Objective To examine the association of DNA copy number variants (CNVs) with pathologic placental lesions (PPLs) in stillborn fetuses. Design A secondary analysis of stillbirth cases in the Stillbirth Collaborative Research Network case-control study. Setting Multicenter, 59 hospitals in 5 geographic regions in the USA. Population 387 stillbirth cases (2006-2008). Methods Using standard definitions, PPLs were categorized by type including maternal and fetal vascular, inflammatory and immune/idiopathic lesions. Using single-nucleotide polymorphism array, CNVs of at least 500 kb were detected. CNVs were classified into two groups: normal, defined as no CNVs>500 kb or benign CNVs, and abnormal, defined as pathogenic or variants of unknown clinical significance. Main outcome measures The proportions of abnormal CNVs and normal CNVs were compared between stillbirth cases with and without PPLs using the Wald Chi-squared test. Results Of 387 stillborn fetuses, 327 (84.5%) had maternal vascular PPLs and 60 (15.6%) had abnormal CNVs. Maternal vascular PPLs were more common in stillborn fetuses with abnormal CNVs compared with those with normal CNVs (81.7% vs. 64.2%; p=0.008). The proportions of fetal vascular, maternal/fetal inflammatory, and immune/idiopathic PPLs were similar among stillborn fetuses with abnormal CNVs compared to those with normal CNVs. Pathogenic CNVs in stillborn fetuses with maternal vascular PPLs spanned several genes with known relevant mechanisms. Conclusions Abnormal placental/fetal CNVs were associated with maternal vascular PPLs in stillborn fetuses. Findings may provide insight on the mechanisms of specific genetic abnormalities associated with placental dysfunction and stillbirth.
Objective To establish pregnancy-specific reference ranges for fasting and postprandial total serum bile acids (TSBA) levels. Design Cross-sectional study. Setting Tertiary care university hospital. Population Healthy pregnant women at term admitted to the Obstetrics Department over one year. Exclusion criteria were an established diagnosis of intrahepatic cholestasis of pregnancy (ICP) or any co-existing condition of increased risk for ICP. Methods and Main Outcome Measures Both fasting and postprandial TSBA levels were measured in 612 women (528 fasting and 377 postprandial samples). Results Reference intervals of 4.4-14.1 µmol/L for fasting TSBA, and 4.7-20.2 µmol/L for postprandial TSBA were established. The postprandial values were significantly higher than the fasting measurements, with a mean increase of 1.77 µmol/L (22%). A correlation between fasting TSBA levels and postprandial levels was found, as well as with fetal gender, parity, and the use of assisted reproductive technologies. A seasonal pattern was noticed for both fasting and postprandial TSBA, with the highest values in the winter season (p < 0.01 and 0.02, respectively). Conclusions Normal pregnancy is a sub-cholestatic state and is associated with a physiological elevation of TSBA levels, therefore a higher threshold should be considered for the diagnosis of ICP. We suggest using the upper reference limit observed in our healthy pregnant population (fasting ≥14 µmol/L and postprandial ≥20 µmol/L). As the fasting measurement is more specific for the diagnosis, and the postprandial is essential for severity assessment, it is recommended to measure both values, rather than use random samplings. Funding No funding to declare.
Objectives: To examine the association between racial origin and preeclampsia(PE) and gestational hypertension(GH) after adjustment for factors in maternal characteristics and medical history in screening study from the Fetal Medicine Foundation (FMF) in England, and to perform a systematic review and meta-analysis of studies on PE. Methods: In the FMF data regression analysis was performed to examine the association between racial origin and PE or GH. Literature search to December 2021 was carried out to identify peer-reviewed publications on race and PE. Main outcome measure: Relative risk of PE and GH in women of black, South Asian and East Asian racial origin by comparison to white women. Results: In the FMF study there were 168,966 singleton pregnancies without major abnormalities delivering at ≥24 weeks’ gestation. In black women the respective risk of total-PE and preterm-PE was 2-fold and 2.5-fold higher and risk of GH was 25% higher, in South Asian women there was a 1.5-fold higher risk of preterm-PE but not total-PE, and in East Asian women there was no significant difference in risk of hypertensive disorders. The literature search identified 19 studies that provided data on several million of pregnancies, but 17 were at moderate or high-risk of bias and only three provided risks adjusted for some maternal characteristics; consequently, these studies did not provide accurate contribution of different racial groups to the prediction of PE. Conclusion: In women of black and South Asian origin the risk of PE, after adjustment for confounders, is higher than in white women
Objective: To explore the duration of the active phase of the second stage of labour in relation to maternal prepregnant body mass index (BMI). Design: Retrospective cohort study. Setting: Labour wards of three Norwegian university hospitals, 2012-2019. Population: Nulliparous and parous women without previous caesarean section with a live singleton foetus in cephalic presentation and spontaneous onset of labour, corresponding to the Ten Group Classification System (TGCS) group 1 and 3. Methods: Women were stratified to BMI groups according to WHO classification, and estimated median duration of the active phase of the second stage of labour was calculated using Kaplan-Meier analyses. We performed stratified analyses in women with and without epidural analgesia and oxytocin augmentation. Finally, we calculated the Hazard Ratio (HR) of delivery using Cox regression analyses. Caesarean sections and operative vaginal deliveries during the active phase were censored. Main Outcome Measures: Estimated median duration of the active phase of second stage of labour. Results: The final study population comprised 23516 women in TGCS group 1 and 27255 in group 3. Increasing BMI was associated with shorter estimated median duration of the active phase in both TGCS groups. The pattern remained when performing stratified analyses for epidural and oxytocin analgesia. The HR for delivery increased significantly with increasing BMI analysed as a continuous variable; HR 1,019 (95%CI 1.016-1.023) in TGCS group 1 and HR 1,017 (95%CI 1.014-1.020) in TGCS group 3. Conclusion: Increasing BMI was associated with shorter estimated median duration of the active second stage Funding: None.
