De novo mutations in children born after medical assisted reproduction.

STUDY QUESTION: Are there more de novo mutations (DNMs) present in the genomes of children born through medical assisted reproduction (MAR) compared to spontaneously conceived children? SUMMARY ANSWER: In this pilot study, no statistically significant difference was observed in the number of DNMs observed in the genomes of MAR children versus spontaneously conceived children. WHAT IS KNOWN ALREADY: DNMs are known to play a major role in sporadic disorders with reduced fitness such as severe developmental disorders, including intellectual disability and epilepsy. Advanced paternal age is known to place offspring at increased disease risk, amongst others by increasing the number of DNMs in their genome. There are very few studies reporting on the effect of MAR on the number of DNMs in the offspring, especially when male infertility is known to be affecting the potential fathers. With delayed parenthood an ongoing epidemiological trend in the 21st century, there are more children born from fathers of advanced age and more children born through MAR every day. STUDY DESIGN, SIZE, DURATION: This observational pilot study was conducted from January 2015 to March 2019 in the tertiary care centre at Radboud University Medical Center. We included a total of 53 children and their respective parents, forming 49 trios (mother, father and child) and two quartets (mother, father and two siblings). One group of children was born after spontaneous conception (n = 18); a second group of children born after IVF (n = 17) and a third group of children born after ICSI combined with testicular sperm extraction (ICSI-TESE) (n = 18). In this pilot study, we also subdivided each group by paternal age, resulting in a subgroup of children born to younger fathers (<35 years of age at conception) and older fathers (>45 years of age at conception). PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole-genome sequencing (WGS) was performed on all parent-offspring trios to identify DNMs. For 34 of 53 trios/quartets, WGS was performed twice to independently detect and validate the presence of DNMs. Quality of WGS-based DNM calling was independently assessed by targeted Sanger sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: No significant differences were observed in the number of DNMs per child for the different methods of conception, independent of parental age at conception (multi-factorial ANOVA, f(2) = 0.17, P-value = 0.85). As expected, a clear paternal age effect was observed after adjusting for method of conception and maternal age at conception (multiple regression model, t = 5.636, P-value = 8.97 × 10-7), with on average 71 DNMs in the genomes of children born to young fathers (<35 years of age) and an average of 94 DNMs in the genomes of children born to older fathers (>45 years of age). LIMITATIONS, REASONS FOR CAUTION: This is a pilot study and other small-scale studies have recently reported contrasting results. Larger unbiased studies are required to confirm or falsify these results. WIDER IMPLICATIONS OF THE FINDINGS: This pilot study did not show an effect for the method of conception on the number of DNMs per genome in offspring. Given the role that DNMs play in disease risk, this negative result is good news for IVF and ICSI-TESE born children, if replicated in a larger cohort. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Netherlands Organisation for Scientific Research (918-15-667) and by an Investigator Award in Science from the Wellcome Trust (209451). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Correlation between the Covalency and the Thermometric Properties of Yb3+/Er3+ Codoped Nanocrystalline Orthophosphates.

Lanthanide-doped NaYF4 nanoparticles are most frequently studied host materials for numerous biomedical applications. Although efficient upconversion can be obtained in fluoride nanomaterials and good homogeneity of size and morphology is achieved, they are not very predestined for extensive material optimization toward enhanced features and functions. Here, we study the impact of rare-earth metals RE = Y, Lu, La, and Gd ions within Yb3+/Er3+ codoped nanocrystalline REPO4 orthophosphates. The enhanced luminescent thermometry features were found to be in relation to the covalency of RE3+-O2- bonds being modulated by these optically inactive rare-earth ion substitutes. Up to 30% relative sensitivity enhancement was found (from ca. 3.0 to ca. 3.8%/K at -150 °C) by purposefully increasing the covalence of the RE3+-O2- bond. These studies form the basis for intentional optimization thermal couple-based luminescent thermometers such as Yb3+-Er3+ upconverting ratiometric thermometer.

Towards robust and versatile single nanoparticle fiducial markers for correlative light and electron microscopy.

Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye-labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold-core rhodamine B-labelled silica particles, 15 nm CdSe/CdS/ZnS core-shell-shell quantum dots (QDs) and 230 nm Y2 O3 :Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold-core rhodamine B-labelled silica NPs and QDs are quenched after a single exposure to 60 ke-  nm-2 with an energy of 120 keV, while Y2 O3 :Eu3+ NPs are robust and still show luminescence after five doses of 60 ke- nm-2 . In addition, the luminescence intensity of Y2 O3 :Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2 O3 :Eu3+ NPs are promising as robust fiducial marker in CLEM. LAY DESCRIPTION: Luminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye-labelled silica particles, quantum dots and lanthanide-doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye-labelled silica NPs and QDs are quenched after a single exposure to 60 ke- nm-2 with an energy of 120 keV, while lanthanide-doped inorganic NPs are robust and still show luminescence after five doses of 60 ke- nm-2 . In addition, the luminescence intensity of lanthanide-doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide-doped NPs are promising as robust fiducial marker in CLEM.

One-step synthesis and luminescence properties of tetragonal double tungstates nanocrystals.

A versatile one-step thermolysis protocol is demonstrated to produce a uniform dispersion of tetragonal double tungstates NaRE(WO4)2 (RE = rare earth) nanocrystals (NCs). Oriented attachment in the [001] direction occurred. Doping with luminescent RE(3+) ions resulted in highly luminescent NCs showing characteristic line emission of the dopant as well as a blue emission assigned to surface adsorbed organic species.

Prediction model for live birth in ICSI using testicular extracted sperm.

STUDY QUESTION: Which parameters have a predictive value for live birth in couples undergoing ICSI after successful testicular sperm extraction (TESE-ICSI)? SUMMARY ANSWER: Female age, a first or subsequent started TESE-ICSI cycle, male LH, male testosterone, motility of the spermatozoa during the ICSI procedure and the initial male diagnosis before performing TESE were identified as relevant and independent parameters for live birth after TESE-ICSI. WHAT IS KNOWN ALREADY: In reproductive medicine prediction models are used frequently to predict treatment success, but no prediction model currently exists for live birth after TESE-ICSI. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study between 2007 and 2015 in two academic hospitals including 1559 TESE-ICSI cycles. The prediction model was developed using data from one centre and validation was performed with data from the second centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included couples undergoing ICSI treatment with surgically retrieved sperm from the testis for the first time. In the development set we included 526 couples undergoing 1006 TESE-ICSI cycles. In the validation set we included 289 couples undergoing 553 TESE-ICSI cycles. Multivariable logistic regression models were constructed in a stepwise fashion (P < 0.2 for entry). The external validation was based on discrimination and calibration. MAIN RESULTS AND THE ROLE OF CHANCE: We included 224 couples (22.3%) with a live birth in the development set. The occurrence of a live birth was associated with lower female age, first TESE-ICSI cycle, lower male LH, higher male testosterone, the use of motile spermatozoa for ICSI and having obstructive azoospermia as an initial suspected diagnosis. The area under the receiver operating characteristic (ROC) curve was 0.62. From validation data, the model had moderate discriminative capacity (c-statistic 0.67, 95% confidence interval: 0.62-0.72) but calibrated well, with a range from 0.06 to 0.56 in calculated probabilities. LIMITATIONS, REASONS FOR CAUTION: We had a lack of data about the motility of spermatozoa during TESE, therefore, we used motility of the spermatozoa used for ICSI after freeze-thawing, information which is only available during treatment. We had to exclude data on paternal BMI in the model because too many missing values in the validation data hindered testing. We did not include a histologic diagnosis, which would have made our data set less heterogeneous and, finally, our model may not be applicable in centres which have a different policy for the indication for performing sperm extraction. The prognostic value of the model is limited because of a low 'area under the curve'. WIDER IMPLICATIONS OF THE FINDINGS: This model enables the differentiation between couples with a low or high chance to reach a live birth using TESE-ICSI. As such it can aid in the counselling of patients and in clinical decision-making. STUDY FUNDING/COMPETING INTERESTS: This study was partly supported by an unconditional grant from Merck Serono (to D.D.M.B. and K.F.) and by the Department of Obstetrics and Gynaecology of Radboud University Medical Center, Nijmegen, The Netherlands, the Department of Obstetrics and Gynaecology, Jeroen Bosch Hospital, Den Bosch, The Netherlands, and the Department of Obstetrics and Gynaecology, Academic Medical Center, Amsterdam, The Netherlands. Merck Serono had no influence in concept, design, nor elaboration of this study. TRIAL REGISTRATION NUMBER: Not applicable.

