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Featured Articles
NIPT: an opportunity for early detection and promising treatment for children with XXY
Inconsistent detection and false-positive rates have plagued traditional screening measures for trisomy, thus encouraging the development of less risky and invasive measures. Through the advent of single-nucleotide polymorphism-based and informatics-based non-invasive prenatal testing, accurate detection of trisomies 13, 18, 21 as well as the X and Y chromosomal aneuploidies of XXY, XYY and XXX in early in pregnancy is now possible. This technology is extremely important in ensuring infants with these disorders are identified in a timely manner so that proper care and treatment can be administered for optimal development.
by Emily J. Stapleton, Dr Megan Hall and Dr Carole A. Samango-Sprouse
Cell-free DNA-based non-invasive prenatal testing
Traditional serum- and ultrasound-based screens have high false-positive rates and less-than-ideal detection rates, resulting in unnecessary and risky invasive procedures and missed diagnoses [1]. The discovery of fetal cell-free DNA (cfDNA) in maternal circulation allowed the development of a more accurate, non-invasive approach for fetal aneuploidy screening [termed non-invasive prenatal testing (NIPT)] [2]. However, cfDNA is highly fragmented and is heavily diluted with maternal cfDNA [3]. Hence, methods to accurately detect fetal aneuploidies using cfDNA analysis had to overcome these technical limitations. Two approaches to-date have accomplished this and have been successfully commercialized. The first-generation quantitative ‘counting’ approaches amplify and sequence non-polymorphic loci and compare absolute quantities of DNA from the chromosome(s) of interest (e.g. chromosome 21) to that of reference chromosomes [4]. The second, next-generation approach specifically amplifies and sequences single-nucleotide polymorphisms (SNPs), identifying both allele identity and distribution [4].
First-generation quantitative counting methods
The most straight forward counting methods non-specifically amplify cfDNA, followed by massively parallel shotgun sequencing (MPSS) [4]. A more recent approach uses targeted amplification and sequencing, thus improving efficiency [4]. Both methods amplify non-polymorphic loci, and identify fetal aneuploidy by detecting abnormally high or low amounts of cfDNA from the chromosome(s) of interest relative to internal reference chromosomes that are presumably euploid in the fetus. If the proportion of reads associated with a particular chromosome relative to the reference chromosome(s) is found to be significantly above the expected proportion for a euploid fetus, the extra reads are presumed to have originated from an extra chromosome present in the fetal genome and fetal trisomy is inferred. Counting methods have shown remarkable improvements over serum screening and ultrasound methods, reporting >97% sensitivity for trisomies 21 and 18, and false positive rates of <0.2% for trisomy 21 [4]. However, the false positive rate can be as high as 1% for other indications [4]. Additionally, counting methods have reduced sensitivity when detecting aneuploidy of chromosomes 13 and X [4]. This is thought to be due to a combination of variable amplification efficiency due to decreased guanosine–cytosine content, as well as unusual biology specific to the X chromosome. Significantly, the requirement for a reference chromosome renders these methods unable to detect triploidy.
A next-generation approach for NIPT: analysing SNPs
The next-generation PanoramaTM test is the only commercialized NIPT that incorporates genotypic information, in the form of SNPs, to accurately identify fetal chromosomal copy number from cfDNA [5, 6]. This allows a more complex and nuanced cfDNA analysis than first-generation methods that do not take into account genotypic information and only consider the number of reads. This SNP-based approach is able to identify both the allele identity and distribution, thus identifying the maternal and fetal cfDNA contribution to the sequence reads. Additionally, Panorama uses a sophisticated bioinformatics algorithm called Next-generation Aneuploidy Testing Using SNPs (NATUS) that leverages advanced Bayesian statistics.
The NATUS algorithm incorporates parental genotypic information to aid analysis of relatively noisy measurements that result from the mixture of maternal and fetal cfDNA. Specifically, NATUS considers the maternal genotype, which is obtained by measuring genomic DNA isolated from white blood cells present in the maternal blood sample, as well as the paternal genotype, if available (though not necessary); the algorithm incorporates crossover frequency data from the human genome project to bioinformatically predict all of the possible fetal genotypes that could arise from the parental genotypes. These billions of hypotheses are then compared to the actual cfDNA measurements, and a likelihood is calculated for each hypothesis. The hypothesis with the maximum likelihood indicates the actual genetic state of the fetus, thus determining the presence or absence of a chromosomal abnormality.
This approach enables the incorporation of many more quality control metrics, improving accuracy over first-generation counting approaches. First, it creates the ability to flag samples with additional abnormalities, including samples with large deletions and duplications, mosaicism, and extra parental haplotypes, which indicate undetected twins, vanishing twins, or triploidy; any of these may result in miscalls with first-generation NIPTs. Second, the algorithm can take into account a number of other indicators of accuracy in addition to fetal fraction, for example the total amount of cfDNA in the sample, and the degree of contamination. This allows the algorithm to determine when the data is insufficiently clear to make an accurate call, even if the fetal fraction is above the minimum threshold of 3.8%; this reduces the number of incorrect calls. Third, this approach does not rely on a reference chromosome, which enables highly accurate detection of abnormalities on chromosomes that do not amplify with reliable efficiency, such as chromosome 13 and the sex chromosomes, as well as the unique ability to detect triploidy [5, 6]. These advantages, therefore, overcome limitations of the first-generation approach.
