Single Reference Laboratory Research Articles

P-539 Chaotic blastocysts in preimplantation genetic testing for aneuploidies: prevalence, characterization and re-biopsy results

Abstract Study question Are we excluding blastocysts with favorable reproductive potential when embryos are designated as chaotic by Preimplantation Genetic Testing for Aneuploidies (PGT-A)? Summary answer The re-biopsy of chaotic blastocysts can rescue embryos with favorable reproductive potential only when there are ≥5 chromosomes displaying intermediate copy number values (CNV). What is known already An embryo is deemed as chaotic when multiple aneuploidies (5, 6 or more) are detected in the biopsied cells of the trophectoderm. Such embryos are classified as aneuploid, therefore their transfer is not recommended. There is evidence that chaotic embryos may result from the biopsy of apoptotic cells, cellular fragments and, more broadly, DNA degraded by multiple factors. Studies involving the re-biopsy and re-testing of a limited number of chaotic embryos have found a relevant percentage of euploid results. Moreover, there has been published a case report of a healthy live birth after the transfer of a chaotic embryo. Study design, size, duration This is a retrospective study of 1,442 cycles of PGT-A performed in twenty-two in vitro fertilization (IVF) clinics from November 2017 to December 2023. A total of 5,801 blastocysts were biopsied and analyzed in a single reference laboratory using Next-Generation Sequencing. Ninety-three chaotic blastocysts donated from patients who provided informed consent were thawed for re-biopsy in two IVF clinics. The subsequent re-analysis was performed in the same PGT laboratory using the same methodology. Participants/materials, setting, methods This study assessed the prevalence of chaotic embryos in all included cycles. Chaotic embryos were considered those with ≥5 chromosomes showing CNV below 1.75 or above 2.25. We sub-classified these embryos into pure chaotic (≥5 uniform aneuploidies), mixed chaotic (≥5 alterations considering uniform aneuploidies and chromosomes with intermediate CNV) and mosaic chaotic (≥5 chromosomes with intermediate CNV). Re-biopsies of 69 chaotic blastocysts were compared to the initial PGT-A results. Main results and the role of chance The prevalence of chaotic embryos was 4.3% (251/5,801); 13.5% (n = 34) pure, 53.0% (n = 133) mixed and 33.5% (n = 84) mosaic. After thawing 93 embryos, the survival rate was 74.2%. The 69 embryos that survived underwent re-biopsy yielding the following PGT-A results: 24.6% (n = 17) euploid, 37.7% (n = 26) chaotic, 26.1% (n = 18) aneuploid non-chaotic, 7.2% (n = 5) mosaic and 4.4% (n = 3) non informative. Nevertheless, outcomes varied substantially when the analysis was conducted based on the sub-category of chaotic embryos. In the pure category (n = 11), 72.7% (n = 8) showed identical results to the initial biopsy while 27.3% (n = 3) were aneuploid non-chaotic confirming at least one aneuploidy identified at the initial biopsy. Among the mixed category (n = 24), 54.2% (n = 13) were also mixed chaotic, and 45.8% (n = 11) were aneuploidy non-chaotic. All re-biopsies in this category also manifested at least one common aneuploidy with the initial biopsy or its opposite (trisomy/monosomy). In the mosaic category (n = 31), 54.8% (n = 17) were euploid, 16.1% (n = 5) were mosaic chaotic, 16.1% (n = 5) were mosaic non-chaotic and 12.9% (n = 4) were aneuploid non-chaotic. The concordance with the initial biopsy in terms of aneuploid result was 100% in the pure and mixed chaotic categories. However, over half of the embryos initially categorized as mosaic chaotic were, in fact, euploid. Limitations, reasons for caution Implantation, miscarriage and live birth rates are not explored. These outcomes are crucial for understanding the clinical significance of euploid embryos initially deemed as chaotic. The survival rate of chaotic embryos should be considered as it is lower than the survival rate of vitrified blastocysts reported in most IVF laboratories. Wider implications of the findings Previous research has suggested that re-biopsy can rescue embryos from PGT-A cycles given that a chaotic result has reduced predictive value. However, our current study reveals that not all chaotic embryos are suitable for re-biopsy, as 100% of pure and mixed chaotic blastocysts were aneuploid when re-tested. Trial registration number Not applicable

Co-occurrence of germline pathogenic variants in a Brazilian cohort of patients with breast cancer.