The surgical management of prolapse has undergone a meandering path, with innovation, controversy and legislation all being encountered en route. Some of the dust is now settling with respect to the role of mesh implant surgery, and whilst it continues to have albeit a contracted role, there is very much a new direction set on native tissue and non-mesh repairs with the advent of techniques such as laparoscopic suture hysteropexy, cervicopexy and colporrhaphy.The authors of this paper present the largest series of women undergoing autologous fascia sacrocolpopexy for the treatment of moderate-severe prolapse. Learning from the past, two key questions that must always be answered when evaluating any new procedure are safety and efficacy. For both these measures, the authors show encouraging results comparable with current gold-standard, mesh augmented repairs.The use of autologous fascia has been well established to treat women with urinary incontinence -the pubovaginal / rectus fascial sling (Mcguire EJ et al . Pubovaginal sling procedure for stress incontinence. J Urol. 1978;119:82–4) – the use of which has resurged following the widespread suspension of synthetic sling procedures. Reports of autologous fascial support of the vaginal vault however are limited to a few short-term case series.This series involves 132 women, followed up for a median of 2.2 years; the authors present five-year data with comparable success rates to those reported in the landmark CARE study (Nygaard I, et al. Long-term outcomes following abdominal sacrocolpopexy for pelvic organ prolapse.JAMA . 2013 May 15;309(19):2016-24) without the complication of mesh erosion.The mixed bag of patient types and concomitant surgery in this study underscores the myriad of pathology and presenting symptoms to the pelvic floor surgeon; sadly, this reality hinders forensic evaluation of the single procedure. It is noted that around three-quarters of the women in the study were having primary prolapse surgery, with a similar proportion undergoing some form of hysterectomy coupled with autologous fascial vault support. Other sacrocolpopexy series have involved women the majority who have already had primary procedures, are without a uterus and represent an already failed and perhaps more difficult to successfully treat group (Maher C, et al. Surgery for women with apical vaginal prolapse. Cochrane Database Syst Rev. 2016 Oct 1;10(10):CD012376.) The addition of a hysterectomy, as well as the harvesting of autologous fascia inevitably means a lengthening of procedure time compared to those usually quoted for women undergoing laparoscopic vault suspension procedures of hysteropexy or sacrocolpopexy.The complexities of pelvic floor patients and their symptoms mean that additionally nearly 2/3 of the patients had Burch colposuspensions performed at the time of index surgery. The unpredictability of pelvic floor surgery on bladder symptoms is amply demonstrated by around 1/3 of women complaining of stress incontinence and a third suffering overactive bladder symptoms following the procedure. It’s clear that functional improvements do not always go hand in hand with anatomical correction for the pelvic floor patient.Many women remain alarmed by the adverse reports of mesh augmentation surgery in gynaecology (Izett-Kay ML, et al ’What research was carried out on this vaginal mesh?’ Health-related concerns in women following mesh-augmented prolapse surgery: a thematic analysis. BJOG . 2021 Jan;128(1):131-139). Contemporary best practice involves distilling out irrefutable principles such as recognising the importance of appropriate apical support which is usually optimally achieved abdominally, as well as an awareness of risks of surgery, careful counselling regarding mesh and being able to offer evidence-based alternatives. This paper provides valuable long-term data for a further promising meshless surgical technique.