This translates to a quantifiable improvement in performance [6]. Specifically, in clinical studies, the NATUS algorithm showed 100% sensitivity when detecting trisomy 21, trisomy 18, trisomy 13, fetal sex, and triploidy, and of 91.7% when detecting monosomy X (Turner syndrome) [5, 6]. Reported specificities were 100% when detecting trisomy 21, trisomy 13, triploidy, and fetal sex, and 99.9% for trisomy 18 and monosomy X [6].
Why NIPT is clinically important
With the advent of SNP-based NIPT, the increase in the number of populations that can affordably and conveniently receive prenatal testing has dramatically increased and, subsequently, so has the identification of children with genetic abnormalities. Through early identification of chromosomal aneuploidies, children can receive early intervention services that are critical to the management of the associated disorders. This is especially true regarding the X and Y chromosomal variations that the NIPT identifies, specifically 47, XXY.
The impact of prenatal testing on 47, XXY
47, XXY (Klinefelter Syndrome) is characterized by the presence of an additional X chromosome and has a frequency of occurrence of 1 in 400 to 1 in 1,000 births [7]. However, due to their mild phenotypic presentation only 25% of boys with the disorder will ever be properly diagnosed. Boys with 47, XXY present neurocognitive deficits in language-based learning disabilities, atypical social development as well as reading disorders [8]. Musculoskeletal findings consist of decreased muscle tonus with joint laxity, pectus excavatum and pescavus. MRI brain imaging in individuals with 47, XXY revealed morphological, volumetric, and gray and white matter differences that are associated with the deficits in neurodevelopmental performance [9].
Androgen insufficiency in XXY has been described in several studies and it has been posited that the androgen deficiency contributes to the neurodevelopmental challenges associated with these disorders, as small research studies report improved brain function in association with androgen replacement [10]. Additionally, recent studies on 47, XXY and 49, XXXXY showed improvement in selected aspects of neurodevelopmental outcome when treated with androgen prior to 24 months of age [11, 12]. The area of greatest difficulty in the disorder is speech and language of which early hormonal treatment (EHT) has shown the most robust improvements in select areas of the verbal domain.
Boys with 47, XXY are susceptible to atypical social interactions, social isolation, and poor self-esteem as a result of the significant language-based learning disorders [9]. Ultimately, these issues may lead to low employment rates, depression and behavioural disruptions if not treated early in life [13]. Although there is a wide variability of cognitive capabilities in 47, XXY individuals, research studies indicate that prenatally diagnosed children demonstrate higher intellectual abilities [9]. Late diagnosis and untreated learning disorders coupled with deficits in executive function may result in significant neurocognitive challenges and behavioural disruptions [13]. School failure is common in these circumstances, which is costly for society in the form of low employment and high risk for psychiatric disturbances of depression and anxiety.
The importance of prenatal diagnosis is critical for the timely implementation of targeted and syndrome-specific treatments, most importantly EHT, and ensuring an optimal developmental trajectory for the child. The development of speech, language and early neurocognitive skills is critical to the growth of later reading proficiency and academic success. These skills are the building blocks for advanced abstract thinking capabilities and as a result allow for job employment and independent living. Research suggests that without timely treatment the growth of these critical neurodevelopmental abilities would be stunted or possibly altogether halted.
Summary
Although this article highlights only one disorder that can be identified through NIPT, the studies presented throughout demonstrate that the neurodevelopmental function of a very common neurogenetic disorder may be improved through early treatment. The importance of NIPT for early identification is imperative in XXY as well as other X and Y chromosomal disorders. The ramifications of prenatal detection and early identification cannot be understated; with knowledge comes the ability to improve a child’s life as well as the family’s well being from the moment of birth onward.
References
1. Invasive prenatal testing for aneuploidy. ACOG Practice Bulletin No. 88. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2007; 110: 1459–1467.
2. Noninvasive prenatal testing for fetal aneuploidy. Committee Opinion No. 545. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2012; 120: 1532–1534.
3. Lo YM, Tein JS, Lau TK, Haines CJ, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for non-invasive prenatal diagnosis. Am J Hum Genet. 1998; 62: 768–775.
4. Levy B, Norwitz E. Non-invasive prenatal aneuploidy testing: technologies and clinical implication. MLO Med Lab Obs 2013; 45: 8,10,12.
5. Samango-Sprouse C, Banjevic M, Ryan A, Sigurjonsson S, et al. SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy. Prenat Diagn. 2013; 33: 1–7.
6. Pergament E, McAdoo S, Curnow K, et al. SNP-based non-invasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y in a high- and low-risk cohort. Manuscript under review.
7. Morris JK, Alberman E, Scott C, Jacobs P. Is the prevalence of Klinefelter syndrome increasing? Eur J Hum Genet. 2008; 16: 163–170.
8. Samango-Sprouse CA, Gropman AL. Introduction: Past, present, and future care of individuals with XXY. Am J Med Genet C Semin Med Genet. 2013; 163C: 1–2.
9. Lee NR, Wallace GL, Clasen LS, Lenroot RK, et al. Executive function in young males with klinefelter (XXY) syndrome with and without comorbid attention-deficit/hyperactivity disorder. J Int Neuropsychol Soc. 2011; 22: 1–9.
10. Patwardhan AJ, Eliez S, Bender B, Linden MG, Reiss AL. Brain morphology in Klinefelter syndrome: extra X chromosome and testosterone supplementation. Neurology 2000; 54(12): 2218–2223.
