Good laboratory practice for PGT-M: Turkish Society of Reproductive Medicine guidelines.

This guideline was prepared by the Turkish Society of Reproductive Medicine to define the conditions and requirements for an outsourced preimplantation genetic testing (PGT) programme in line with the experience and needs of practitioners. This guideline is intended to be a reference document for assisted reproductive technology centres, genetic diagnosis centres, non-governmental organizations working on reproductive health, legal experts, consultants working on laboratory accreditation, academicians specializing in ethical issues, and policy makers. The Consortium aims to provide recommendations addressing the challenges of genetic testing, especially PGT for monogenic diseases (PGT-M) due to the high rate of consanguineous marriage in Turkey. For this purpose, this summary document specifically includes challenges and recommendations regarding PGT-M practice, and aims to identify and aid in prevention of errors leading to misdiagnosis. The recommendations can be modified to fit other locations.

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.

PGT for structural chromosomal rearrangements in 300 couples reveals specific risk factors but an interchromosomal effect is unlikely.

RESEARCH QUESTION: What factors affect the proportion of chromosomally balanced embryos in structural rearrangement carriers? Is there any evidence for an interchromosomal effect (ICE)? DESIGN: Preimplantation genetic testing outcomes of 300 couples (198 reciprocal, 60 Robertsonian, 31 inversion and 11 complex structural rearrangement carriers) were assessed retrospectively. Blastocysts were analysed either by array-comparative genomic hybridization or next-generation sequencing techniques. ICE was investigated using a matched control group and sophisticated statistical measurement of effect size (φ). RESULTS: 300 couples underwent 443 cycles; 1835 embryos were analysed and 23.8% were diagnosed as both normal/balanced and euploid. The overall cumulative clinical pregnancy and live birth rates were 69.5% and 55.8%, respectively. Complex translocations and female age (≥35) were found to be risk factors associated with lower chance of having a transferable embryo (P < 0.001). Based on analysis of 5237 embryos, the cumulative de-novo aneuploidy rate was lower in carriers compared to controls (45.6% versus 53.4%, P < 0.001) but this was a 'negligible' association (φ < 0.1). A further assessment of 117,033 chromosomal pairs revealed a higher individual chromosome error rate in embryos of carriers compared to controls (5.3% versus 4.9%), which was also a 'negligible' association (φ < 0.1), despite a P-value of 0.007. CONCLUSIONS: These findings suggest that rearrangement type, female age and sex of the carrier have significant impacts on the proportion of transferable embryos. Careful examination of structural rearrangement carriers and controls indicated little or no evidence for an ICE. This study helps to provide a statistical model for investigating ICE and an improved personalized reproductive genetics assessment for structural rearrangement carriers.

Maternal exome analysis for the diagnosis of oocyte maturation defects and early embryonic developmental arrest.

RESEARCH QUESTION: Can a methodology be developed for case selection and whole-exome sequencing (WES) analysis of women who are infertile owing to recurrent oocyte maturation defects (OOMD) and/or preimplantation embryo lethality (PREMBL)? DESIGN: Data were collected from IVF patients attending the Istanbul Memorial Hospital (2015-2021). A statistical methodology to identify infertile endophenotypes (recurrent low oocyte maturation rate, low fertilization rate and preimplantation developmental arrest) was developed using a large IVF dataset (11,221 couples). Twenty-eight infertile women with OOMD/PREMBL were subsequently enrolled for WES on their genomic DNA. Pathogenic variants were prioritized using a custom-made bioinformatic pipeline set to minimize false-positive discoveries through resampling in control cohorts (the Human Genome Diversity Project and 1343 whole-exome sequences from oocyte donors). Individual single-cell RNA sequencing data from 18 human metaphase II (MII) oocytes and antral granulosa cells was used for genome-wide validation. WES and bioinformatics were performed at Igenomix and the National Research Council, Italy. RESULTS: Variant prioritization analysis identified 265 unique variants in 248 genes (average 22.4 per sample). Of the genes harbouring high-impact variants 78% were expressed by MII oocytes and/or antral granulosa cells, significantly higher than for random sample of controls (odds ratio = 5, Fisher's exact P = 0.0004). Seven of the 28 women (25%) were homozygous carriers of missense pathogenic variants in known candidate genes for OOMD/PREMBL, including PATL2, NLRP5 (n = 2),TLE6, PADI6, TUBB8 and TRIP13. Furthermore, novel gene-disease associations were identified. In fact, one woman with a low oocyte maturation rate was a homozygous carrier of high-impact variants in ENSA, an essential gene for prophase I meiotic transition in mice. CONCLUSIONS: This analytical framework could reveal known and new genes associated with isolated recurrent OOMD/PREMBL, providing essential indications for scaling this strategy to larger studies.