e22532 Background: The estimated prevalence of germline pathogenic variants (PV) or likely pathogenic variants (LPV) in high and moderate penetrance genes is around 7-10% among women with breast cancer (BC). The emergence of next-generation sequencing (NGS) technology allowed multi-gene panel testing (MGPT) analysis at a lower cost. However, the detection of incidental findings with unknown significance (VUS) and challenges with variant interpretation increase substantially with MGPT. The effect of co-existing PV/LPV in two or more genes is still understudied. Here, we present the prevalence of concurrent germline LPV/PV in a cohort of Brazilian women with BC who underwent a MGPT in a single reference laboratory. Methods: Retrospective observational study using a laboratory cohort (Oncoclínicas Precision Medicine). This study includes women with BC who performed a germline MGPT (able to call SNVs, indels and CNVs) from 2019 to 2023. All patients were tested in a single reference laboratory (OC Precision Medicine) and originated from several Brazilian centers within the Oncoclínicas network. Two distinct germline NGS assays were used: breast and ovarian cancer (BOC) panel (36 genes) and a pan-cancer (PC) panel (105 genes). Results: In the overall cohort, 2,651 women with BC were included in this analysis. The median age at genetic testing was 51 years. 1,867 pts (70.4%) underwent a PC panel testing and the remaining 784 pts (29.6%) a BOC panel.Overall, 314 (12%) pts carrier of LP/PV in high- and/or moderate-penetrance genes. Two or more heterozygous LP/PV were found in 35 pts (1.32%), including 8 pts (1.02%) in the BOC panel and 27 pts (1.44%) in the PC panel subgroups. There were no differences in age of onset of BC in single vs co-occurring double mutants. The most frequent co-existing PV/LPV were found in BRCA2 (11) / BRCA1 (8) / PALB2 (4) / TP53 (3) and MUTYH (7) / CHEK2 (3) in the high and moderate penetrance genes, respectively. Six pts (0.23%) had a double heterozygous involving high and/or moderate penetrance genes. Interestingly, there was a case with four heterozygous variants in TP53, MUTYH, MUTYH and APC. Conclusions: In times of rapid incorporation of MGPT in the clinical practice, our real-world data showed that a small proportion of BC patients could be carriers of two or more germline heterozygous LP/PV in cancer genes. However, this represents a challenging scenario for patient and family management, especially in a co-occurrence of high and moderate penetrance genes.

Retrospective analysis of change in frequency of STK11 mutation in lung adenocarcinomas over a 10-year period.

8032 Background: Previous studies have linked the presence of inactivating mutations in the tumor suppressor gene STK11 with reduced efficacy of immune checkpoint inhibitor (ICI) treatments in lung adenocarcinoma (LA). We queried whether there has been a change in the STK11 mutation frequency over the last 10-year period that could potentially reflect the emergence of additional acquired resistance mutations. Methods: Over a 10-year period from 2013 through 2022, 109,763 clinically advanced LA underwent hybrid capture based comprehensive genomic profiling (CGP) to assess all classes of genomic alterations (GA) at a single reference laboratory (Foundation Medicine). MSI-high status and tumor mutational burden (TMB) were determined from the sequencing data. PD-L1 status was measured by immunohistochemistry (IHC) using the Dako 22C3 kit with TPS scoring. Statistical comparisons utilized the 2-tailed Chi Square method with the Yates’ correction. Results: The frequency of STK11 inactivating GA in all cases of LA significantly increased during the study period from 2013 to 2022 (16.1% vs 20.1%; p=0.0005). This increase appeared to be predominantly in KRAS wild type LA (10.1% vs 15.5%; p<0.0001). The STK11 GA frequency slightly declined in the KRAS mutated LA over the 10-year period (29.9% vs o 27.0%, P=0.25). During the same time period, the KEAP1 GA frequency increased from 12.6% to 14.9% (p= 0.076) and the frequency of combined STK11 and KEAP1 GA increased from 4.8% to 8.0% (p=.0006). The frequencies of MSI-high status, TMB and PD-L1 expression did not significantly change over the observation period. Conclusions: The continuously increasing approved indications for ICI-based treatments for both early stage and clinically advanced LA appears to be accompanied by a significant increase in the frequency of inactivating STK11 GA predominantly in the KRAS mutation negative LA population. This inactivation is associated with an attenuated response rate and progression-free survival in this population. Given the development of novel therapies focused on potential restoration of STK11 function by HDAC/CoREST inhibition, further study of the STK11 GA distribution in LA appears warranted. [Table: see text]

A portrait of germline pathogenic variants from a large real-world cohort of Brazilian patients with breast cancer.