11. Samango-Sprouse CA, Gropman AL, Sadeghin T, Kingery M, et al. Effects of short-course androgen therapy on the neurodevelopmental profile of infants and children with 49,XXXXY syndrome. Acta Paediatrica 2011; 100(6): 861–865.
12. Samango-Sprouse CA, Sadeghin T, Mitchell FL, Dixon T, et al. Positive effects of short course androgen therapy on the neurodevelopmental outcome in boys with 47, XXY syndrome at 36 and 72 months of age. Am J Med Genet A. 2013; 161A: 501–508.
13. Simpson JL, Graham JM, Samango-Sprouse CA, Swerdloff R. 2005. Klinefelter Syndrome. In Cassidy SB, Allanson JE (editors) Management of Genetic Syndromes, pp.323–334, 2nd edn. New York: Wiley-Liss.
The authors
Emily J. Stapleton1* BSc, Megan Hall2 PhD, and Carole A. Samango-Sprouse1, 3 EdD
1The Focus Foundation, Davidsonville, MD, USA.
2Natera Inc., San Carlos, CA, USA
3George Washington University of the Health Sciences, Washington, D.C., USA
*Corresponding author
E-mail: ndckids@gmail.com
Autoantibodies against MDA-5: very important serological markers in amyopathic dermatomyositis with rapidly progressive interstitial lung disease
by Assoc. Prof. Y. Muro, Assoc. Prof. K. Sugiura and Prof. M. Akiyama Autoantibodies against MDA-5 are serologically important biomarkers because they are mainly detected in patients with amyopathic dermatomyositis complicated with rapidly progressive interstitial lung disease (ILD). Anti-MDA-5 antibodies are useful not only for diagnosis but possibly also for monitoring disease activity in ILD.
Fecal immunochemical testing in colorectal cancer screening
Most of the colorectal cancer (CRC) screening programmes are based on fecal occult blood tests (FOBT). However, there is little information on the utility of FOBT in familial-risk CRC screening. A recently published study has evaluated the diagnostic accuracy of fecal immunochemical tests (FIT) for CRC and advanced colonic neoplasia detection in this population.
by Dr Inés Castro and Dr Joaquín Cubiella
Relevance of colorectal cancer screening
Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer deaths in developed countries [1]. Several factors have been related to the risk of developing CRC. Age, gender and CRC familial history, especially if there are first-degree relatives (FDR) with CRC, are the strongest associated risk factors [2]. Furthermore, CRC risk is directly related to the number of FDRs and inversely related to the age of youngest FDR [3, 4].
Evidence from several studies has shown that CRC screening is effective for CRC prevention in the average-risk population (asymptomatic individuals between 50 and 69 years) [2]. Indeed, both flexible sigmoidoscopy and fecal occult blood tests (FOBT) have been shown to reduce CRC-specific mortality and incidence in randomized controlled trials [2, 5, 6]. This reduction is based on CRC early detection and prevention through adenoma detection and endoscopic resection [2]. Accordingly, these two strategies, along with colonoscopy, have been universally accepted and recommended for CRC screening [2]. In Europe, annual or biennial FOBT is the most widespread CRC screening strategy.
Approximately 95% of CRCs develop on advanced adenomatous polyps (larger than 10 mm, with high-grade dysplasia or villous architecture). These lesions, and early CRC, are characterized by intermittent microscopic blood loss in the stool that can be detected with FOBTs before they are clinically apparent.
Advantages and disadvantages of the different FOBTs
FOBTs detect through blood or blood products (such as globin) in feces with different methods. Mainly, there are two types of FOBTs: chemical (cFOBT) or immunological (FIT).
Chemical fecal occult blood tests
cFOBTs are simple, qualitative tests that use different indicators such as guaiac resin (Hemoccults, Hemoccult II®, SmithKline Diagnostics, Sunnyvale, California, USA), orthotolidine (Hematest®, MilesLaboratories, Elkhart, Indiana, USA) or benzidine (Hemofec®, Med-Kjemi AS, Asker, Norway). By an oxidative reaction and in the presence of a developer solution, hemoglobin seudoperoxidase activity produces a colour change in the paper impregnated with guaiac resin, orthotolidine or benzidine.
However these tests have multiple drawbacks: first, they are not specific to human hemoglobin. Oxygenation reactions may occur with foods with peroxidase activity such as vegetables or uncooked red meat, so it is recommended to withdraw them from the diet for 3 days before sample collection to reduce false positive results. In addition, hemoglobin from the upper gastrointestinal digestive tract is also detected. So, gastrolesive drugs (NSAIDs or aspirin) should be interrupted 7 days before testing. Besides, two stool samples on three consecutive bowel movements are required, limiting adherence to screening programmes [7]. Furthermore, as it is a qualitative test, its result is subjected to interpretation. Finally, their sensitivity for CRC and advanced adenoma detection is low. This is associated with an interval CRC rate ranging between 30 and 50%.
Fecal immunochemical tests
FITs are based on the reaction of monoclonal or polyclonal antibodies specific for human hemoglobin, albumin or other fecal blood components. Thus, they require no dietary or pharmacological restriction, as long as they do not react with blood from upper digestive tract or with any food component. The greatest advantage is determined by its ability to detect and quantify fecal hemoglobin concentrations 7 to 15 times lower than those detected by chemical tests, significantly improving CRC and advanced adenoma detection sensitivity. Besides, FITs allows a reliable and accurate automated analysis, avoiding subjective interpretation. Up to 50 samples an hour can be processed, making this test ideal for population-based screening [7].
