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

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!

Bee and wasp venom allergy is a potentially life-threatening condition and diagnostic errors can therefore have serious consequences. Currently, the diagnosis of allergy to stinging insects relies on patient case history and quantification of specific IgE antibodies and skin prick testing to identify the responsible insect. However, the diagnosis can sometimes be problematic as patients may have very low levels of specific IgE and also because many patients show positive test results to several venom species. Moreover it is often difficult for the patient to identify the offending insect. Component based specific IgE testing helps to increase the sensitivity of testing as well as to resolve which stinging insect species the patient is sensitized to. By applying these new component-specific IgE tests and including testing for serum tryptase, the certainty in identifying patients that will benefit from relevant and safe venom immunotherapy treatment increases greatly.

by Magnus Borres, MD, PhD, MPH

Background
Venoms from stinging insects such as bees and wasps (Hymenoptera) can induce anaphylaxis in susceptible people, and stinging insects are the second most common cause of anaphylaxis in Europe and USA (prevalence of 0.3 to 7.5% in Europe).  Most of the severe and fatal reactions to insect stings in Europe are caused by members of the Vespidae family – commonly known as wasps. In contrast to many other IgE mediated allergic reactions, venom allergies may arise very unexpectedly as they can affect also individuals that do not have a genetic pre-disposition to make IgE antibodies. 

The reactions elicited by a bee or wasp sting can range from mild/local immediate reactions, to larger often late local reactions up to immediate systemic reactions, eventually leading into life threatening conditions requiring emergency treatment.

Markers and risk factors in venom allergies
The presence of specific IgE antibodies to venoms supports the diagnosis of an allergic reaction. In many patients however, the levels are low and there is no direct correlation between the levels of specific IgE antibodies and the risk for reactions. In fact, it is not uncommon that severe reactions occur in patients with very low or sometimes even undetectable venom-specific IgE levels. This exemplifies the need for highly sensitive diagnostic tests that can detect and quantify very low specific IgE levels.
The risk of developing severe reactions after a Hymenoptera sting is dependent on several factors, such as the patient’s history of previous reactions, serum tryptase levels, age and specific IgE-sensitization. People who have already suffered from severe systemic reactions due to stings are predisposed for future reactions – up to 80% will develop severe reactions following a subsequent sting.  However, in 50% of the fatal cases no previous systemic reaction has occurred. Serum tryptase is an important marker for evaluating the risk for systemic reactions, where elevated baseline tryptase levels indicate a higher risk for severe anaphylactic reactions. Approximately a fourth of patients who experience severe venom reactions have elevated baseline levels of this marker. The risk for severe reaction to venom stings also increases with age, and is higher in adults than in children and adolescents. This may be explained by an increased number of mast cells in addition to other contributing clinical conditions in older people.

Identify the little beast!
For patients who are highly allergic to insect stings the treatment option is to undergo venom immunotherapy (VIT) aiming at inducing tolerance. For selecting the most effective treatment, correct identification of the Hymenoptera species that causes reactions in the patient is crucial. This is however not trivial as many patients do not know what insect stung them, and as approximately 60% of the patients show up positive to both bee and wasp in venom extract-based tests.

Diagnostic in vitro tests in venom allergies
Patient history forms the basis in diagnosing a venom allergy and specific IgE antibody test results can support the doctor in the diagnosis and in choosing the appropriate treatment. Whether the reaction in a patient is IgE mediated or not needs to be established. This is usually done by in vitro testing for specific IgE and/or skin prick testing, but as many as 10-20% who seek medical care for sting-induced reactions are negative in these extract-based tests. The reason may be that the reaction was due to another pathogenic mechanism than an allergic reaction, or it could have been caused by an underlying mast cell disease. When using conventional extract based test, which due to the preparation procedure may be low in content of certain allergenic proteins, the sensitivity may not be high enough to pick up certain sensitizations. In addition patients reacting for the first time to a sting may initially have levels of venom-specific IgE below the detection limit.
On the other hand, it is common that patients appear to be sensitized to both bee and wasp venoms when using extractbased specific IgE tests, even in cases when proven non-reactive to one of the species.  Diagnostic tests capable of discriminating between clinically relevant and irrelevant sensitizations, while reliably detecting true co-sensitization to both species greatly improves proper diagnosis and selection of therapeutic interventions. 
There has thus been a need to increase both the sensitivity to detect low levels of IgE antibodies and the specificity to distinguish between sensitization to different Hymenoptera species. Recently this has become possible through the introduction of component-resolved diagnostics, or molecular allergology.