Pre-eclampsia screening studies - overcoming intervention biasHS CuckleFaculty of Medicine, Tel Aviv University, Ramat Aviv, IsraelThe ASPRE trial established beyond doubt the efficacy of aspirin prophylaxis in women with positive multi-marker first trimester preeclampsia (PE) screening test results (Rolnik et al. N Eng J Med 2017;50:613-22). Screening combined maternal characteristics, blood pressure, uterine artery Doppler, maternal serum pregnancy associated plasma protein (PAPP)-A and placental growth factor (PlGF). Screen-positive women were randomised to aspirin or placebo and there was a 62% reduction of pre-term PE in the aspirin arm.Subsequently, a practical question has arisen regarding the maternal serum markers: which is superior, PlGF or PAPP-A? This is best answered by non-intervention studies of PE screening when all markers are measured prospectively. There are four such studies and all show that the detection rate for a fixed 10% false-positive rate was higher when PlGF was included compared with PAPP-A; the increase ranged from 5% to 7% (Cuckle. Ultrasound Obstet Gynecol 2022;??:??-??).However, a non-intervention study, despite not revealing the PE screening test report to clinicians and patients, does not preclude the use of aspirin in some women; for example, those with high risk characteristics are likely to be recommended treatment. Moreover, these occasional interventions might bias the PlGF versus PAPP-A comparison. This would occur as a consequence of simultaneous Down syndrome screening using the Combined Test, since that test report includes the PAPP-A level. If this marker was low, treatment might be recommended, leading to the prevention of some pre-term PE cases, a proportion of which are screen-positive. In the absence of intervention these screen-positive cases would be true-positive but actually become false-positive, hence reducing the detection-rate and slightly increasing the false-positive rate. These effects will be stronger for PAPP-A combinations. The standard Combined Test does not include PlGF yielding a bias towards superior PE screening performance for PlGF combinations.In the current analysis, data from two of the four non-intervention studies are reanalysed to adjust for this potential bias (Wright et al. BJOG 2022;??:??-??). In both combined 4.2% (1066/25,226) had taken aspirin, although for nearly all the treatment was sub-optimal compared with the ASPRE regimen of 150mg/night at <16-36 weeks. The reanalysis was by statistical modeling using the original ‘competing risks’ method. But additionally superimposed were simulations from an ‘imputation’ model, which re-assigned false-positives among the 1066 treated women to true- or false-positive according to the probability of reduction in pre-term PE found in ASPRE.The model predicted that the increase in detection rate for a 10% false-positive rate when PlGF was included compared with PAPP-A was 7.0% and this reduced to 6.4% following imputation. Hence, even adopting the extreme assumption that intervention was at an optimal level, the bias in favour of PlGF was small. The authors also modeled combinations without blood pressure or uterine artery Doppler and the bias was proportionally similar or smaller.Clinical studies are often marred by subtle bias, and once discovered it is vital to assess whether the results were materially affected. The current publication is exemplary in using imputation modeling to confirm the superiority of PlGF over PAPP-A.(500 words)
Objective: To determine the peri-operative characteristics associated with an increased risk of post-operative urinary retention (POUR) following vaginal pelvic floor surgery. Design: A retrospective cohort study using multivariable prediction modelling. Setting: A tertiary referral urogynaecology unit. Population: Patients undergoing vaginal pelvic floor surgery from January 2015 to February 2020. Methods: Eighteen variables (24 parameters) were compared between those with and without POUR and then included as potential predictors in statistical models to predict POUR. The final model was chosen as the one with the largest c-index from internal cross-validation. This was then externally validated using a separate data set (n=94) from another surgical centre. Main Outcome Measures: diagnosis of POUR following surgery while the patient was in hospital. Results: Among the 700 women undergoing surgery, 301 (43%) experienced POUR. Pre-operative variables with statistically significant univariate relationships with POUR included age, menopausal status, prolapse stage, and uroflow parameters. Significant peri-operative factors included estimated blood loss, amount of intravenous fluid administered, operative time, length of stay, and specific procedures including vaginal hysterectomy with intraperitoneal vault suspension, anterior colporrhaphy, posterior colporrhaphy, and colpocleisis. The lasso logistic regression model had the best combination of internally cross-validated c-index (0.73) and accurate calibration curve. Using this data, a POUR risk calculator was developed (https://pourrisk.shinyapps.io/POUR/). Conclusions: This POUR risk calculator will allow physicians to counsel patients pre-operatively on their risk of developing POUR after vaginal pelvic surgery and help focus discussion around potential management options.