The most used and better evaluated methods nowadays for CRC screening are those based on latex agglutination (OC-Sensor®, OC-Micro®, Eiken Chemical Co., Ltd, Japan; FOB-Gold® Sentinel Diagnostics®, Milan) or magnetized gelatin particles (Magstream1000®, Fujirebio Inc.,Tokyo, Japan).
Effectiveness of FOBT-based CRC screening in the the average-risk population
As previously commented, multiple studies have demonstrated that CRC screening with cFOBT in the average-risk population significantly reduces CRC mortality [8]. So far, there are no data available on the effect of FIT in CRC mortality or incidence. However, several diagnostic test studies have compared cFOBT and FIT for CRC and advanced adenomas detection. These studies have shown that FIT is more sensitive and specific for CRC and advanced adenomas detection and is a cost effective screening test [9]. On this basis, CRC screening programmes are based mainly on FIT.
Efficacy of FOBT CRC screening in the familial-risk population
Evidence on the best screening strategy in this population is limited and its quality is low [10, 11]. At present, it is unclear which is the best screening strategy in individuals with a FDR with CRC [12]. Clinical practice guidelines recommend more aggressive screening than in the average-risk population, based on studies that have shown that endoscopic screening reduces CRC incidence and mortality in individuals with a FDR with CRC [2, 6]. Screening is recommended from 40 years or 10 years before the youngest proband. However, this approach has an empirical basis and no prospective controlled study has compared different screening strategies in this population. Furthermore, colonoscopy is an invasive technique that requires sedation, is associated with rare but serious complications (perforation or hemorrhage), requires a substantial financial investment in equipment and expertise and has a limited adherence.
Data on the utility of FOBT for CRC screening in this population are scarce. Our group has recently evaluated FIT diagnostic accuracy for advanced colorectal neoplasia (CRC or advanced adenoma) in asymptomatic individuals with at least one first-degree relative with CRC submitted to a colonoscopy [13]. Patients with an advanced neoplasia had a fecal hemoglobin concentration statistically higher than those without an advanced neoplasia. Fecal hemoglobin concentration was significantly higher among individuals with CRC or advanced adenomas when compared with subjects with non-advanced adenomas or without neoplasia. In contrast, no significant differences were found between individuals with CRC and advanced adenomas as shown in Figure 1. Diagnostic accuracy for CRC detection was high, reaching 100% at a fecal hemoglobin threshold below 115 ng/ml. On the other hand, depending on the number of determinations and the positivity threshold cut-off used sensitivity for advanced neoplasia detection ranged between 34.38 and 50% and specificity between 92.66 and 98.72%. In fact, analysing two samples did not improve diagnostic accuracy and, instead, increased the number of colonoscopies needed to detect a CRC or an advanced neoplasia and the costs per lesion detected [Fig. 2]. These data suggest that FIT is an adequate CRC screening test in this population. However, FIT as a screening method must be evaluated in prospective controlled studies designed to determine CRC mortality reduction in the long term.
Conclusion
The reduction in mortality in FOBT-based CRC screening programmes and the improved sensitivity of immunochemical tests for CRC and advanced adenomas detection makes FIT CRC screening programmes a cost effective strategy. Preliminary data show that FIT is equally effective in the familial-risk population. Thus, FIT could be an adequate CRC screening technique in this population.
References
1. Ferlay J, Shin H-R, Bray F, Forman D, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; 127(12): 2893–2917.
2. Levin B, Lieberman DA, McFarland B, Andrews KS, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology 2008; 134: 1570–1595.
3. Baglietto L, Jenkins MA, Severi G, Giles GG, et al. Measures of familial aggregation depend on definition of family history: meta-analysis for colorectal cancer. J Clin Epidemiol. 2006; 59(2): 114–124.
4. Butterworth AS, Higgins JPT, Pharoah P. Relative and absolute risk of colorectal cancer for individuals with a family history: a meta-analysis. Eur J Cancer 2006; 42(2): 216–227.
5. Mandel JS, Church TR, Bond JH, Ederer F, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer. NEJM 2000; 343(22): 1603–1607.
6. Atkin WS, Edwards R, Kralj-Hans I, Wooldrage K, et al. Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 2010; 375(9726): 1624–1633.
7. Quintero E. [Chemical or immunological tests for the detection of fecal occult blood in colorectal cancer screening?]. Gastroenterología y hepatología 2009; 32(8): 565–576.
8. Hewitson P, Glasziou P, Watson E, Towler B, Irwig L. Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update. Am J Gastroenterol. 2008; 103(6): 1541–1549.
9. Guittet L, Bouvier V, Mariotte N, Vallee JP, et al. Comparison of a guaiac based and an immunochemical faecal occult blood test in screening for colorectal cancer in a general average risk population. Gut 2007; 56(2): 210–214.
10. Dove-Edwin I, Sasieni P, Adams J, Thomas HJW. Prevention of colorectal cancer by colonoscopic surveillance in individuals with a family history of colorectal cancer: 16 year, prospective, follow-up study. BMJ 2005; 331(7524): 1047.