10593 Background: The estimated prevalence of germline pathogenic/likely pathogenic variants (P/LP) in high and moderate penetrance (HMP) genes is around 7-10% among women with breast cancer (BC). Up to 50% of all hereditary BC cases are due to germline variants in BRCA1/2, but other HMP genes are increasing in relevance. The prevalence and spectrum of BC P/LP variants are affected by age at diagnosis, race/ethnicity, geographical region, BC molecular subtype. Methods: Retrospective cohort study to evaluate the germline profile of P/LP variants in HMP genes ( BRCA1, BRCA2, PALB2, TP53, CDH1, NF1, PTEN, STK11, CHEK2, ATM, BARD1, RAD51C and RAD51D) in women diagnosed with BC in Brazil. All patients were tested in a single reference laboratory (Oncoclinicas [OC] Precision Medicine) using multi-gene NGS panels covering from 35 to 105 genes. Primary endpoint was the prevalence of P/LP variants and VUS in BRCA1/2as well as in other HMP BC genes, in overall population and according to age and BC subtype. Clinical and pathological data were retrieved from curated Real-World Database of OC Group and used as reference for demographics comparisons. Results: This cohort involved 2,207 women with BC from 2019 to 2023. The majority of pts (79.7%) were from Southeastern Brazil, followed by Midwestern (5.6%), Southern (5.0%) and Northeastern (4.8%) Brazil. The median age at genetic testing was 47 years and most patients (59.4%) were ≤ 50 years. As reference, median age is 57 years in the overall BC population treated at OC (> 20,000 during the study period). BC subtype was available in 641 cases, 264 pts (41.2%) were HR+/HER2-, 116 (18.1%) were HER2+ and 261 (40.7%) were triple negative (TNBC). In the OC Database, TNBC represents only 15% of all new BC cases. Overall, 217 (9.8%) had a P/LP in HMP genes, including 131 (5.9%) pts who had a BRCA1/2 P/LP variant and 86 (3.9%) who had a P/LP variant in other HMP BC genes. The most frequent LP/PV variants were found in BRCA2 (31%) / BRCA1 (29%) / TP53 (13%) / PALB2 (9%) / CHEK2 (9%). A TP53founder pathogenic variant (R337H) accounted for 75% of all TP53 LP/PV. The deleterious variants were more common in pts ≤50 years (12%) than in pts > 50 (7%) as well as in patients with TNBC (14%) followed by HER2+ (9%) and HR+/HER2- (7%). The BRCA1/2 P/LP variants were higher in TNBC pts (8%) than in HR+/HER2- (4%) and HER2+ (3%). The overall VUS rate in the HMP genes was 20%. The most frequent VUS were found in ATM, BRCA2, NF1 and CHEK2genes. Conclusions: Studies involving germline profile of P/LP variants in cancer genes have provided a broader understanding of BC risk and have opened doors to new therapeutic or surveillance strategies across all subtypes. In Brazil, the prevalence of deleterious variants in HMP is comparable to published US and EU cohorts. However, in the real-world community practice, testing is still limited to populations with higher baseline risk for hereditary BC, such as young patients with TNBC.

P-034 Clinical experience with expanded carrier screening results on a sperm donor applicant population

Abstract Study question What is the frequency of clinically significant results from expanded carrier screening (ECS) in a sperm donor applicant population and how should these be managed? Summary answer ECS results revealed clinically significant information for approximately 1 in 51 donor applicants, which warrant specific management considerations in the context of third-party reproduction. What is known already The American Society of Reproductive Medicine (ASRM) Practice Committee published guidance in January 2021 that outlines the recommended carrier screening approach for gamete donors. However, there is no additional direction related to results management including donor suitability, counseling, and informed consent. Prior studies have illustrated the high frequency of donors who are identified as carriers of one or more recessive disorders on an ECS panel; thus, excluding all prospective gamete donors identified as carriers for recessive conditions is not feasible or appropriate given the availability of reciprocal screening for the reproductive partner. Study design, size, duration A retrospective review of donor genetic screening records from July 2017 to December 2021 was performed. Relevant data was extracted and categorized by carrier screening result. Genetic counselors evaluated the health and reproductive risks to the potential donors (PDs) associated with being a carrier of a pathogenic mutation in each gene using published data, reference laboratory interpretations, and the professional health management guidelines. Participants/materials, setting, methods ECS was performed on sperm donor applicants as part of the routine donor qualification process. Testing was performed at an outside reference laboratory after participants provided written consent for genetic testing. The genes included on the ECS panel were analyzed using multiple methodologies. Specific methodologies varied based upon laboratory offerings at the time in which the potential donor entered the donor program. Main results and the role of chance A total of 966 PDs had ECS during the specified timeframe. Of these applicants, 19 total PDs (1.97%) were identified as having potentially significant health risks based on their ECS results. Of those 19 PDs, eleven were found to be either heterozygous or hemizygous for conditions that may convey significant health risks to carriers. Of these, nine were positive for a variant in a gene typically associated with an AR condition and two carried variants in genes associated with X-linked conditions. Eight additional PDs were found to be either compound heterozygous or homozygous for variants in a gene associated with an autosomal recessive condition. Limitations, reasons for caution Donor recruitment was limited to select major metropolitan areas in the U.S.; thus, donor applicants may not represent all ethnic groups or socioeconomic backgrounds. Additionally, this study described results from a single reference laboratory and does not illustrate the variation in ECS panels across genetic testing laboratories. Wider implications of the findings While this study examined ECS results in a donor applicant population, findings are also applicable to the general reproductive population and illustrate the necessity of informed consent and post-test counseling. In some cases, ECS may provide insight on potential future health risks and health management opportunities. Trial registration number Not applicable

O-063 Accurate mitochondrial DNA quantification clarifies the clinical value of measuring mtDNA in trophectoderm biopsy specimens

Abstract Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p < 0.0001), extent of blastocyst expansion (p < 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p < 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

A high prevalence of neutrophil‐specific antibodies in ELANE‐mutated severe congenital neutropenia

An assay for neutrophil-specific antibodies is frequently used in the workup of chronic severe neutropenia and is suggestive of autoimmune, or sporadically alloimmune neutropenia, rather than severe congenital neutropenia (SCN). We analyzed a neutropenia consortium database for the outcomes of antibody testing initiated before receiving genetic diagnosis in Polish SCN cohort. Test results, performed in a single reference laboratory, were available for 14 patients with ELANE-mutated SCN or cyclic neutropenia, and were frequently positive (36%). We note that the trigger for genetic studies in severe neutropenia should not be affected by antibody-positivity and should be clinically driven.