BJOG-21-1256 Systematic reviews and meta-analyses: bigger is not necessarily betterSystematic reviews and often concomitant meta-analyses are designed to summarize the existing and sometimes overwhelming amount of published literature on a specific topic. Ideally, a systematic review and meta-analysis is reproducible, rigorous and strengthens the evidence on the specific topic, but there are potential pitfalls in designing such studies.O’Byrne et al. (BJOG 2021;) performed a systematic review and meta-analysis regarding the pregnancy outcomes of pregnant women with chronic inflammatory disease exposed to biologics. In the past years some other summarized data have been published on this relevant topic. The authors of the current study tried to distinguish their study from these previous systematic reviews and meta-analyses by using broad inclusion criteria and by expanding the control group population. For reference, a study from Tsao et al. (Rheumatology2020;59:1808–1817) included 24 individual studies and approximately 5600 pregnancies in women with inflammatory systemic diseases exposed to biologics. They compared the outcomes with a disease-matched control group. Komaki et al. (Journal of Autoimmunity 2017;76:38-52) studied 5600 pregnancies of women with immune mediated diseases and the use of anti-tumor necrosis factor-α agents. They included 13 studies and both a disease-matched control group and a general population control group. O’Byrne et al. in contrast, also included case series and case reports in addition, resulting in a study population of more than 11000 exposed cases.Then, the methods of data analysis were fitted to the wide design of the study. The most applicable method of measuring an effect of an exposure on maternal and fetal outcomes is with relative effect measures, also known as odds ratios. In the current study the authors used proportions in their meta-analyses. Proportions represent the absolute risk of an outcome in the exposed and non-exposed groups and are not perfectly suited to draw conclusions about the association of an exposure and an outcome. But the wide study design, in particular the inclusion of case series and case reports, left the authors with no choice but to use proportions.Further, this broad inclusion has two more major drawbacks. The meta-analysis showed in most analyses a high heterogeneity. This can be attributed to the inclusion of different study designs and comparisons of different diseases and medications, as the authors mentioned. In addition, there is a serious risk of bias, firstly due to the high selectivity applied when choosing the population of case series and case reports, and secondly due to the use of proportions, making it impossible to adjust for confounders.Well-designed systematic reviews and meta-analyses can generate a clear overview and provide valuable information for clinicians and patients alike. However, although including a large number of cases is recommended to strengthen the evidence of a systematic review/meta-analysis, this should not be done at any cost - it can sometimes weaken the quality of the study. The most appropriate method should be applied and individualized to the specific study.
Mechanisms of adverse pregnancy outcomes in women with PCOS and ways to prevent these outcomes by knowing more about these mechanismsSedigheh Hantoushzadeh (1), Maasoumeh Saleh* (2), Sepehr Aghajanian (3), Mahboubeh Saleh (4)1-Professor of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Maternal-Fetal Neonatal Research Center, Tehran University of medical sciences, Valiasr Hospital, Tehran, Iran.2-Fellowship of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Tehran University of Medical Sciences, Shariati hospital, Tehran, Iran.3-Department of Community Medicine, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran.4-MD, Department of Urogynecology, Fasa University of Medical Sciences, Fars, Iran.Corresponding author*: Maasoumeh Saleh, E-Mail: firstname.lastname@example.orgDear editor,We have read the study by Valgeirsdottir et al. (1), and wanted to congratulate the authors for this prosperous article on risk of stillbirth in polycystic ovarian syndrome (PCOS) women and make some minor contributions.PCOS is a complex disorder with unclear exact etiology. It involves cardiovascular (CV), metabolic, endocrine, and reproductive system. Several studies have reported an increase in adverse maternal and fetal outcomes for women with PCOS, independent of the use of reproductive technology or body mass index (BMI). PCOS is strongly associated with preeclampsia (PE), gestational diabetes mellitus (GDM), very preterm birth (PTB), large for gestational age fetuses (LGA), and asphyxia during labor (2). In the study by Valgeirsdottir et al. (1), PCOS had a 50% increased risk of stillbirth compared to women without PCOS, especially at term. In this study, the mechanisms of stillbirth are well explained, which include the following: 1-Hyperandrogenism and associated insulin resistance 2-Obesity 3-Hypertensive disorders 4-Placental diseases, fetal anomalies and umbilical cord abnormalities 5-Fetal growth restriction (FGR) 6-Gestational diabetes 7-Chronic inflammation, oxidative state and mitochondrial dysfunction.Serum concentration of homocysteine (Hcy) decreases during pregnancy due to physiologic fall in albumin, as well as folic acid supplementation (3). High serum Hcy level in PCOS women is an independent risk factor for atherosclerotic vascular disease and thromboembolic disorders (4) and it is associated with insulin resistance (5) and adverse pregnancy outcomes, including PE, PTB, and low birth weight (LBW) (6). Elevated C-reactive protein (CRP) levels indicate a low-grade inflammation in PCOS, it is the most sensitive predictor of CV morbidity (7) and also it is associated with adverse pregnancy outcomes (8). These two important mechanisms, including high Hcy serum level and inflammation can play a role in the adverse pregnancy consequences. So, the measurement of serum Hcy level and CRP may be helpful in PCOS patient’s risk stratification for pregnancy complications.Another important issue is to suggest ways to prevent these adverse pregnancy consequences in PCOS patients. Possible options are: 1-Lifestyle modification 2-pharmacological options: metformin, statins, aspirin and high dose folic acid. The possible mechanisms by which aspirin prevents adverse pregnancy outcomes are: 1-improvement in the placentation 2-inhibition of platelet aggregation and its antithrombotic effect 3-antiinflammatory effects and endothelial stabilization (9). Metformin has beneficial effects on endothelial function and improve insulin resistance (10). Statins reverses the pregnancy-specific angiogenic imbalance and improve endothelial function (11). High dosage of folic acid reduces Hcy concentration (12), and through this mechanism may be effective in reducing pregnancy complications. Various studies have been performed with these goals. But in the future, more extensive studies on methods of preventing adverse pregnancy consequences by focusing on their mechanisms are recommended in PCOS women.