11. Gimeno-García AZ, Quintero E, Nicolás-Pérez D, Hernández-Guerra M, et al. Screening for familial colorectal cancer with a sensitive immunochemical fecal occult blood test: a pilot study. Eur J Gastroenterol Hepatol. 2009; 21(9): 1062–1067.
12. Winawer S, Fletcher R, Rex D, Bond J, et al. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003; 124(2): 544–560.
13. Castro I, Cubiella J, Rivera C, González-Mao C, et al. Fecal inmunochemical test accuracy for colorectal cancer and advanced neoplasia detection in familial risk colorectal cancer screening. Int J Cancer 2013; doi: 10.1002/ijc.28353 (Epub ahead of print).
The authors
Inés Castro MD and Joaquín Cubiella* PhD
Department of Gastroenterology, Complexo Hospitalario Universitario de Ourense, Ourense, Spain
*Corresponding author
E-mail: Joaquin.cubiella.fernandez@sergas.es
Preimplantation genetic screening and related issues
Preimplantation genetic screening is a diagnostic approach dedicated to patients undergoing IVF with the proper indications (advanced maternal age, recurrent implantation failure, recurrent pregnancy loss) in order to increase pregnancy rates per transfer via euploid embryo selection. This strategy, and all the associated techniques, are in constant evolution and will shed more light on unexplored aspects of embryology, such as female meiosis or chromosomal mosaicism, creating new criteria for embryo selection.
by Dr D. Cimadomo, Dr A. Capalbo, Dr L. Rienzi and Dr F. M. Ubaldi
Background
Preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) are two diagnostic approaches increasingly exploited in recent decades within assisted reproduction facilities in the presence of specific indications. PGD is used to identify unaffected embryos in couples at high reproductive risk of a hereditary disease. Usually, these couples conceive naturally and undergo prenatal genetic testing, i.e. villocentesis or amniocentesis; procedures that are invasive and carry a high risk of subsequent miscarriage. The ultimate aim of PGD is, therefore, to prevent the conception of a fetus affected specifically and uniquely by a pathology whose causative mutations have been identified and characterized in the parental genomes before conception. Consequently, PGD depends on a preliminary ad hoc work-up for each couple approaching to an IVF cycle. PGS, instead, is meant to identify only chromosomally normal embryos, thus looking for the presence of chromosomal abnormalities. Since the development of aneuploidies is a de novo event directly linked to maternal age, this diagnostic method is independent from any specific preliminary set-up, thus being identical for each PGS cycle. The indications for this analysis are mainly advanced reproductive maternal age (more than 35 years old; AMA), recurrent implantation failure (more than three failed IVF attempts; RIF) and recurrent pregnancy loss (more than three miscarriages; RPL). From an embryological perspective there is no difference between PGD and PGS. Indeed, strategy and planning of the cycle and biopsy techniques are similar, whereas the genetic technical aspects are significantly different.
Testing for aneuploidy
Interestingly, the data collected by the ESHRE PGD consortium IX showed a constant increase in the number of the PGD cycles approached uniquely for euploid embryo selection. In particular, more than 60% of PGD cycles were actually PGS for AMA, RIF or RPL patients, and this percentage is still currently increasing. There is, in fact, a striking impact of aneuploidies on human reproduction. In particular, their incidence in newborns is around 0.3%, mostly represented by trisomies of chromosomes 13, 18 and 21 and sex chromosome aneuploidies. However, tracking backwards through the developmental stages sees this incidence sharply increase, involving other chromosomes and reaching an incidence of up to 60% in preimplantation embryos and 70% in eggs or polar bodies [1]. On the contrary, this incidence in sperm is definitely less severe, as it is never greater than 3–4%. Moreover, a significant number of spontaneous abortions are linked to aneuploidies (more than 60% of products of conception follow chromosomal abnormalities), both increase exponentially with maternal age and fertility rate collapses (Fig. 1) [2].
From a biological perspective, the origin of high trisomy rates found in clinically recognized pregnancies (which sharply increases in patients older than 35 years) resides mainly in maternal meiosis I and II [3]. Recent data obtained through array comparative genomic hybridization (aCGH) on polar bodies (PBs) showed that chromatid errors in female meiosis, such as premature separation of sister chromatids, definitely outnumber impairments involving whole chromosomes as previously thought [4, 5]. Capalbo et al. [5] performed analyses on biopsies at sequential stages of development, in particular the two PBs, a single blastomere at day 3 of embryo development and also a trophectoderm (TE) sample at the blastocyst stage (Fig. 2). This study design allowed the determination of PB analysis accuracy and the impact of male and mitotic errors as well as the evaluation of the occurrence of correction mechanisms throughout preimplantation development. It came to light that 76 out of 78 (97.4%) abnormal meiotic segregations concerned errors involving chromatids rather than whole chromosomes at meiosis I. Furthermore, it unveiled not only a false positive rate in PB biopsy analysis of 20.5%, as just 79.5% (62/78) of meiotic segregation errors identified in PB biopsies were confirmed in blastomeres, but also a false negative rate of 47.6%, as 10 out of 21 embryos showed mitotic or male-derived aneuploidies confirmed at day 3 and at the blastocyst stage of development, which are, obviously, not observable in PBs. This evidence subverts our previous scenario of chromosomal aneuploidy genesis, as well as undermining the reliability of the PB analysis strategy.