P556: PGT-M for hereditary cancer conditions: A 12-year testing experience at a single reference laboratory

P556: PGT-M for hereditary cancer conditions: A 12-year testing experience at a single reference laboratory

PMON61 Evaluation of elevated IGF-1 in patients without clinical evidence of acromegaly

Abstract Introduction Insulin-like growth factor-1 (IGF-1) acts downstream of growth hormone mediating many of its effects. It is the recommended screening test for acromegaly, in evaluating pituitary incidentalomas and possible hypopituitarism. Case-based observations suggest that elevated IGF-1 may occur in patients without acromegaly. The aims were to 1) identify the frequency of elevated IGF-1 without evidence of GH excess, and 2) to examine potential differences in relevant medications and comorbidities between people with an elevated IGF-1 compared to a control group matched for age, sex, gonadal and pituitary status. Methods All people whose IGF-1 was measured at a single reference laboratory between Dec 1st 2018 – Dec 1st 2020 were identified. The electronic records of those with at least one IGF1 >1.1x the upper limit of the age-matched reference range were appraised to determine; 1) documentation of acromegalic features, 2) presence of relevant comorbidities and medication use, and 3) further investigation to exclude pathological GH excess. Results There were 2759 IGF-1 levels measured in 1963 people over the specified period. Out of these, 204 were in patients over 18 years of age and were >1.1 times the upper limit of the age-matched reference range. We excluded 48 with diagnosed acromegaly, 4 who had further workup for acromegaly planned, 9 on GH substitution for proven GH deficiency, 3 with Cushing's syndrome and 5 who were pregnant. A further 33 were excluded based on insufficient data available. A total of 102 cases (61M, 41F) were included in the analysis. Of these 102 people, 67 had pituitary MRI scans, 24 had a 75g oral glucose tolerance test, 28 had a subsequent repeat IGF-1 that had normalised and 13 had random GH <1ug/L. The characteristics of these 102 cases were compared to those of the matched control group (n=102) with normal IGF-1 assayed in the same period. There was no significant difference between the elevated and normal IGF-1 groups in age (mean 46.7 vs 47.0 years, P=0.90), sex (P>0.99), gonadal status (P=0.87) or pituitary status (P=0.15). Variables that were significantly different between cases and controls included dopamine agonist therapy (19/102 cases vs 6/102 controls, OR=3.66, 95% CI: 1.45-9.29, P=0.009) and chronic kidney disease (14/102 cases vs 4/102 controls, OR = 3.90, 95% CI: 1.28-11.14, P=0.024). Conclusion Out of 1963 patients having IGF-1 measured at a reference laboratory, a significantly elevated IGF-1 occurred in 204. In 102 (50%) of these patients, there was no known acromegaly, GH replacement or glucocorticoid excess, and no documented definitive acromegalic features. There were significant associations with dopamine agonist use and CKD. While assay-related issues and accuracy of the age-matched reference ranges probably represent the main influences on the prevalence of elevated IGF-1, we have identified two additional factors which should be considered. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Noninvasive Prediction of Congenital Cytomegalovirus Infection After Maternal Primary Infection.

To develop and internally validate a noninvasive method for the prediction of congenital cytomegalovirus (CMV) infection after primary maternal CMV infection. We conducted a secondary analysis of a multicenter randomized placebo-controlled trial of CMV hyperimmune globulin to prevent congenital infection. Women were eligible if they had primary CMV infection, defined as detectable plasma CMV-specific immunoglobulin (Ig)M and CMV-specific IgG with avidity less than 50% before 24 weeks of gestation or IgG seroconversion before 28 weeks, and were carrying a singleton fetus without ultrasonographic findings suggestive of CMV infection. Antibody assays were performed in a single reference laboratory. Congenital infection was defined as CMV detection in amniotic fluid, neonatal urine or saliva, or postmortem tissue. Using backward elimination, we developed logit models for prediction of congenital infection using factors known at randomization. The performance of the model was assessed using leave-one-out cross-validation (a method of internal validation). Of 399 women enrolled in the trial, 344 (86%) had informative data for this analysis. Congenital infection occurred in 68 pregnancies (20%). The best performing model included government-assisted insurance, IgM index 4.5 or higher, IgG avidity less than 32%, and whether CMV was detectable by polymerase chain reaction in maternal plasma at the time of randomization. Cross-validation showed an average area under the curve of 0.76 (95% CI 0.70-0.82), indicating moderate discriminatory ability. More parsimonious one-, two-, and three-factor models performed significantly less well than the four-factor model. Examples of prediction with the four-factor model: for a woman with government-assisted insurance, avidity less than 32%, IgM index 4.5 or higher, and detectable plasma CMV, probability of congenital infection was 0.69 (95% CI 0.53-0.82); for a woman with private insurance, avidity 32% or greater, IgM index less than 4.5, and undetectable plasma CMV, probability of infection was 0.03 (95% CI 0.02-0.07). We developed models to predict congenital CMV infection in the presence of primary maternal CMV infection and absence of ultrasonographic findings suggestive of congenital infection. These models may be useful for patient counseling and decision making.