Re: Pregnancies in women with Turner Syndrome: A retrospective multicentre UK studyNiels Holmark Andersen1, Claus Højbjerg Gravholt2-31. Department of Cardiology, Aalborg University Hospital, Hobrovej 18-22, DK-9000 Aalborg2. Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark DK-8200 Aarhus N3. Department of Molecular Medicine, Aarhus University Hospital, DK-8200 Aarhus N, DenmarkCorresponding author:Niels Holmark AndersenDepartment of Cardiology, Aalborg University Hospital, Hobrovej 18-22, DK-9000 Aalborgholmark@ki.au.dkORCID:NHA: 0000-0002-5394-3016CHG: 0000-0001-5924-1720Sir,We read with interest the article by Cauldwell et al., which concluded that pregnancy in women with Turner syndrome is associated with major maternal cardiovascular risks and cardiovascular assessment and counselling prior to assisted or spontaneous pregnancy should be managed by a specialist team.These conclusions are based on data from 127 pregnancies in 81 women with Turner syndrome.Cauldwell et al. describe data from 16 different centres with one to 42 pregnancies per centre over an observation period of 20 years. Details on pregnancies per centre are not presented but there must have been many centres where a Turners syndrome pregnancy was a rarity not even happening once a year. When analysing the numbers, it seems that the different centres beyond the centre with 42 cases will have seen an average of 6 pregnant women with Turner syndrome over 20 years. This must have had significant impact on the experience and expertise of the obstetricians in the different centres. Could this be the reason why the caesarean section rates where markedly higher than other wise reported?In the present article the rate was 61-67 % compared to data from Sweden where it was 38 % and 47 % in France. 1-2It is also stated that pregnancies in Turner syndrome are associated with major cardiovascular risks based upon three events. One woman suffered a type A dissection at 18 weeks gestation despite a normal aortic size but a bicuspid aortic valve. No other details about hypertension, aortic morphology or growth rates are presented. The two other events were women with severe aortic disease. It is unclear whether these women were counselled and offered prophylactic aortic surgery before pregnancy. So, in two out of three cases it is possible that the adverse outcomes were due to inadequate clinical care.In this cohort only 57.4% had seen a cardiologist within 24 months before getting pregnant, which is far from what is necessary in the caretaking of women with Turner syndrome and a childbearing potential.Data from Scandinavia, France, and from the Alliance for Adult Research in Congenital Cardiology tells another story. 2-4 In these cohorts the number of pregnancy related dissections was very low due to pre-pregnancy counselling, centralised follow-up, and timely prophylactic aortic surgery before considerations about normal pregnancy or egg donation. This simply generates better results.A safe pregnancy and childbirth are very important for women with Turner syndrome. What we can learn from this article is what not to do. Pregnancy and delivery in women with Turner syndrome must be centralised and a timely cardiovascular assessment of women with Turner syndrome and a pregnancy wish should be mandatory. Otherwise, we will end with results as described in this article.In other words, the conclusion of the article should have been lack of centralisation and appropriate cardiovascular pre-pregnancy assessment leads to increased morbidity and cardiovascular risk. If such pre-pregnancy assessment is in place and caretaking is centralised, cardiovascular risk during pregnancy is very low in Turner syndrome.1-41. Hagman A, Källén K, Barrenäs M-L, Landin-Wilhelmsen K, Hanson C, Bryman I, et al. Obstetric outcomes in women with Turner karyotype. J Clin Endocrinol Metab. 2011; 96: 3475–82.2. Bernard V, Donadille B, Zenaty D, Courtillot C, Salenave S, Brac de la Perrière A, et al. Spontaneous fertility, and pregnancy outcomes amongst 480 women with Turner syndrome. Hum Reprod 2016; 31:782–8.3. Hagman A, Loft A, Wennerholm U-B, Pinborg A, Bergh C, Aittomäki K, et al. Obstetric and neonatal outcome after oocyte donation in 106 women with Turner syndrome: a Nordic cohort study. Hum Reprod 2013; 28: 1598–609.4. Grewal J, Valente AM, Egbe AC, Wu FM, Krieger EV, Sybert VP et al. Cardiovascular outcomes of pregnancy in Turner syndrome. Heart 2021;107 :61–6.Conflicts of interest: None to declare
Sexual Minority Individuals and Pregnancy Outcomes: A CommentaryJulie Croll, MPHa,b; Laura Sanapo, MD, MSHS, RDMSa,c; Ghada Bourjeily, MD, FCCPa,caWomen’s Medicine Collaborative at Lifespan, The Miriam Hospital, 146 West River Street, Providence, RI 02904, USAbRCSI University of Medicine and Health Sciences, School of Medicine, 123 St Stephen’s Green, Dublin 2, IrelandcWarren Alpert Medical School of Brown University, Department of Medicine, 222 Richmond Street, Providence, RI, USA
A commentary on the discrepancy between blood and tumorBRCA testing: an open question Elisa De Paolis1, Claudia Marchetti2;3, Paola Concolino1, Giovanni Scambia2;3, Andrea Urbani1,3, Anna Fagotti2;3, Angelo Minucci1*1Molecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy;2Division of Oncological Gynecology, Department of Women’s and Children’s Health, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy;3Catholic University of the Sacred Heart, Rome, Italy.Running title: Understanding discordant BRCA test cases.