Chromosomal mosaicism
From a diagnostic perspective in PGS, post-zygotic mitotic segregation errors are definitely more troubling than meiotic ones, as, whereas the latter involve the same aberrant chromosomal layout in the whole developing embryo, the former entail the phenomenon of chromosomal mosaicism. In the last decade several publications focused on the problem of mosaicism and its influence on PGD/PGS, claiming an incidence fluctuating between 25% [6, 7] and up to more than 70% [8]. Even when these data are analysed with a critical approach, it still emerges that mosaicism is a substantial source of misdiagnosis when the embryo is biopsied at day 3 post-fertilization. This evidence encouraged a shift of the biopsy strategy toward the blastocyst stage and, to this end, different studies were conducted in order to thoroughly describe its cytogenetic constitution and the impact of biopsy itself on embryonic developmental competence. In particular, Capalbo et al. [9] published data outlining the impact of chromosomal mosaicism on a diagnosis at day 5/6 of embryo development as well as the aneuploid cells setting between inner cell mass (ICM) and TE. To this end, a novel method of ICM biopsy was conceived [as described in 9], characterized via KRT18 staining [as described in 10] and its efficiency tested. It led to the absence of TE contamination in 85.7% of the ICM biopsy products, and a low TE contamination rate (only 2% of TE cells) in the rest of them. These data attest the reliability of this biopsy procedure to test the influence of mosaicism at the blastocyst stage. The study design entailed a preliminary aCGH analysis on a TE biopsy during blastocyst-stage PGS clinical cycles, followed by FISH re-analysis of three further fragments of TE and of the ICM from those blastocysts found to be carriers of copy-number chromosomal errors as well as euploid embryos. This revealed that at the blastocyst stage of development, 79.1% of the aneuploidies were constitutional, while 20.9% of them were mosaic. However, only 4% of the blastocysts were found to be mosaic diploid/aneuploid, being at risk of misdiagnosis due to mosaicism when testing at the blastocyst stage. These data strengthen the theory that the impact of mosaicism could be critical at day 3 of embryo development, but it has definitely less influence at the blastocyst stage, thus strongly presenting the latter as the most reliable candidate biopsy stage to perform PGS. Importantly, in the same paper, Capalbo et al. demonstrated that, after excluding low grade mosaicism (<20% of aneuploid cells) and mosaicism confined to one or two TE sections, in 97.1% of cases concordance for all chromosomes re-analysed by FISH between ICM and TE was observed. On a per embryo analysis, instead, complete concordance in TE-based prediction of ICM chromosomal complement was reported (Fig. 3) [9]. Northrop et al. [11] conducted a similar analysis exploiting a single nucleotide polymorphism (SNP) array, which is a comprehensive chromosomal screening technique. This method was found to detect aneuploidy in samples possessing more than 25% aneuploidy, thus when as few as 2 of the 5 cells within a TE biopsy contain the same chromosomal error. Their data showed no preferential aneuploid cell migration to the TE layer, as aneuploidy was observed in 31% of ICM samples (15 out of 48 ICM products) and 32% of TE ones (46 of 144 TE products). Furthermore, a mosaicism rate of 24% was attested, since 12 out of 50 blastocysts screened showed more than a single diagnosis in all of the multiple sections that were re-analysed.
Does the biopsy procedure affect embryo reproductive competence?
One concern about PGS is that biopsy could affect embryo reproductive competence. To investigate this possibility, Scott et al. [12] designed a randomized and paired clinical trial. They selected two of the best quality embryos from the same patient to be transferred and randomized them, one to undergo biopsy, either at day 3 or at day 5 of embryo development, and the other as a control. The biopsy was submitted to SNP array analysis. If only one embryo implanted, buccal DNA obtained from the neonate after delivery was analysed by SNP array to determine whether the implanted embryo was the control one or not. The data collected clearly showed that conducting the biopsy at the cleavage stage affects the clinical outcome, as an absolute reduction in implantation rate of 19.6% with respect to the control was reported. On the contrary, blastocyst biopsy led to a non-significant overall reduction of implantation of 3%; thus an implantation rate equivalent to the control. It is still unclear whether this is due to a smaller proportion of the embryo’s total number of cells being removed, or to the fact that only extra-embryonic cells are involved, or to a higher stress-tolerance of the blastocyst; however, it is still additional important evidence supporting TE biopsy as the ‘gold standard’ for PGS. From a clinical perspective, the same authors also published a randomized controlled trial [13] comparing the clinical outcomes of single euploid blastocyst transfer versus double untested blastocyst transfer. Ongoing pregnancy rates per randomized patient were similar between the two groups (60.7% in the study group vs 65.1% in the control group), whereas a higher multiple pregnancy rate in the control group was recorded (54% vs 0% in the study group). Ultimately then, PGS on TE biopsy associated with a single euploid blastocyst transfer elicits the same clinical outcomes as conventional IVF, but reduces its risks.
Conclusion
In conclusion, PGS is an important diagnostic approach for patients with the proper indications (AMA, RIF or RPL), performed in order to boost implantation rate per transfer. Euploid embryo selection prevents useless and potentially detrimental embryo transfers. Consequently, further advantages of performing PGS are a lower time-to-pregnancy and a higher cost-effectiveness of each single treatment. Moreover, by adopting a biopsy strategy at day 5/6, it is possible to take advantage of a more robust genetic analysis, a high clinical predictive value, the absence of impact of the biopsy on embryo quality, a low influence of mosaicism, as well as a reduced number of embryos to analyse per cycle, as only developmentally competent ones would reach the blastocyst stage. These last aspects will help in reducing costs, thus extending the patients population that can benefit from this technology. Finally, novel comprehensive chromosomal screening techniques, i.e. aCGH, SNP array and quantitative real-time PCR (qPCR), provide us with reliable, sensible and accurate analysis methods, making of PGS also a technically solid approach.