Prediction model for obtaining spermatozoa with testicular sperm extraction in men with non-obstructive azoospermia.

STUDY QUESTION: Can an externally validated model, based on biological variables, be developed to predict successful sperm retrieval with testicular sperm extraction (TESE) in men with non-obstructive azoospermia (NOA) using a large nationwide cohort? SUMMARY ANSWER: Our prediction model including six variables was able to make a good distinction between men with a good chance and men with a poor chance of obtaining spermatozoa with TESE. WHAT IS KNOWN ALREADY: Using ICSI in combination with TESE even men suffering from NOA are able to father their own biological child. Only in approximately half of the patients with NOA can testicular sperm be retrieved successfully. The few models that have been developed to predict the chance of obtaining spermatozoa with TESE were based on small datasets and none of them have been validated externally. STUDY DESIGN, SIZE, DURATION: We performed a retrospective nationwide cohort study. Data from 1371 TESE procedures were collected between June 2007 and June 2015 in the two fertility centres. PARTICIPANTS/MATERIALS, SETTING, METHODS: All men with NOA undergoing their first TESE procedure as part of a fertility treatment were included. The primary end-point was the presence of one or more spermatozoa (regardless of their motility) in the testicular biopsies.We constructed a model for the prediction of successful sperm retrieval, using univariable and multivariable binary logistic regression analysis and the dataset from one centre. This model was then validated using the dataset from the other centre. The area under the receiver-operating characteristic curve (AUC) was calculated and model calibration was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: There were 599 (43.7%) successful sperm retrievals after a first TESE procedure. The prediction model, built after multivariable logistic regression analysis, demonstrated that higher male age, higher levels of serum testosterone and lower levels of FSH and LH were predictive for successful sperm retrieval. Diagnosis of idiopathic NOA and the presence of an azoospermia factor c gene deletion were predictive for unsuccessful sperm retrieval. The AUC was 0.69 (95% confidence interval (CI): 0.66-0.72). The difference between the mean observed chance and the mean predicted chance was <2.0% in all groups, indicating good calibration. In validation, the model had moderate discriminative capacity (AUC 0.65, 95% CI: 0.62-0.72) and moderate calibration: the predicted probability never differed by more than 9.2% of the mean observed probability. LIMITATIONS, REASONS FOR CAUTION: The percentage of men with Klinefelter syndrome among men diagnosed with NOA is expected to be higher than in our study population, which is a potential selection bias. The ability of the sperm retrieved to fertilize an oocyte and produce a live birth was not tested. WIDER IMPLICATIONS OF THE FINDINGS: This model can help in clinical decision-making in men with NOA by reliably predicting the chance of obtaining spermatozoa with TESE. STUDY FUNDING/COMPETING INTEREST: This study was partly supported by an unconditional grant from Merck Serono (to D.D.M.B. and K.F.) and by the Department of Obstetrics and Gynaecology of Radboud University Medical Center, Nijmegen, The Netherlands, the Department of Obstetrics and Gynaecology, Jeroen Bosch Hospital, Den Bosch, The Netherlands, and the Department of Obstetrics and Gynaecology, Academic Medical Center, Amsterdam, The Netherlands. Merck Serono had no influence in concept, design nor elaboration of this study. TRIAL REGISTRATION NUMBER: Not applicable.

Eu(2+) luminescence in strontium aluminates.