*Corresponding author:Angelo MinucciMolecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.email@example.comMolecular evaluation of BRCA1/2 (BRCA ) genes represents a well-known example of precision oncology. The availability of Poly ADP Ribose Polymerase inhibitors (PARPi) as target therapy option for several BRCA mutated cancers types (e.g. ovarian, breast, prostate, and pancreatic)1 changed the course ofBRCA testing over the last years. In this context, an emerging path of molecular evaluation is represented by the BRCA testing performed directly on tumor tissue (tBRCA ): this increased the chance to identify more patients with higher likelihood of benefiting from PARPi treatment. This approach leads to the simultaneous identification of both constitutional and somatically acquired variants, with a lower turnaround time: the identification of BRCApathogenic variants (PVs) could lead to a secondary “reflex” germlineBRCA (gBRCA) testing in order to assess personal and familiar risks. In contrast, performing gBRCA as first molecular test causes the loss of a relevant proportion of patients with tissue acquired BRCA PVs, needing of a following tumor test2;3.However, challenges exist in tBRCA that may lead to inefficient germline variant call. A recently published paper by Kordes et al. reported a pancreatic adenocarcinoma patient with a germlinenovel BRCA2 c.516+4A>G variant classified as deleterious by the authors based on in silico and functional data4. Also the tumor tissue was sequenced in order to achieve the enrolment criteria for a clinical trial of Olaparib in combination with pembrolizumab (KEYLYNK-007). Unexpectedly, the germline variant was not stated in the final report. The authors took into account all the relevant basis of the experienced discrepancy, without identifying a confident reason.In our opinion it is crucial to investigate about the reliability of tBRCA in the identification of both somatic and germline variants. Inspired by the recently published commentary of Gourley5 and taking into account that several troubling cases of discrepancy between blood and tBRCA testing have been reported in literature, we collected the recent relevant studies covering the comparison between gBRCA and tBRCA to give a critical opinion about some shared key points of the somatic testing that could affect the final genotyping and reporting (Table 1).Major reasons of discrepancies are related to: (1) differences in input DNA quality, (2) type of BRCA gene alteration, (3) inherent limitations of the Next Generation Sequencing (NGS), (4) bioinformatics pipeline features (e.g. the ability to predict the occurrence of Copy Number Alterations (CNAs) and the evaluation of the intron/exon boundaries), and finally (5) the issues related to the BRCAvariants interpretation and classification.To date, tBRCA testing is mainly performed on two sample types: Fresh Frozen Tissue (FFT) and Formalin-Fixed Paraffin-Embedded (FFPE). Here, we focused on tBRCA performed on FFPE being the most common tissue type used for clinical diagnostic purpose. Pre-analytical procedures regarding fixation step, tissue section size, and neoplastic cell content assessment, are well-known crucial aspects of the tBRCA testing reliability. In fact, sub-optimal DNA quality represents a relevant reason of inaccuracy of tBRCA and also it is the cause of around 5% of FFPE tBRCA NGS testing fails, with the consequent need of additional new samples9. In Bekos et al. only the retesting of a newly extracted tumor DNA solved two cases of discrepancies with gBRCA : the BRCA1c.1881_1884del variant was not recognized due to poor NGS quality data related to the input materials, as well as for theBRCA2 c.8537_8538del variant2. Also in Careet al. the test failure rate was related to fixation methods or storage of FFPE material8. Ad hocrecommendations for the “ideal” starting tissue material are available9;14.Furthermore, different approaches should be used in the analytical step for the BRCA genes amplification and sequencing, with several types of sequencing chemistries (e.g. amplicon-based, capture-based), platforms (e.g. Illumina, IonTorrent) and data analysis pipelines (e.g. full-coding regions or hot spot analysis, different size of splice site region analysed, CNAs detection). Each one of these could be characterized by specific pitfalls that affect the downstream bioinformatics variants filtering and calling. For example, in amplicon-based approaches, a reason leading to the missing of a variant detection may be related to the experimental design of the primers distribution along the genomic region of interest. Variants located at the 3’ or 5’ ends of overlapping amplicons could be covered by only one read and could be consequently identified with a “strand bias” flag and filtered out at the bioinformatics quality check3.Also the use of different bioinformatics pipeline for the NGS data analysis derived from the germline and the somatic tests of the same patient could be the cause of apparent inconsistent results. For example, in a large cohort of patients affected by several types of malignancies and analysed for the evaluation of the utility of germline test following tumor test, Lincoln et al. identified several cases of discrepancies between the two tests (n=4)15. Among these, the germline BRCA2 c.8967_8973del variant was not detected in tumor sequencing due to the characteristic of somatic panel (hot spot type), not comparable to the germline one. Moreover, in case of discrepancy involving splice site variants could be useful to check the concordance of the splice site region size included in the germline and somatic bioinformatics pipelines3. Regarding data analysis, it should be acknowledged that some tumor testing platforms filter out germline variants in the final reports in order to improve the accuracy of somatic variant calling.A well-known cause of gBRCA /tBRCA non-concordance resulted from the challenge in the bioinformatics calling of CNAs in tissue samples2;3;15. NGS sensitivity in CNAs detection mostly depends on DNA quality, tumor heterogeneity, library preparation, type of algorithms, and size of rearrangement. As a consequence, the somatic bioinformatics pipeline must require computational algorithms developed ad hoc and specific characteristics of sequencing raw data (e.g. maximum amount, coverage