References
1. Nagaoka SI, Hassold TJ, Hunt PA. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet. 2012; 13(7): 493-504.
2. Heffner LJ. Advanced maternal age–how old is too old? N Engl J Med. 2004; 351(19): 1927-1929.
3. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2001; 2(4): 280-291.
4. Handyside AH, Montag M, Magli MC, Repping S, et al. Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation. Eur J Hum Genet. 2012; 20(7): 742-747.
5. Capalbo A, Bono S, Spizzichino L, Biricik A, et al. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod. 2013; 28(2): 509-518.
6. Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet. 2000; 106(2): 210-217.
7. Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod. 2000; 6(11): 1055-1062.
8. Mertzanidou A, Wilton L, Cheng J, Spits C, et al. Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos. Hum Reprod. 2013; 28(1): 256-264.
9. Capalbo A, Wright G, Elliott T, Ubaldi FM, et al. FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod. 2013; 28(8): 2298-2307.
10. Cauffman G, De Rycke M, Sermon K, Liebaers I, Van de Velde H. Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos. Hum Reprod. 2009; 24(1): 63-70.
11. Northrop LE, Treff NR, Levy B, Scott RT Jr. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts. Mol Hum Reprod. 2010; 16(8): 590-600.
12. Scott RT Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013; 100(3): 624-630.
13. Forman EJ, Hong KH, Ferry KM, Tao X, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013; 100(1): 100-107.
The authors
Danilo Cimadomo BSc, Antonio Capalbo* PhD, Laura Rienzi MS, Filippo Maria Ubaldi MS
G.EN.E.R.A. Centre for Reproductive
Medicine, Clinica Valle Giulia, Via G. De Notaris 2b, 00197 Rome, Italy
*Corresponding author
E-mail: capalbo@generaroma.it
Molecular allergology – probing deeper into the triggers of allergies
Molecular allergology is a cutting edge technology that enables the triggers of allergies to be characterized to a new level of detail. Two new component-resolved immunoblot test systems provide in-depth profiling of allergic reactions against birch and grass pollens and against bee and wasp venoms. The molecular tests supplement the established Euroline allergy range, which comprises a comprehensive spectrum of application-oriented profiles designed for use in any diagnostic laboratory.
by Dr Jacqueline Gosink
Advanced diagnostic approach
Molecular allergology or component-resolved diagnostics is a novel approach to allergy diagnostics, whereby single purified allergen components (SPAC) are used for specific IgE detection in place of the usual whole extracts. This powerful technology introduces a new dimension to differential allergy diagnostics.
Precise, in-depth profiling
The raw allergen preparations of substances such as pollen that are traditionally used for in vitro allergy diagnostics are generally not well characterized and are thus difficult to standardize. In contrast, the allergenic targets used in molecular allergology tests are defined recombinant proteins, which are capable of delivering precise information about the source of sensitization.
The in-depth profiling enables allergologists to:
- Identify disease-causing allergens
- Assess the risk of cross reactions
- Determine patients’ suitability for specific immunotherapy
Multiple pollen sensitizations
Pollen allergies are the most frequently occurring inhalation allergies, with sensitizations to birch and grass pollen as the most common ones. Typically, patients with multiple pollen sensitizations suffer from rhinitis, conjunctivitis and allergic asthma. The allergen extract-based determination of specific IgE antibodies encompasses sensitizations against major allergens and cross reacting minor allergens.
The Euroline SPAC Pollen 1 profile (Figure 1) combines the major and minor allergens of birch (Bet v1, Bet v2, Bet v4, Bet v6) and timothy grass (Phl p1, Phl p5, Phl p7, Phl p12), allowing the differentiation of pollen cross reactions from true multiple pollen sensitizations.
The efficacy of the assay has been confirmed by clinical studies. In one study the test successfully confirmed sensitizations to birch or grass pollen in 77 patients with clinically and anamnestically diagnosed allergies (1), and in a further study the test verified allergic reactions in 44 patients with birch and grass pollen double sensitizations (2). Furthermore, the test system correlated well with comparable commercial assays, demonstrating an EAST class correlation of 95-100% for each of the allergen components.
Bee and wasp venom allergies
Bee and wasp venom stings can pose a problem in the summer months. Whereas a normal reaction to a sting involves local swelling, itching and reddening, persons with an allergy can develop severe systemic reactions, including anaphylactic shock. Bee and wasp venom reactions can be identified using the single allergen components i208 (bee venom) and i209 (wasp venom). i208 represents the main bee venom marker rApi m1 from the honey bee (Apis mellifera) and i209 is the main allergen rVes v5 from the common wasp (Vespula vulgaris). Both preparations are free of cross-reactive carbohydrate determinant (CCD), providing higher reliability in result interpretation. The SPAC analysis allows true double sensitization to be distinguished from cross reactions between insect venoms. The Euroline SPAC Insect Venoms 1 profile (Figure 1) provides the recombinant antigens i208 and i209 together with the corresponding extracts i1 (bee venom) and i3 (wasp venom), allowing an efficient and comprehensive investigation of bee and wasp venom sensitizations with one test.