Large Intra-Age Group Variation in Chromosome Abnormalities in Human Blastocysts

Research Question: Is maternal age only a gross predictor of chromosome abnormalities in human embryos? Design: Here, we evaluated the less-studied variation in chromosome abnormality rates in embryos of patients within the same age group. Patients undergoing IVF and PGD for chromosomal abnormalities in ~127 different IVF clinics were included. PGT-A analysis was performed by a single reference laboratory using array CGH or NGS. To get an estimate of the range of abnormalities observed, the aCGH and NGS data were studied both independently and together. Results: The overall results showed the typical increase in aneuploidy rates with advancing maternal age (AMA) but extensive variability within each age group. Conclusions: Increasing aneuploidy with maternal age has been demonstrated in live births, unborn fetuses, IVF embryos and oocytes. In contrast, post-meiotic and other abnormalities that might lead to mosaicism, polyploidy and haploidy, are commonplace (around 30%), regardless of maternal age. Here we conclude that age is only a gross predictor of chromosome abnormalities in IVF embryos. In contrast to the existing standard of offering PGT-A to AMA patients, the high rate and extreme variation of chromosomal abnormalities in human embryos may warrant PGT-A for further IVF cycles even in younger age groups, especially if a history of increased levels of aneuploidy is evident. Furthermore, better indicators are needed to determine which patients are at a higher risk of producing increased levels of aneuploid embryos.

The incidence and prevalence of patients with paroxysmal nocturnal haemoglobinuria and aplastic anaemia PNH syndrome: A retrospective analysis of the UK's population-based haematological malignancy research network 2004-2018.

A retrospective population-based study to determine the incidence and prevalence of patients with the rare blood disease paroxysmal nocturnal haemoglobinuria (PNH). All patients were identified by flow cytometric detection of blood cells deficient in glycosylphosphatidylinositol (GPI) linked proteins at a single diagnostic reference laboratory that serves the Yorkshire based, Haematological Malignancy Research Network (HMRN) with a population of 3.8million. One hundred and ninety-seven patients with detectable PNH clones at a level of >0.01% in at least two lineages of cells (neutrophils, monocytes and/or red cells) were identified over a 15-year period (2004-2018). Of these, 88% had aplastic anaemia (AA), 8% classical PNH and 3% myelodysplastic syndrome. The overall incidence rate was estimated at 0.35 cases per 100000 people per year. This equates to 220 cases newly diagnosed in the United Kingdom each year. The overall prevalence rate was 3.81 per 100000, this equates to an estimated 2400 prevalent cases in the UK. The overall and relative 5-year survival rates were 72% and 82.7%, respectively. This study showed that classical haemolytic PNH is a rare disease and represents only a small proportion overall of patients with detectable PNH cells, the majority of which have aplastic anaemia.

A comparative study of platelet factor 4‐enhanced platelet activation assays for the diagnosis of heparin‐induced thrombocytopenia

BackgroundFunctional platelet activation assays, such as the serotonin release assay (SRA), are the gold standard for the diagnosis of heparin‐induced thrombocytopenia (HIT). Recently, platelet activation assays using added platelet factor 4 (PF4) have been described and suggest improved sensitivity. Direct comparisons of these assays have not been performed. ObjectiveWe compare the performance characteristics of three PF4‐enhanced platelet activation assays, the PF4/heparin‐SRA (PF4/hep‐SRA), the PF4‐SRA, and the P‐selectin expression assay (PEA), at a single reference laboratory. MethodsSerum samples from two cohorts of patients were used. The referral cohort (n = 84) included samples that had previously undergone routine diagnostic testing for HIT and tested positive or negative using the SRA. The clinical cohort (n = 101) consisted of samples from patients with clinically confirmed HIT whose serum contained platelet‐activating antibodies. We simultaneously tested all samples in PF4‐enhanced SRA‐based assays (PF4/hep‐SRA, PF4‐SRA) and the flow cytometry‐based PEA. ResultsIn the referral cohort, the three PF4‐enhanced assays identified all samples that were previously determined to be positive in the SRA. However, specificity of the PF4/hep‐SRA was 96.6%, the PF4‐SRA was 84.7%, and the PEA was 67.8%. In the clinical cohort of samples, all SRA‐based assays displayed high performance characteristics (>92.1% sensitivity, >98.4% specificity). Sensitivity and specificity of the PEA was the lowest, 65.8% and 63.5%, respectively; but improved to 92.1% and 96.8% using preselected platelet donors. ConclusionsAll PF4‐enhanced assays demonstrated good performance characteristics when platelet donors were preselected. Further comparisons across multiple laboratories should be conducted for consensus on optimal HIT diagnostic testing.

Trophectoderm biopsy protocols may impact the rate of mosaic blastocysts in cycles with pre-implantation genetic testing for aneuploidy.