The birth of a baby with mosaicism resulting from a known mosaic embryo transfer: a case report.

Mosaic embryos have the potential to implant and develop into healthy babies. The transfer of mosaic embryos is now considered to be a possible option for women undergoing ART with preimplantation genetic testing for aneuploidies and in the absence of euploid embryos, particularly those with diminished ovarian reserve and/or advanced maternal age. It can aid in avoiding the discard of potentially viable embryos, which might otherwise result in healthy babies. In over 500 studies on mosaicism, there have been no reports of mosaicism in babies born following the transfer of mosaic embryos. Here, we present a case report of a 39-year-old woman with diminished ovarian reserve with only one blastocyst available for trophectoderm biopsy. The transfer of the embryo, which showed 35% mosaicism of monosomy 2, resulted in pregnancy. Amniocentesis revealed a mosaic trisomic mos46,XX(98)/47,XX,+2(2) karyotype. There were no pathological findings in detailed ultrasonography, and the fetus showed a normal fetal growth with no evidence of intrauterine growth retardation. A healthy female baby was born at Week 37. The peripheral blood chromosome analysis validated with fluorescence in situ hybridization showed 2% mosaic monosomy 2 [mos45,XX,-2(2)/46,XX(98)]. This is the first reported case of true fetal mosaicism resulting in a live birth following the transfer of a known mosaic embryo. Worldwide, prenatal diagnosis has shown the depletion of mosaicism in embryos transferred after they have been reported as mosaics. Our case demonstrates the need for close prenatal monitoring and diagnosis by early amniocentesis, preferably at >14 weeks gestation.

High rates of aneuploidy, mosaicism and abnormal morphokinetic development in cases with low sperm concentration.

PURPOSE: The aim of our study was to evaluate the impact of severe male infertility (SMF) on the chromosomal status of embryos and any possible correlation between chromosomal status and embryo morphokinetics in younger women using data obtained from comprehensive preimplantation genetic tests. METHODS: The trial was conducted in an ART and Reproductive Genetics Centre between 2011 and 2018. A total of 326 cycles in cases with SMF where the female partner's age was ≤ 35 years were evaluated. SMF is defined as sperm concentration below 5 mil/ml (million per milliliter) and divided into three subgroups according to sperm concentrations: 1-5 mil/ml, < 1mil/ml and testicular sperm. The control group of 190 cycles had normal sperm parameters. RESULTS: Significantly lower chromosomal euploidy rates were found in the testicular sperm group compared with the normal sperm controls when the female age was ≤ 35 years. In SMF, statistically significantly affected chromosomes were 2, 10, 11, 17, 21 and sex chromosomes. The mosaicism and abnormal morphokinetic development rates were higher in the SMF group than in control group, and this difference was significant when testicular sperm was used. CONCLUSION: Lower euploidy rates, higher mosaicism rates and a higher incidence of abnormal morphokinetic development were observed in cases with testicular sperm with female partners ≤ 35 years compared with normal sperm controls. These findings suggest that PGT-A may be advisable in severe male infertility cases. Furthermore, the correlation between morphokinetics and chromosomal status was greatly reduced or absent in these most severe forms of male infertility, thus the need for new morphokinetic models.

Is there a correlation between follicle size and gene expression in cumulus cells and is gene expression an indicator of embryo development?