uniformity and sufficient reads depth)1. Even if the majority of methods are optimized for somatic CNAs identification6;8, attention should be given in the comparison of blood and tissue tests results13. As an example, Bekos et al. failed to identify in the tumor sample a verified pathogenic germline deletion of BRCA1 exon 20. Only a careful re-evaluation of the bioinformatics variant calls finally revealed the deletion and leaded to the correction of the report2.Relevant role in the evaluation of non-concordant results is played by the post-analytical step involving the BRCA variants interpretation. Complex issues underlying the classification ofBRCA variants exist. The American Collage of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) have established the best practice for germline variant interpretation providing the well-known classification using a five-tier system16. Conversely, the interpretation of somatic variants should be focused on their impact on clinical care. Specifically, evidence-based categorization of somatic variants released by the AMP, the American Society of Clinical Oncology (ASCO), and the College of American Pathologists (CAP) includes a four-tier system: (1) variants of strong clinical significance (level A and B of evidence); (2) variants of potential clinical significance (level C and D of evidence); (3) variants of unknown clinical significance; (4) benign of likely benign variants17. To date, with the publication of an increasing number of large-scale tumor sequencing projects, a plenty of information is being collected into several public databases useful for the querying about the significance of aBRCA variant. Cancer-specific variant databases are available as: BRCAexchange, OncoKB, Catalog of Somatic Mutations in Cancer, My Cancer Genome, cBioPortal, Memorial Sloan Kettering Cancer Center, International Cancer Genome Consortium, and VARSOME. Likewise, constitutional variant databases available are mainly: ClinVar, Human Gene Mutation Database, ENIGMA, Leiden Open Variation Database, and VARSOME. Differences in the germline- and somatic-based annotation may exist between the abovementioned tools. Consequently, the risk of non-concordant annotations of a BRCA variant could occur. This is crucial in the comparison between the same molecular test performed by different labs and it is exacerbated in the case of tBRCA and gBRCA concordance evaluation: variants that met germline guidelines16 to be considered pathogenic may not meet the criteria17 to be considered oncogenic in the somatic test. This situation could more likely affect the missense Variants of Unknown Significance (VUSs)15. As reported by Bekos et al. , after the inclusion of BRCA VUSs in the secondary data analyses, the concordance rate of tumor testing compared to germline one decreased, mainly due to VUSs classification2. Moreover, in a large study investigating the differences in variant interpretation between germline and somatic variants accounted in several cancer-related genes, Moodyet al. highlighted a relevant percentage of discrepancies in variants classification. Among these, the authors reported fourBRCA2 variants with discordant somatic/germline annotations15.In a retrospective cohort of 57 subjects tested for both germline and somatic BRCA status, Kim et al. highlighted one case of a germline variant not identified in the tissue evaluation10. This discrepancy derived from a true reversion of the germline BRCA1 variant accounted via restoration of the wild-type allele in the tissue cells. Finally, tBRCAshould follow specific criteria that maximize molecular information, improving the clinical relevance of the test and giving a more comprehensive interpretation of each variant. With these purposes, peculiar role is played by the “naturally occurring” BRCAsplicing isoforms. As we recently described for the BRCA1c.788G >T variant, complex considerations should be done for rare variants that not only are different germline and somatic annotations, but also are characterized by variability in final effect and annotation in the context of all gene relevant transcripts19.In conclusion, we underline as the systematic and careful checking of tumor tissue suitability could prevent and solve non-concordance cases. Moreover, the robust identification of BRCA variants in FFPE sample correlates with the confidence of the bioinformatics pipeline adopted for the variant filtering and calling, especially for the CNAs detection. In addition, translation of variant calls into clinical decisions relies on proper annotations and discrepancies in classifications of specific variants between tumor and germline contexts could represent a relevant pitfall.We argue that only harmonized guidelines encompassing the abovementioned methodological and post-analytical steps could solve the BRCAgermline and somatic testing bias. In our laboratory, BRCAgenetic testing is routinely performed on blood, FFT and FFPE samples1. In many cases, we routinely analyze matched blood and tissue samples belonging from the same patient, in order to perform an efficient BRCA test comprehensive of both germline and somatic evaluation. This approach pointed out also the relevance of multi-disciplinary and skilled resources for a solid molecular characterization of the tumor. Together with the need of standardization, we suggest as performing BRCA molecular test at both germline and somatic levels in the same laboratory could improve the reliability of the entire molecular path taken by the patient and his clinicians.Disclosure of interests: none declared.Contribution to Authorship: E.D.P. and A.M. conceived of the presented paper and wrote the manuscript with support from P.C. and C.M.. G.S., A.U. and A.F. supervised the project. All authors discussed, edited and contributed to the final manuscript.Details of Ethics Approval: not applicable.Funding: no funding was received for this commentary.