The luminescence properties of Eu(2+) doped strontium aluminates are reported and reviewed for a variety of aluminates, viz. SrAl12O19, SrAl4O7, Sr4Al14O25, SrAl2O4 and Sr3Al2O6. The aim of the research is to investigate the role of local coordination and covalency of the aluminate host lattice, related to the Sr/Al ratio, on the optical properties of the Eu(2+) ion. The UV and VUV excited luminescence spectra as well as luminescence decay curves were recorded to characterize the luminescence properties of the investigated aluminates. The emission of Eu(2+) ions varies over a wide spectral range, from ultraviolet (UV) to red, for the series of aluminates. The variation in emission color can be related to the crystal-field splitting of the 5d levels and the covalent interaction with the surrounding oxygen anions. In the least covalent material, viz. SrAl12O19:Eu(2+), narrow line emission due to the (6)P7/2-(8)S7/2 transition occurs at 4 K, indicating that the 4f(6)5d excited state is situated above the (6)P7/2(4f(7)) excited state around 360 nm. The most alkaline material, viz. Sr3Al2O6:Eu(2+) is the most covalent host and exhibits several d-f emission bands in the yellow to red spectral range due to the Eu(2+) ions located on different crystallographic Sr(2+) sites. The Eu(2+) emission spectra in the other aluminates confirm the trend that with increasing Sr/Al ratio the Eu(2+) emission shifts to longer wavelengths. Interesting differences are observed for the Eu(2+) from different crystallographic sites which cannot always be related with apparent differences in the first oxygen coordination sphere. The discussion gives insight into how in a similar class of materials, strontium aluminates, the emission color of Eu(2+) can be tuned over a wide spectral region.

Er(3+)/Yb(3+) upconverters for InGaP solar cells under concentrated broadband illumination.

The inability of solar cell materials to convert all incident photon energy into electrical current, provides a fundamental limit to the solar cell efficiency; the so called Shockley-Queisser (SQ) limit. A process termed upconversion provides a pathway to convert otherwise unabsorbed low energy photons passing through the solar cell into higher energy photons, which subsequently can be redirected back to the solar cell. The combination of a semi-transparent InGaP solar cell with lanthanide upconverters, consisting of ytterbium and erbium ions doped in three different host materials (Gd2O2S, Y2O3 and NaYF4) is investigated. Using sub-band gap light of wavelength range 890 nm to 1045 nm with a total accumulated power density of 2.7 kW m(-2), a distinct photocurrent was measured in the solar cell when the upconverters were applied whereas a zero current was measured without upconverter. Furthermore, a time delay between excitation and emission was observed for all upconverter systems which can be explained by energy transfer upconversion. Also, a quadratic dependence on the illumination intensity was observed for the NaYF4 and Y2O3 host material upconverters. The Gd2O2S host material upconverter deviated from the quadratic illumination intensity dependence towards linear behaviour, which can be attributed to saturation effects occurring at higher illumination power densities.

Direct band gap wurtzite gallium phosphide nanowires.

The main challenge for light-emitting diodes is to increase the efficiency in the green part of the spectrum. Gallium phosphide (GaP) with the normal cubic crystal structure has an indirect band gap, which severely limits the green emission efficiency. Band structure calculations have predicted a direct band gap for wurtzite GaP. Here, we report the fabrication of GaP nanowires with pure hexagonal crystal structure and demonstrate the direct nature of the band gap. We observe strong photoluminescence at a wavelength of 594 nm with short lifetime, typical for a direct band gap. Furthermore, by incorporation of aluminum or arsenic in the GaP nanowires, the emitted wavelength is tuned across an important range of the visible light spectrum (555-690 nm). This approach of crystal structure engineering enables new pathways to tailor materials properties enhancing the functionality.

Influence of cell-internalization on relaxometric, optical and compositional properties of targeted paramagnetic quantum dot micelles.