BACKGROUND: In an article published in 2017, we discussed the results of the first part of our study into the morphokinetic development of embryos in relation to follicle diameter and homogeneity of follicular development. Our findings showed that embryos coming from small follicles in heterogeneous cycles had significantly higher rates of arrest or failure to reach blastocyst than embryos coming from large follicles in homogenous cycles. The aim of this further study was to investigate the relationship between follicular size and gene expression of cumulus cells (CCs) and evaluate whether gene expression could be an indicator of embryo development. METHODS: This study was based on 2495 COCs from 184 patients. CC expressions of five genes (TNFAIP6, PTGS2, HAS2, PTX3 and GDF9) were studied by generalized linear mixed models (GLMMs) regarding follicular size. CC expressions were then separately analysed regarding patient-specific variables (age, BMI, AMH and follicular size) in relation to embryos reaching blastocyst (eRB) or top or good quality blastocysts (TQ + GQ) using GLMMs with logit link. RESULTS: Follicular size significantly correlated with the potential of an oocyte to develop into a blastocyst: oocytes developing from large follicles were more than twice as likely to develop into an eRB than oocytes from small follicles (p < 0.001). Gene expression of HAS2 and GDF9 correlated with blastocyst quality when separately evaluated with follicular size and the patient specific variables of age, BMI and AMH. However, no such correlation was found in other gene expressions studied. CONCLUSIONS: Our findings suggest that differences in the expression of genes studied could be related to follicular size rather than to embryo quality. Although gene expression of HAS2 and GDF9 correlated with blastocyst quality, the only variable correlating with eRB and TQ and GQ blastocysts for each of these five models was follicular size. TRIAL REGISTRATION: This prospective cohort study was registered at clinicaltrials.gov (NCT02230449).

The effect of follicle size and homogeneity of follicular development on the morphokinetics of human embryos.

PURPOSE: Our aim was to investigate follicular size (large, ≥17 mm and small, <17 mm) at the time of OPU and homogeneity of follicular development (homogenous development: follicles being present in a homogenous spread of all sizes; heterogeneous: a predominance of small and large follicles) by analysing the morphokinetics of embryo development. METHODS: In this prospective cohort study, 2526 COCs belonging to 187 patients were cultured to day 5. Embryos were evaluated morphokinetically. Four subgroups were defined: large follicles from heterogeneous cycles (LHet) and homogenous cycles (LHom) and small follicles from heterogeneous cycles (SHet) and homogenous cycles (SHom). RESULTS: Rates of fertilization, blastocyst formation and top and good quality blastocysts were found to be significantly higher in embryos from the LHom group (p < 0.001; p < 0.001; p < 0.001). Small follicles from both homogenous and heterogeneous cycles had significantly lower blastocyst formation and top and good quality blastocyst rates (p < 0.001; p < 0.001). Embryos from SHet had significantly more direct cleavages (p = 0.011). Time to reach blastocyst was shorter in SHom than LHet and LHom (p = 0.002; p = 0.027, respectively). However, once the blastocyst stage was achieved, implantation rates were not significantly different between subgroups, the highest rate being observed in the LHom group. Multivariable analysis revealed that homogeneity of follicular development and follicular size had a significant effect on blastocyst development and quality (p = 0.049; p < 0.001, respectively). CONCLUSION: Follicular dynamics, illustrated by follicular size and homogeneity of follicular development, influence early human embryo development. Patterns of follicular growth have an impact on embryo quality and viability which is reflected in morphokinetic variables.

Relative kinetic expressions defining cleavage synchronicity are better predictors of blastocyst formation and quality than absolute time points.