PurposeThis study aimed to analyze the impact of different biopsy protocols on the rate of mosaic blastocysts.MethodsThis is a retrospective cohort study which included 115 cycles with pre-implantation genetic testing for aneuploidy (PGT-A). Two groups were allocated based on the biopsy protocols: method 1 group, the zona pellucida (ZP) was drilled on day 3 embryos followed by trophectoderm (TE) biopsy; and method 2 group, the ZP was opened on day 5 or 6 blastocysts followed by TE biopsy. All biopsy samples were assessed using next-generation sequencing (NGS) at a single reference laboratory. The euploid, aneuploid, and mosaic blastocyst rates and clinical outcomes were compared.ResultsThe mosaicism rate in the method 1 group was 19.58%, significantly higher than the method 2 group (8.12%; P < 0.05). No statistically significant difference was observed in euploid, aneuploid blastocyst rates, and clinical pregnancy rates between the two groups. Logistic regression analysis indicated that the biopsy protocols were independently associated with the mosaicism rates among all the variables.ConclusionsThe present study showed that different biopsy protocols may have an impact on the mosaic blastocyst rate. ZP opening on day 3 combined with TE biopsy might increase the incidence of mosaic blastocysts.

Bovine Tuberculosis: A Review of Molecular Diagnostic Methods and Impact on Public Health

Bovine tuberculosis (BTB) is a zoonotic infectious disease of cattle, other domesticated animals, and certain wildlife populations. It has been widely distributed throughout the world, and it has been a cause for great economic loss in animal production. In developed countries, the eradication programs have reduced or eliminated TB in cattle. Many factors contribute to the persistence of the disease, such as the limitations of diagnostic tests, larger herd sizes, increase in animal movements and trade, and limited options for control, such as restrictions on whole herd depopulation. The available advanced TB diagnostic techniques can detect and differentiate the causative mycobacterial species, and help an early confirmation of the diagnosis, which is useful to design appropriate control measures in the national BTB control programs. Molecular diagnostics, such as polymerase chain reaction, spoligotyping, restriction fragment length polymorphism (RFLP), variable number tandem repeats typing (VNTR), and polymorphism GC-rich repeat sequence (PGRS) are the techniques used for concurrent detection and typing of Mycobacterium species at the strain level. The molecular epidemiology is also being used to identify the source of contamination, to determine the risk factors of BTB transmissions, to investigate the drug resistance pattern, and to track the geographic distribution and spread of clones of Mycobacteria species. The molecular diagnosis is the tool to check whether active transmission or reactivation of BTB, hence, it is better to adopt these methods for the epidemiological survey of BTB. Since BTB is a major public health problem, at least a single reference laboratory should be available for molecular based diagnosis as part of its control.

DNA fragmentation of sperm: a radical examination of the contribution of oxidative stress and age in 16 945 semen samples.

What is the relationship between sperm DNA fragmentation and oxidative stress (OS) with increasing male age? Sperm DNA fragmentation increases with age and is likely related to both defective spermatogenesis and increasing OS levels. Sperm quality declines with age. The presence of DNA damage in a high fraction of spermatozoa from a raw semen sample is associated with lower male fertility in natural conception and intrauterine insemination. A retrospective cohort study of 16 945 semen samples analysed at a single reference laboratory between January 2010 and December 2018. All males were undergoing an infertility evaluation. The cohort was divided into seven age categories: <30, 30-34, 35-39, 40-44, 45-49, 50 to <54 and ≥55 years. The mean age was 37.6 years (SD 6.8). Sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) were calculated using flow cytometry. OS levels were measured using the oxidative stress adducts (OSA) test, by spectrophotometry. ANOVA with weighted polynomial contrast analysis was used to evaluate trends for DFI, OSA and HDS values across age categories. Mean DFI significantly increased across all age groups (Ptrend < 0.001). OSA was lowest in patients <30 years old (mean 3.6, SD 1.0) and also increased as age increased (Ptrend < 0.001). There was a statistically significant difference between age groups for each of the three parameters (P < 0.001). There was a significant linear trend for DFI, OSA and HDS across the seven age categories (P < 0.001). Among patients with high DFI, there was a decreasing age-dependent trend in the patients observed with high OSA (P < 0.001). This is a retrospective study. All males included in the study were undergoing a work-up for infertility and may not be representative of a fertile population. Additional patient demographics and clinical data were not available. DNA and/or oxidative damage in sperm may be just as important to understand as the chromosomal aberrations that are carried in the oocyte. Further studies are needed to evaluate the effect of advancing paternal age on the male genome and, ultimately, on the health of the offspring. No funding was obtained for this study. V.D. is an employee of Reprosource/Quest Diagnostics. D.S. reports he was a Scientific Advisor to Cooper Surgical. N/A.