Quantum dot micelles (pQDs) with a paramagnetic coating are promising nanoparticles for bimodal molecular imaging. Their bright fluorescence allows for optical detection, while their Gd payload enables visualization with contrast-enhanced MRI. A popular approach in molecular MRI is the targeting of abundantly expressed cell surface receptors. Ligand-receptor binding often results in cell internalization of the targeted contrast agent. The interpretation of molecular imaging with pQDs therefore requires knowledge about the consequences of cellular internalization for their relaxometric, optical and compositional properties. To study these, Cd-containing core-shell-shell QDs coated with a monolayer of lipids, of which 50 mol% was a Gd-containing lipid, were incubated with human umbilical vein-derived endothelial cells (HUVECs) for up to 24 h. α(ν) ß(3)-integrin targeted (RGD) and non-targeted (NT) pQDs were compared. pQDs uptake was monitored by fluorescence microscopy, FACS, ICP-MS, relaxometry and MRI. Cell-associated pQDs displayed longitudinal relaxation rates and fluorescent intensities which were linear with the cell-associated Gd and Cd concentrations, implying that the Gd and Cd uptake by HUVECs can be quantified using relaxometric and optical measurements, respectively. However, the Gd-to-Cd molar ratio in pellets of pQD-incubated cells was consistently higher than the Gd-to-Cd molar ratio of the pQDs as prepared. It is proposed that this increase in Gd-to-Cd molar ratio was due to non-specific lipid-transfer between the pQDs and the cellular membranes. These findings show that, in the case of contrast agents that are formed by non-covalent interactions, experimental procedures are needed with which representative components of the probes can be monitored.

The effect of temperature and dot size on the spectral properties of colloidal InP/ZnS core-shell quantum dots.

Visual color changes between 300 and 510 K were observed in the photoluminescence (PL) of colloidal InP/ZnS core-shell nanocrystals. A subsequent study of PL spectra in the range 2-510 K and fitting the temperature dependent line shift and line width to theoretical models show that the dominant (dephasing) interaction is due to scattering by acoustic phonons of about 23 meV. Low temperature photoluminescence excitation measurements show that the excitonic band gap depends approximately inversely linearly on the quantum dot size d, which is distinctly weaker than the dependence predicted by current theories.

High-frequency EPR and ENDOR spectroscopy on semiconductor nanocrystals.

EPR and ENDOR experiments at 95 GHz on ZnO nanoparticles reveal the presence of shallow donors related to interstitial Li and Na atoms. The experiments allowed, for the first time, to probe the effect of confinement on the shape of the electronic wave function. In addition, it is observed that the 67Zn nuclear spins become polarized upon saturation of the EPR transition. This Overhauser effect is induced by the zero-point vibrations of the phonon system in the nanoparticles.

Reappearance of fine structure as a probe of lifetime broadening mechanisms in the 4f(N) --> 4f(N-1)5d excitation spectra of Tb3+, Er3+, and Tm3+ in CaF2 and LiYF4.

High-energy transitions in the 4f(N) -->4f(N-1)5d excitation spectra of lanthanide ions in host crystals are usually broadened due to the short excited-state lifetimes, whereas low-energy transitions, with longer excited-state lifetimes, may show fine structure. We report the surprising observation that for some materials fine structure is observed not only for the low-energy excitation bands but also for some high-energy transitions. The excited states that display fine structure are those for which the 5d electron is in the lowest crystal-field level but the 4f(N-1) core is in a highly excited state, indicating that the broadening depends only on the energy of the 5d electron and not on the total energy of the 4f(N-1)5d excited state.

Visible quantum cutting in LiGdF4:Eu3+ through downconversion

For mercury-free fluorescent lamps and plasma display panels, alternative luminescent materials are required for the efficient conversion of vacuum ultraviolet radiation to visible light. Quantum cutting involving the emission of two visible photons for each vacuum ultraviolet photon absorbed is demonstrated in Eu3+-doped LiGdF4 with the concept of downconversion. Upon excitation of Gd3+ with a high-energy photon, two visible photons can be emitted by Eu3+ through an efficient two-step energy transfer from Gd3+ to Eu3+, with a quantum efficiency that approaches 200 percent.