PURPOSE: Morphology alone is not enough for the selection of the embryo (s) with the highest implantation potential and time-lapse imaging has added embryo development kinetics as another selection criterion. Therefore, a combination of morphology with kinetics has inspired a new field termed "morphokinetics", providing a new way of evaluating and selecting embryos. The aim of the study was to identify a criterion solely based on morphokinetic data and available up to the 8-cell stage (t8) to predict blastocyst formation and quality. METHODS: The study included 3,354 embryos, with annotations up to t8, and cultured until day 5 from 626 infertile patients. A total of 17 kinetic expressions, either absolute cleavage timings and time intervals or time ratios were tested retrospectively for the prediction of blastocyst formation and quality. RESULTS: Relative timings (t8-t5, the cleavage synchronicity from 4 to 8 cells and from 2 to 8 cells) were found to be better indicators of blastocyst formation and quality when compared to absolute time-points. Especially, the cleavage synchronicity from 2 to 8 cells (CS2-8) = ((t3-t2) + (t5-t4))/(t8-t2)) was found to be the best predictor available on day 3 for blastocyst formation and quality (AUC:0.786; sensitivity: 83.43; specificity: 62.46). CONCLUSIONS: Time intervals and relative ratios based on selected cleavage cycles defining synchronicity allowed a specific analysis providing high predictivity of blastocyst formation and quality.

Chromosomal, gestational, and neonatal outcomes of embryos classified as a mosaic by preimplantation genetic testing for aneuploidy.

OBJECTIVE: To understand the clinical risks associated with the transfer of embryos classified as a mosaic using preimplantation genetic testing for aneuploidy. DESIGN: Analysis of data collected between 2017 and 2023. SETTING: Multicenter. PATIENTS: Patients of infertility treatment. INTERVENTION: Comparison of pregnancies resulting from embryos classified as euploid or mosaic using the 20%-80% interval in chromosomal intermediate copy numbers to define a mosaic result. MAIN OUTCOME MEASURES: Rates of spontaneous abortion, birth weight, length of gestation, incidence of birth defects, and chromosomal status during gestation. RESULTS: Implanted euploid embryos had a significantly lower risk of spontaneous abortion compared with mosaic embryos (8.9% [n = 8,672; 95% confidence interval {CI95} 8.3, 9.5] vs. 22.2% [n = 914; CI95 19.6, 25.0]). Embryos with mosaicism affecting whole chromosomes (not segmental) had the highest risk of spontaneous abortion (27.6% [n = 395; CI95 23.2, 32.3]). Infants born from euploid, mosaic, and whole chromosome mosaic embryos had average birth weights and lengths of gestation that were not statistically different (3,118 g and 267 days [n = 488; CI95 3,067, 3,169, and 266, 268], 3052 g and 265 days [n = 488; CI95 2,993, 3,112, and 264,267], 3,159 g and 268 days [n = 194; CI95 3,070, 3,249, and 266,270], respectively). Out of 488 infants from mosaic embryo transfers (ETs), one had overt gross abnormalities as defined by the Centers for Disease Control and Prevention. Most prenatal tests performed on pregnancies from mosaic ETs had normal results, and only three pregnancies produced prenatal test results reflecting the mosaicism detected at the embryonic stage (3 out of 250, 1.2%; CI95 0.25, 3.5). CONCLUSION: Although embryos classified as mosaic experience higher rates of miscarriage than euploid embryos (with a particularly high frequency shortly after implantation), infants born of mosaic ETs are similar to infants of euploid ETs. Prenatal testing indicates that mosaicism resolves during most pregnancies, although this process is not perfectly efficient. In a small percentage of cases, the mosaicism persists through gestation. These findings can serve as risk-benefit considerations for mosaic ETs in the fertility clinic.

Silk fiber mechanics from multiscale force distribution analysis.

Here we decipher the molecular determinants for the extreme toughness of spider silk fibers. Our bottom-up computational approach incorporates molecular dynamics and finite element simulations. Therefore, the approach allows the analysis of the internal strain distribution and load-carrying motifs in silk fibers on scales of both molecular and continuum mechanics. We thereby dissect the contributions from the nanoscale building blocks, the soft amorphous and the strong crystalline subunits, to silk fiber mechanics. We identify the amorphous subunits not only to give rise to high elasticity, but to also ensure efficient stress homogenization through the friction between entangled chains, which also allows the crystals to withstand stresses as high as 2 GPa in the context of the amorphous matrix. We show that the maximal toughness of silk is achieved at 10-40% crystallinity depending on the distribution of crystals in the fiber. We also determined a serial arrangement of the crystalline and amorphous subunits in lamellae to outperform a random or a parallel arrangement, putting forward what we believe to be a new structural model for silk and other semicrystalline materials. The multiscale approach, not requiring any empirical parameters, is applicable to other partially ordered polymeric systems. Hence, it is an efficient tool for the design of artificial silk fibers.