SETTING EXPECTATIONS FOR COUPLES UNDERGOING PREIMPLANTATION GENETIC TESTING (PGT) FOR TWO SEPARATE INHERITED SINGLE GENE DISORDERS WITH CONCURRENT 24 CHROMOSOME ANEUPLOIDY SCREENING

Outcomes of in vitro fertilization (IVF) cycles with preimplantation genetic testing for monogenic/single gene disorders (PGT-M) and aneuploidy (PGT-A) are well documented (Gutierrez-Mateo, 2009; Konstantinidis M, 2015). With increased availability and expansion of preconception genetic testing, PGT-M is now being requested for multiple single gene conditions. However, there is little information available regarding cycle outcomes and likelihood of obtaining unaffected and euploid embryo(s) for transfer after undergoing PGT-M for two single gene disorders with PGT-A. To report the rates of unaffected and euploid embryo results for patients undergoing PGT-M for two single gene disorders with concurrent PGT-A. Retrospective analysis of trophectoderm (TE) biopsy results from IVF patients who were referred for PGT-M for two disorders with PGT-A. The TE biopsies were performed according to each clinic’s standard procedures. Samples were shipped to a single reference laboratory for genotyping using Illumina Cyto12 SNP-based microarray and an informatics technique (Johnson, 2010). Twenty-seven couples underwent 48 IVF cycles for PGT-M for two single gene disorders and PGT-A. A total of 303 biopsy sample were tested with an average of 6.3 embryos tested per cycle (range 1-17). PGT-M testing was performed for 38 single gene disorders. Of the 27 couples, 12 (44.4%) had both conditions identified due to personal and/or family history of an affected individual, 10 (37.0%) had one condition identified by personal/family history and the other condition detected by genetic carrier screening, and 5 (18.5%) had both conditions identified by genetic carrier screening. The average maternal age for the patient cohort was 33.8 years (range 25–39 years). Seventeen couples (63.0%) had at least one euploid and unaffected (for both single gene conditions) embryo available to transfer after a single IVF cycle. Seven couples (25.9%) pursued additional IVF cycles, six of which eventually had at least one euploid and unaffected embryo identified for transfer. For these couples, an average of 2.5 IVF cycles was necessary (range 2-5 IVF cycles).Table 1Conditions for which PGT-M test development was requested by caseDisorder 1Disorder 1 identified byDisorder 2Disorder 2 identified by2p11.2 duplicationPersonal/family historyCOL6A2 VUS*Personal/family historyATRX-related disorder VUS*Personal/family history18q22.1 microduplicationPersonal/family historyBRCA1-related breast and ovarian cancerPersonal/family historyCombined oxidative phosphorylation deficiency-31Personal/family historyBRCA1-related breast and ovarian cancerCarrier screeningNBN-related cancer susceptibilityCarrier screeningBRCA1-related breast and ovarian cancerCarrier screeningCDH1-related hereditary diffuse gastric cancerCarrier screeningBRCA1-related breast and ovarian cancerPersonal/family historyFabry diseaseCarrier screeningBRCA1-related breast and ovarian cancerPersonal/family historyFragile X syndromeCarrier screeningBRCA1-related breast and ovarian cancerPersonal/family historyCystic fibrosisCarrier screeningBRCA2-related breast and ovarian cancerPersonal/family historyvon Hippel-Lindau syndromePersonal/family historyBRCA2-related breast and ovarian cancerPersonal/family historyGlucose-6-phosphate dehydrogenase (G6PD) deficiencyCarrier screeningBRCA2-related breast and ovarian cancerPersonal/family historyFamilial Mediterranean feverCarrier screeningCDH1-related hereditary diffuse gastric cancerPersonal/family historyPRKAG2-related Wolf Parkinson White syndrome/hypertrophic cardiomyopathyPersonal/family historyCharcot-Marie-Tooth neuropathy, type X1Personal/family historyAlport syndromePersonal/family historyCystic fibrosisCarrier screeningHuntington disease (non-disclosure)Personal/family historyCystinosisPersonal/family historyFragile X syndromePersonal/family historyFamilial Mediterranean feverPersonal/family historyAnkylosing spondylitis (HLA B27)Personal/family historyFamilial Mediterranean feverCarrier screeningL1CAM syndromePersonal/family historyGJB2-related non-syndromic hearing lossCarrier screeningSmith-Lemli-Opitz syndromeCarrier screeningGJB2-related non-syndromic hearing lossCarrier screening21-hydroxylase-deficient congenital adrenal hyperplasiaCarrier screeningHolt-Oram syndromePersonal/family historyFactor XI deficiencyCarrier screeningInclusion body myopathy 2Carrier screeningBiotinidase deficiencyCarrier screeningPolycystic kidney disease VUS*Personal/family historyLong-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD)Carrier screeningPRRT2-related disorderPersonal/family historySCN2A-related benign familial neonatal infantile seizures VUS*Personal/family historyPTPN11-related disorderPersonal/family historySLC6A1-related disorderPersonal/family historySCN11A-related disorderPersonal/family historyFLNC-related disorderPersonal/family historySpinal muscular atrophyPersonal/family historyFragile X syndromeCarrier screeningTTN-related dilated cardiomyopathyPersonal/family historyAnkylosing spondylitis (HLA B27)Personal/family history*VUS = variant of unknown significance, as categorized by the genetic testing laboratory Open table in a new tab *VUS = variant of unknown significance, as categorized by the genetic testing laboratory In our study, 15/27 (55.6%) of couples had at least one condition detected by carrier screening. This study highlights the importance of carrier screening even in individuals pursuing PGT-M due to a personal and/or family history of a genetic disorder. When undergoing PGT-A and PGT-M for two inherited genetic conditions, a euploid and unaffected embryo may be possible to achieve in a single IVF cycle. Overall, this information may be useful in counseling patients who wish to consider PGT-M for two single gene disorders to set realistic expectations and plan for the potential need for multiple IVF cycles.