Obstetric anal sphincter injury by maternal origin and length of residence: a letterIt gives us great pleasure to read the study entitled “Obstetric anal sphincter injury (OASI) by maternal origin and length of residence: a nation-wide cohort study” by Sorbye and Bains et al1. We appreciate the authors for conducting a large scale multicentric cohort study on this newer aspect of OASI. However, we wish to make certain observations to further help in comprehending the results.Firstly, the eligibility criteria for the recruitment of participants needs clarification as to why foreign-born women with Norwegian-born parents were excluded from enrolment. Keeping them as a separate group could have been beneficial in assessing whether environmental factors due to migrating out of Norway had an impact on the incidence of OASI. Futhermore, the greater odds of OASI among women with foreign-born partners has to be digested with a pinch of salt. A subgroup analysis comparing the newborn birth weight (NBW) and head circumference (HC) could be instrumental in solving this dilemma. Prior studies by Vik et al from Norway had demonstrated similar outcomes in neonatal survival as well2. In the absence of significant difference in NBW and HC, social issues need due consideration. It probably opens up the arena for potential future research in this very field.The study does mention that the mean HC of newborns to foreign-born women with OASI did not differ from Norwegian-born counterparts without OASI. But the p value mentioned alongside in the text is 0.000, which would amount to high significance. This area needs clarification.Table 3 has stratified the association between OASI and the length of residence in Norway. We appreciate this robust comparison as this outcome was vital in hypothesizing the impact of environment and lifestyles on the incidence of OASI. But, it is quite strange to note that women who had childbirth before their lawful residence (probably had immigrated recently) had lesser odds of OASI compared to those who had legal residence upto 4 years. Discrete analysis of this subgroup of patients might give us a better comprehension. Another analysis which can be done is to assess whether the place of delivery (government or private setup) was significantly affecting the prevalence of OASI. It can be thought of as an auxiliary outcome. This will ultimately help in addressing the barriers to optimal utilization of resources and will probably stimulate the health care policy to achieve equitable care across the nation.References:Sorbye IK, Bains S, Vangen S, Sundby J, Lindskog B, Owe KM. Obstetric anal sphincter injury by maternal origin and length of residence: a nation-wide cohort study. BJOG. 2021 Oct 28. doi: 10.1111/1471-0528.16985. Epub ahead of print. PMID: 34710268.Vik ES, Aasheim V, Nilsen RM, Small R, Moster D, Schytt E. Paternal country of origin and adverse neonatal outcomes in births to foreign-born women in Norway: A population-based cohort study. PLoS Med. 2020 Nov;17(11):e1003395.
Objective To investigate the epidemiological changes in extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) vaginal colonization in high-risk pregnant women and identify independent risk factors. Further, the differences in perinatal outcomes according to maternal ESBL-E vaginal colonization were analyzed. Design Retrospective cohort study. Setting Republic of Korea Population 1,460 women admitted to our high-risk pregnancy unit between 14+0 and 35+6 gestational weeks. Methods The study period was divided into periods 1 (January 2010 to July 2015) and 2 (August 2015 to December 2020). The main outcomes were analyzed according to each period and ESBL-E vaginal colonization. Main Outcome Measures ESBL-E vaginal colonization rate, risk factors for ESBL-E vaginal colonization, and perinatal outcomes. Results The ESBL-E vaginal colonization rate was higher in period 2, which was attributed to a significantly higher proportion of ESBL-producing Escherichia coli. Cerclage (odds ratio [OR]: 3.248; 95% confidence interval [CI]: 1.744–6.049) and prior antibiotic treatment (OR: 3.044; 95% CI: 1.713–5.410) were found as independent risk factors for ESBL-E vaginal colonization. Earlier gestational age at delivery, and higher proven early-onset neonatal sepsis (EONS) rate were observed in the ESBL-E-positive group. Conclusions The ESBL-E vaginal colonization rate in high-risk pregnant patients has increased over the past decade, and the independent risk factors for colonization are cerclage and prior antibiotic treatment. Additionally, maternal ESBL-E vaginal colonization is associated with higher proven EONS rates. Funding This study received no funding. Keywords Extended-spectrum β-lactamase, Enterobacteriaceae, vaginal colonization, antibiotics use, cerclage, neonatal sepsis