Bottom-up computational modeling of semi-crystalline fibers: from atomistic to continuum scale.

A bottom-up computational approach involving Molecular Dynamics (MD) of silk fiber subunits and Finite Element (FE) simulations of whole spider silk fibers is presented. The approach is discussed with an emphasis on the benefits and bottlenecks of incorporating the atomistic and continuum models of crystalline and disordered domains in the fibers. The approach does not require any empirical parameters and it is applicable to similar semi-crystalline systems.

Prevalence of oral abnormalities in a Turkish newborn population.

OBJECTIVE: There are a few studies that describe the oral findings in newborn children in various populations but none conducted for a Turkish population. Hence, this study determined the prevalence of intraoral findings in a group of newborns and examined the correlation among these findings with the mother's systemic and gestational medical complications, cigarette consumption during pregnancy and consanguinity between the parents. METHODS: 2,021 full-term, newborn children were examined. Oral cysts, ankyloglossia, attached upper midline frenum and other medical diagnoses at birth were investigated. Medical information for each child and parent was recorded via standard questionnaire. Obtained data was analysed using the Pearson Chi-Square test (P≤0.05). RESULTS: The most common findings were of oral inclusion cysts situated palatally. CONCLUSIONS: There was a statistically significant relationship between the presence of oral inclusion cysts with the congenital diabetes and also insulin treatment and cigarette consumption during pregnancy. Moreover, a significant relationship was found between the presence of oral inclusion cysts and gestational diabetes and with the presence of consanguinity between the parents (P=0.004).

How sequence determines elasticity of disordered proteins.

How nature tunes sequences of disordered protein to yield the desired coiling properties is not yet well understood. To shed light on the relationship between protein sequence and elasticity, we here investigate four different natural disordered proteins with elastomeric function, namely: FG repeats in the nucleoporins; resilin in the wing tendon of dragonfly; PPAK in the muscle protein titin; and spider silk. We obtain force-extension curves for these proteins from extensive explicit solvent molecular dynamics simulations, which we compare to purely entropic coiling by modeling the four proteins as entropic chains. Although proline and glycine content are in general indicators for the entropic elasticity as expected, divergence from simple additivity is observed. Namely, coiling propensities correlate with polyproline II content more strongly than with proline content, and given a preponderance of glycines for sufficient backbone flexibility, nonlocal interactions such as electrostatic forces can result in strongly enhanced coiling, which results for the case of resilin in a distinct hump in the force-extension curve. Our results, which are directly testable by force spectroscopy experiments, shed light on how evolution has designed unfolded elastomeric proteins for different functions.

Mechanical response of silk crystalline units from force-distribution analysis.

The outstanding mechanical toughness of silk fibers is thought to be caused by embedded crystalline units acting as cross links of silk proteins in the fiber. Here, we examine the robustness of these highly ordered beta-sheet structures by molecular dynamics simulations and finite element analysis. Structural parameters and stress-strain relationships of four different models, from spider and Bombyx mori silk peptides, in antiparallel and parallel arrangement, were determined and found to be in good agreement with x-ray diffraction data. Rupture forces exceed those of any previously examined globular protein many times over, with spider silk (poly-alanine) slightly outperforming Bombyx mori silk ((Gly-Ala)(n)). All-atom force distribution analysis reveals both intrasheet hydrogen-bonding and intersheet side-chain interactions to contribute to stability to similar extent. In combination with finite element analysis of simplified beta-sheet skeletons, we could ascribe the distinct force distribution pattern of the antiparallel and parallel silk crystalline units to the difference in hydrogen-bond geometry, featuring an in-line or zigzag arrangement, respectively. Hydrogen-bond strength was higher in antiparallel models, and ultimately resulted in higher stiffness of the crystal, compensating the effect of the mechanically disadvantageous in-line hydrogen-bond geometry. Atomistic and coarse-grained force distribution patterns can thus explain differences in mechanical response of silk crystals, opening up the road to predict full fiber mechanics.