What is molecular allergology?
Molecular allergology allows the measurement of specific IgE antibodies to single, pure allergen molecules, thereby helping to identify the exact allergenic molecule (component) that a patient is sensitized to. All allergen sources contain several allergenic molecules, and the ability to produce these by recombinant means and assay them individually greatly increases the precision of specific IgE measurements. Using this component-resolved testing it is possible to discriminate between species-specific sensitizations, where the patient is genuinely sensitized to the allergen source, and sensitizations due to cross-reactivity. Cross-reactivity occurs when antibodies directed against one molecule cross-recognize a very similar but yet distinct  protein. Such cross-reactivity may arise due to high similarity between some components in bees and wasps, but may also be caused by carbohydrate structures (CCDs) on proteins in plants and invertebrates. CCD antibodies do not cause symptoms and are thus clinically irrelevant, but may confuse test results greatly. Recombinant components used in molecular allergology are free of CCD structures and are therefore very specific. In addition, tests that identify antibodies to CCD are available to further increase diagnostic accuracy.

Molecular allergology improves the allergy diagnosis
Results from extract-based tests give the first, although crude answers that guide the diagnosis, while further analyses using component-based testing take the diagnosis to completely new levels by offering improved test sensitivity, resolving ambiguous extract test results and guiding the selection of optimal treatment. In cases where the patient has a convincing history of bee or wasp allergy, but extract -based tests turn out negative, allergen component testing offers increased sensitivity to detect relevant sensitizations. These tests contain only one single pure allergen component therefore the sensitivity to detect antibodies directed to this unique protein is increased as compared to extract-based tests. However the strength of the extract-based test is that they do contain all relevant allergenic proteins in the allergen source, including minor allergens. 

Component-based testing enables the discrimination between true co-sensitization and sensitization due to cross-reactivity. Extract-based test results may indicate sensitization to both bee and wasp venoms, but using component testing it is possible to investigate if these sensitizations are clinically irrelevant or truly suggest allergy to both species. The recombinant markers for bee (Api m 1), common wasp (Ves v 1 and Ves v5) and/or paper wasp (Pol d 5) should be used to determine unambiguously if the sensitization is species-specific or not. CCD-antibodies can also give rise to double positive test results in the absence of specific Hymenoptera venom sensitization since these antibodies often are induced by grass sensitization.

When extract-based test turn out positive to either bee or wasp only, there is little questioning about what species that patient reacts to. Even though this indicates a true Hymenoptera venom sensitization, additional testing with component-based tests can confirm if the patient is sensitized to a major allergen in the relevant species. Venom immunotherapy treatment may be more effective in patients who are sensitized to these major allergens.

New diagnostic tools in Hymenoptera venom allergy are now available for clinicians
The recent development of IgE tests against species-specific allergen components in Hymenoptera venom allergy offers diagnostic tools that greatly improve the ability to differentiate between sensitization to bees and wasp, and helps in discriminating between clinically relevant and irrelevant sensitizations.
Identification of which molecules that triggers the severe reaction is of vital importance for the clinician when considering venom immunotherapy. The combined use of venom components and Tryptase optimize the diagnosis and management of patients with a suspicion of venom allergy.  Currently only Thermo Fisher Scientific, formerly known as Phadia, Uppsala, Sweden, have both tryptase and allergen component test available on the same technology platform.

The author
By Magnus Borres, MD, PhD, MPH
Pediatric Allergist, Uppsala University Hospital, Uppsala, Sweden
Medical Director, ImmunoDiagnostics, Thermo Fisher Scientific, Uppsala, Sweden