Fast and easy test procedure
The molecular allergology immunoblot tests are fast and simple to perform and are suitable for use in any diagnostic laboratory. The test procedure is based on established Euroline technology and consists of three basic steps: serum incubation (60 min), conjugate incubation (60 min) and chromogen substrate incubation (10 min). The in-between washing steps are short, and the entire procedure can be completed in 2.5 to 3 hours. All reagents are ready to use, saving time and reducing the risk of errors.
Only small amounts of sample material, typically 400 μl, are required per test. In a special volume-optimized version of the protocol the test can be performed with as little as 100 μl of patient sample, making it ideal for use in pediatrics.
Since the allergens are configured as a line blot with related allergens grouped together, the evaluation of profiles is effortless. Results are classified according to the RAST/EAST system. All profiles additionally include an indicator band of CCD to aid interpretation of the relevance of specific IgE results, for example in cases where positive IgE reactions are inconsistent with the clinical picture.
Fully automated processing
The standardized design of Euroline test strips allows automated processing using immunoblot incubators such as the EUROBlotOne (Figure 2). This advanced system automates the entire Euroline procedure from sample entry to report release. The compact, tabletop device has a high walkaway capacity: up to 44 strips can be incubated per run, and different tests can be combined in one run. All dilution, incubation and washing steps are performed automatically, and the integrated barcode scanner ensures that the correct samples are pipetted. User-friendly menus provide easy navigation, and error-detection features ensure high reliability. Test strips are subsequently digitalized using a special camera module.
Results are then automatically evaluated and archived using the worldwide-established and user-friendly EUROLineScan software. The software automatically identifies, quantifies and assigns bands, and a full results report is available within minutes of completing the incubation (Figure 3). The extensive individual data is administered and documented by the system, and all images and data are electronically archived, eliminating the need to store potentially infectious blot strips. The software can be easily integrated into LIS software, for example the EUROLabOffice system, for a smooth daily laboratory routine.
Comprehensive Euroline allergy range
The new molecular allergy tests are part of the established Euroline allergy range, which provides efficient multiparameter analysis of IgE antibodies against up to 36 different allergens in parallel. The immunoblots are composed from a wide portfolio of allergens, comprising both SPAC and native extracts which have been extensively purified and carefully quality controlled to ensure consistency. All profiles are application-oriented, each one being designed to address a particular diagnostic inquiry.
The Euroline system offers a very competitive price per allergen, making this system the ideal choice for laboratories wanting to perform state-of-the art allergy diagnostics on a small budget.
Perspectives
The advent of molecular allergology technology represents a quantum leap for allergy diagnostics. Component-resolved allergy test systems are unrivalled in the depth of diagnostic information they deliver and hence the level of support they provide for therapeutic decision-making. The Euroline SPAC range will soon be expanded to include further test systems based on this cutting-edge technology.
References
1. Weimann et al. 30th Annual Congress of the EAACI, Istanbul, Turkey, June 2011.
2. Weimann et al. 20th IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine (EuroMedLab), Milan, Italy, May 2013.
The author
Jacqueline Gosink PhD
Euroimmun AG
Luebeck, Germany
Sugar: a bad and good drug
Recently a senior Dutch health official claimed that sugar is ‘the most dangerous drug of the times’ and called for cigarette packet-type warnings stating that ‘sugar is addictive and bad for health’ to be mandatory on the labels of products such as soft drinks and sweets.
A plethora of studies has examined the effects of overconsumption of sugar. Many are based on consumers reporting the amount of sugar in their diet; under-reporting is very common in such surveys, though a recently discovered biomarker based on the ratio of Carbon 12 and 13 can now measure long-term sugar intake from a single blood or hair sample. Other studies don’t distinguish between free monosaccharides and disaccharides added to food products and those occurring naturally in food. While recognising the limitations of many studies, most of us would accept that overconsumption of sugar is linked to obesity, dental caries, macular degeneration and Alzheimer’s disease in older age, cardiovascular disease and diabetes. And hypoglycemia (defined as a blood glucose level of < 2.5mmol/L), a frequent problem in diabetes patients receiving treatment, can also occur in non-diabetic subjects as a result of a diet that is too high in refined sugars and too low in complex carbohydrates. And the treatment for hypoglycemia is the sugar dextrose (= glucose), given orally or by intravenous drip depending on how low the glucose level is and how alert the patient.
Hypoglycemia is unfortunately becoming more common in neonates. Around one in three suffer from the condition in the West, reflecting the increase in gestational and maternal diabetes as well as the rising number of pre-term births. Careful management of the newborn is necessary to avoid seizures and serious brain injury, and this normally involves extra feeding with formula (in addition to breast milk, which often interrupts normal breastfeeding) and repeated blood glucose tests involving heel pricks. If a seriously low glucose level persists, babies are admitted to intensive care and intravenous dextrose is administered. However the good news is that a New Zealand study has just been published in ‘The Lancet‘ involving 514 neonates considered at high risk of hypoglycaemia. The babies diagnosed with the condition were randomly assigned to one of two groups. One hundred and eighteen were treated with six applications of 40% dextrose gel over two days, applied to the inside of the cheek, and 119 were treated with placebo gel. The blood glucose levels of the former group stabilised quicker, fewer babies needed extra formula feeds and fewer were admitted to intensive care. Sugar may be a ‘dangerous drug’ but it can also be invaluable!