Multiscale Materials Modeling in an Industrial Environment.

In this review, we sketch the materials modeling process in industry. We show that predictive and fast modeling is a prerequisite for successful participation in research and development processes in the chemical industry. Stable and highly automated workflows suitable for handling complex systems are a must. In particular, we review approaches to build and parameterize soft matter systems. By satisfying these prerequisites, efficiency for the development of new materials can be significantly improved, as exemplified here for formulation polymer development. This is in fact in line with recent Materials Genome Initiative efforts sponsored by the US government. Valuable contributions to product development are possible today by combining existing modeling techniques in an intelligent fashion, provided modeling and experiment work hand in hand.

Recent advances in nanoscale bioinspired materials.

Natural materials have been a fundamental part of human life since the dawn of civilization. However, due to exploitation of natural resources and cost issues, synthetic materials replaced bio-derived materials in the last century. Recent advances in bio- and nano-technologies pave the way for developing eco-friendly materials that could be produced easily from renewable resources at reduced cost and in a broad array of useful applications. This feature article highlights structural and functional characteristics of bio-derived materials, which will expedite the design fabrication and synthesis of eco-friendly and recyclable advanced nano-materials and devices.

Segmented molecular design of self-healing proteinaceous materials.

Hierarchical assembly of self-healing adhesive proteins creates strong and robust structural and interfacial materials, but understanding of the molecular design and structure-property relationships of structural proteins remains unclear. Elucidating this relationship would allow rational design of next generation genetically engineered self-healing structural proteins. Here we report a general self-healing and -assembly strategy based on a multiphase recombinant protein based material. Segmented structure of the protein shows soft glycine- and tyrosine-rich segments with self-healing capability and hard beta-sheet segments. The soft segments are strongly plasticized by water, lowering the self-healing temperature close to body temperature. The hard segments self-assemble into nanoconfined domains to reinforce the material. The healing strength scales sublinearly with contact time, which associates with diffusion and wetting of autohesion. The finding suggests that recombinant structural proteins from heterologous expression have potential as strong and repairable engineering materials.

DNAJC6 is responsible for juvenile parkinsonism with phenotypic variability.

Familial parkinson's disease is both clinically and genetically heterogeneous. By mapping the disease locus with a lod score of 5.13 to a < 3.5 Mbp region at 1p31.3 in a consanguineous family and subsequent exome sequencing analysis, we identified homozygous truncating mutation p.Q734X in DNAJC6. Four members of the family were afflicted with juvenile parkinsonism that presented with mental retardation, pyramidal signs and epilepsy, as well as varying degrees of a progressive neurological disease. Recently a splicing mutation in the same gene was reported in two brothers with juvenile parkinsonism that was not L-Dopa responsive and not accompanied by pyramidal signs or mental retardation. Also, an 80-kb deletion that included DNAJC6 sequences was identified in a boy reported as having obesity, epilepsy and mental retardation but not any signs of parkinsonism. The phenotype of our study family resembles both of those families, which among themselves do not share any clinical features. Our findings further establish DNAJC6 as a juvenile parkinsonism gene, and expand the spectrums of the parkinsonism phenotype and DNAJC6 mutation. DNAJC6 encodes the neuronal co-chaperone auxilin. We found that its transcript is highly significantly more abundant in brain as compared to the non-neural tissues assayed.

Ultrasensitive detection of a protein by optical trapping in a photonic-plasmonic microcavity.

Microcavity and whispering gallery mode (WGM) biosensors derive their sensitivity from monitoring frequency shifts induced by protein binding at sites of highly confined field intensities, where field strengths can be further amplified by excitation of plasmon resonances in nanoparticle layers. Here, we propose a mechanism based on optical trapping of a protein at the site of plasmonic field enhancements for achieving ultra sensitive detection in only microliter-scale sample volumes, and in real-time. We demonstrate femto-Molar sensitivity corresponding to a few 1000 s of macromolecules. Simulations based on Mie theory agree well with the optical trapping concept at plasmonic 'hotspots' locations.