases and controls. Vitamin A levels were higher in the study group compared to those in controls. CONCLUSION: The results suggest that various nutrient deficiencies have no correlation with the occurrence of spontaneous abortion. The role of increased vitamin A levels needs to be studied further.

PIP: The association between maternal vitamin status and spontaneous abortion was investigated in a case-control study conducted at the Government Maternity Hospital in Hyderabad, India. 115 women with first- or second-trimester spontaneous abortion comprised the study group, while another 115 pregnant women matched for age, parity, and gestational age, attending the outpatient department of the same hospital, served as controls. Both serum levels of riboflavin and folic acid and the prevalence of deficiencies of these two vitamins (84% and 25%, respectively) were similar between the two groups. Serum vitamin A levels were significantly higher (p 0.01) among cases than controls (53.6 and 44.3 mcg/dl, respectively), with a progressive increase among cases with increasing gestational age. The incidence of anemia (hemoglobin level under 11 gm/dl) was low (11%) in both groups. Although the role of increased vitamin A levels requires further investigation, these results suggest that various nutrient deficiencies are unrelated to the occurrence of spontaneous abortion.

Antithrombin III levels in pregnancy induced hypertension.

BACKGROUND: Antithrombin III deficiency is common as well as severe in both consumptive coagulopathy and conditions such as pregnancy induced hypertension. We determined antithrombin III levels in women suffering from pregnancy induced hypertension to determine its usefulness in assessing severity of the disease and outcome. METHODS: Forty-five pregnant women with mild and severe forms of pregnancy induced hypertension and 18 women with normal pregnancies matched for gestational age formed the study population. Fasting blood samples were collected and the plasma separated. Antithrombin III levels were estimated by the kinetic Berichrom antithrombin III method. RESULTS: The mean (SD) antithrombin III levels [0.76 (0.233) IU/ml] were significantly lower in pregnancy induced hypertension compared to the control group [0.97 (0.234) IU/ml]. Low antithrombin III and high diastolic blood pressure (> 110 mmHg) were related to poor pregnancy outcome in primigravidae. CONCLUSION: Mean antithrombin III levels were lower in pregnancy induced hypertension compared to a control group of women especially those who were primigravidae and had a diastolic blood pressure > 110 mmHg. Since low antithrombin III levels and a high diastolic blood pressure result in adverse pregnancy outcome monitoring of antithrombin III levels in pregnancy induced hypertension may help in assessing foetal jeopardy.

Iron nutritional status of adolescent girls from rural area and urban slum.

Iron nutritional status of adolescent girls belonging to an urban slum and rural areas was assessed by measuring serum ferritin levels. Overall anemia was observed in 25% of the girls irrespective of their urban rural residence. A higher percentage of rural girls (37.5%) especially below the age of 12 years showed evidence of anemia. Thereafter, the prevalence was similar in both urban and rural girls who had not attained menarche. With increasing age, urban girls who had attained menarche showed an increase in the prevalence of anemia. The prevalence of iron deficiency (serum ferritin < 12 micrograms/dl) showed a progressive increase from 60% at < 12 to 28% at > 14 yrs especially in the girls not attained menarche in the girls not attained menarche in the rural area. Overall iron deficiency was of much higher order in the rural girls irrespective of the menarcheal status. Distribution of iron/folate tablets to cover girl population may go a long way to correct the anemia and iron deficiency in the vulnerable groups.

Seasonal trends in the occurrence of eclampsia.

BACKGROUND: The occurrence of eclampsia may be related to season and altitude. METHODS: We studied the influence of environmental factors such as range of temperature and relative humidity on the occurrence of eclampsia by collecting data on consecutive cases of eclampsia admitted to the Government Maternity Hospital, Hyderabad, from September 1987 to August 1988. The mean range of temperature and relative humidity were calculated for each month. The data on eclampsia and adverse outcome in terms of still-births were analysed in relation to variations in temperature and humidity. RESULTS: The results support the speculated relationship between increasing humidity and a lower temperature range and the increased incidence of eclampsia. Still-births due to eclampsia also showed a positive correlation with relative humidity. CONCLUSION: A better understanding of the relationship of weather to eclampsia may provide further insight into the poorly understood pathophysiology of the disease.

Comparative evaluation of calf, thigh and arm circumferences in detecting low birth weight infants--Part II.

In an earlier study usefulness and validity of calf circumference in the identification of low birth weight (LBW) infants was reported. To evolve a simple indicator in identifying LBW in community, comparative evaluation of three simple measurements, i.e., circumferences of calf (CC), thigh (TC) and arm (AC) was done with respect to their sensitivity and specificity. Though all the three measurements showed a high degree of correlation with the birth weight, calf circumference tended to be most sensitive in identifying almost 95% of LBW infants. Having established the superiority of CC, a two color tape demarcating LBW zone from normal birth weight using 10 cm CC as cut off point was tested by two independent investigators and two ANMs. It was observed that only 5% of cases were misclassified either as LBW or normal weight by the tape with hardly any inter individual variation. Measurement of calf circumference being simple and easy even in the hands of paramedics, it would be used as indicator of LBW and neonatal mortality in the community.

Hyperferritinemia in pregnancy induced hypertension and eclampsia.

A study of serum iron and ferritin levels in Indian women with pregnancy induced hypertension (PIH) (mild/severe) and eclampsia compared with controls of similar gestational ages, revealed that mean serum iron was elevated slightly in PIH and significantly in eclampsia as compared to controls. Mean ferritin levels were significantly elevated both in PIH and eclampsia as compared to controls indicating that ferritin measurement in PIH and eclampsia would not reflect iron nutritional status. Lack of significant changes in liver enzymes and in hemoconcentration in PIH and eclampsia indicate that liver damage and altered hemodynamics only play a partial role in causing hyperferritinemia. Possible role of placental ferritin, being released as a result of placental damage in these conditions seems to be an area needing further research.

Iron nutritional status of preschool children.

Low hemoglobin and low MCHC levels were indicative of high incidence of iron deficiency in preschool children. The extent of iron deficiency as assessed by serum ferritin and free erythrocyte protoporphyrin showed a different trend. While FEP levels were highly suggestive of extensive iron deficiency (in 40-45% of children below the age of 5 years), low serum ferritin was seen in only 16-20% of children. The discrepant finding of high serum ferritin, and high erythrocyte protoporphyrin despite low MCHC in the present study, possibly reflects iron deficiency status along with chronic infection resulting in hyperferritinemia and hyperprotoporphyrinemia. It may be also due to associated folate deficiency resulting in non utilization of iron leading to the elevated levels of protoporphyrin.