267: Non-invasive prediction of congenital cytomegalovirus (CMV) infection after maternal primary infection

To develop and validate a non-invasive method for the prediction of congenital CMV infection after primary maternal CMV infection. Secondary analysis of a multicenter randomized placebo controlled trial of CMV hyperimmune globulin to prevent congenital CMV infection. Women were eligible if they had primary CMV infection, defined as positive IgM antibody and IgG antibody with avidity < 50% before 24 weeks or IgG seroconversion before 28 weeks, and were carrying a singleton fetus without ultrasound findings suspicious for CMV infection. Antibody assay was performed in a single reference laboratory. Congenital infection was defined as CMV detection in amniotic fluid, neonatal urine or saliva, or post-mortem tissue. Using backward elimination, we developed a logit model for prediction of congenital infection from factors known at randomization. Factors included maternal age, parity, race, ethnicity, BMI, children at home, gestational age at serologic testing, IgM antibody index≥4.5, IgG avidity < 32%, and treatment group. The performance of the model was assessed using leave-1-out cross validation (a method of internal validation). 399 women were randomized and 362 had informative data for this analysis. The primary outcome occurred in 71 (20%). The final model (Table) included black race (yes/no), IgM ≥4.5 (yes/no) and IgG avidity < 32% (yes/no). Cross validation showed an average AUC of 0.71(95% CI 0.65-0.78), indicating moderate discriminatory ability. Examples of prediction: For a black woman with avidity < 32% and IgM≥4.5, probability of congenital infection = 0.75 (0.58-0.86); for a non-black woman with avidity ≥32% and IgM< 4.5, probability of infection = 0.04 (0.02-0.08). We developed and validated a model that may be useful for decision making in the face of primary maternal CMV infection and absence of ultrasound findings suspicious for CMV infection.

2: The effect of treatment of maternal CMV infection on the development of placental syndrome

Cytomegalovirus (CMV) is known to infect trophoblasts and result in placental dysfunction. Our objective was to evaluate whether treatment of maternal CMV infection prevents “placental syndrome”, a composite of adverse pregnancy outcomes that involve placental dysfunction. Secondary analysis of a multicenter randomized placebo-controlled trial of CMV hyperimmune globulin (HIG) to prevent congenital CMV infection. Women were eligible for the trial if they had primary CMV infection—positive IgM antibody and positive low avidity (< 50%) IgG antibody before 24 weeks, or IgG seroconversion before 28 weeks—as analyzed in a single reference laboratory, and were carrying a singleton fetus without ultrasound abnormalities suspicious for congenital CMV infection. Trained research nurses abstracted the outcomes which were prespecified according to standard criteria. Placental syndrome was defined as any preeclampsia, gestational hypertension (GHTN), small for gestational age (SGA; birthweight < 10%ile), placental abruption, preterm delivery (PTD before 37 weeks), or perinatal death. We also evaluated the effect of treatment on severe placental syndrome defined as any of the following: preeclampsia or GHTN requiring delivery < 37 weeks, birthweight < 5%ile, PTD < 34 weeks, or perinatal death. P < 0.05 was used to denote statistical significance. The primary analysis showed that HIG did not prevent congenital infection. Of 399 women randomized, 390 had informative data for this analysis. Baseline characteristics were balanced between the treatment groups. Placental syndrome was significantly more common in the HIG group (Table). Each of the individual components of the placental syndrome, as well as severe placental syndrome and its components, occurred more often in the HIG group, but the differences were not statistically significance (Table). In this largest randomized trial to date, treatment of maternal CMV with hyperimmune globulin not only did not prevent congenital infection, but also increased the frequency of placental syndrome.

Bad Tests Die Slowly: The Myelin Basic Protein Example.

Our national reference laboratory sought to improve stewardship for multiple sclerosis (MS) testing, which included orders for myelin basic protein (MBP) and oligoclonal bands (OCB). From 2011 to 2012, we performed 2 interventions for MS testing: one gentle-strength intervention of a publication designed to educate others about the lack of utility for MBP results and a second medium-strength intervention that included removal of MBP from the panel of MS tests. The ordering trends and practice variation were examined for OCB and MBP to retrospectively observe the effect of the interventions. Data from clients within academic and community hospitals were examined (n = 1710 clients). Ordering patterns for OCB and MBP were investigated from 2008 to 2018 by calculating the %OCB: %OCB = (OCB)/(OCB + MBP). Practice variation was examined by comparing the distribution of clients with different %OCB statistics before and after the interventions in 5-year blocks (2008-2012 vs 2014-2018). From 2000 to 2011, the %OCB was approximately 50%, but gradually increased to 67% in 2018. For practice variation, analysis of the distribution of clients by %OCB also demonstrated a shift toward clients favoring OCB alone vs OCB + MBP for MS testing for the later time period of 2014-2018. Our 2 interventions had a measurable, beneficial effect on ordering trends for MS testing over a 10-year period at a single reference laboratory. However, given that MBP has questionable clinical utility, stronger interventions are likely needed to bring about larger changes in ordering behavior.