Abstract Study question Is it possible to apply RNA-sequencing to assisted reproductive technology (ART) to predict blastocyst quality? Summary answer We identified 14 commonly identified differentially expressed genes by grouping blastocysts according to three distinct parameters such as developmental potential, maternal age, and Gardner score What is known already In ART, while the selection of a suitable embryo for transfer is critical for a successful pregnancy, neither preimplantation genetic testing nor morphological and chronological evaluation of the embryo can fully guarantee a successful pregnancy. Recently, transcriptional events in early human embryonic development have been analyzed using RNA-sequencing (RNA-seq) and researchers are attempting to apply this information to ART. We have reported that 96 differentially expressed genes (DEGs) were identified using RNA-seq of each inner cell mass (ICM) and trophectoderm (TE) in blastocysts classified according to the developmental potential which correlates with pregnancy rate at the ESHRE 37th annual meeting. Study design, size, duration After retrospectively analyzing 1,890 cases undergoing freeze-thaw blastocyst transfer from March 2018 to December 2020 to examine the correlation between blastocyst developmental potential and pregnancy rate, a total 13 blastocysts cryopreserved between February 2011 and September 2018, then scheduled for disposal and with consented, were subjected to RNA-seq to identify genes associated with pregnancy expectation. RNA-seq data were then examined whether common DEGs could be found when classified by maternal age and Gardner score, respectively. Participants/materials, setting, methods Blastocysts were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic with informed consent under ethical approval. TE cells and ICM cells were collected from blastocysts classified by developmental potential and subjected to RNA-seq to identify DEGs. In addition, RNA-seq data were regrouped by maternal age and Gardner score to find common DEGs. DEGs (q-value < 0.01) were identified using the R package “DESeq2” (version 1.32.0). Main results and the role of chance When the RNA-seq data obtained from blastocysts classified according to pregnancy expectation were re-grouped by maternal age and re-analyzed, we identified 7 and 17 genes that were down- and up-regulated in the elder group, respectively, in ICM. In TE, 2 and 12 genes were down- and up-regulated in the elder group, respectively. On the other hand, when re-grouped by Gardner score ( Limitations, reasons for caution Although we established an expected pregnancy rate concerning the degree of blastocyst development from retrospective clinical outcomes and used it as a surrogate marker for assigning biopsied blastocysts to different analysis groups, it remains unknown whether the gene expression profiles accurately reflect the pregnancy outcomes. Wider implications of the findings UCHL1 expression was commonly increased with lower blastocyst developmental potential, higher maternal age, and lower Gardner score. Since UCHL1 has been reported to be essential for blastocyst development in mice, our results suggest that UCHL1 may also be a marker of blastocyst quality in humans. Trial registration number not applicable

Abstract Study question Is it possible to identify the molecular factors that contribute to the implantation potential of blastocysts? Summary answer Genes correlated with expected pregnancy rate in trophectoderm (TE) and inner cell mass (ICM) respectively were identified, and aneuploidy alone couldn’t predict the pregnancy expectation. What is known already The selection of suitable embryos for transfer is critical for achieving successful pregnancy outcomes in assisted reproductive technology (ART). Although pre-implantation genetic testing for aneuploidy (PGT-A) as well as morphological and chronological evaluation of embryos, have been conducted in clinical practice, they do not fully guarantee successful pregnancy. Recently, transcriptional events in early human embryonic development have been analyzed using RNA-sequencing (RNA-seq) and researchers are attempting to apply this information to ART. Study design, size, duration To determine the correlation between blastocyst evaluation and pregnancy rate, we retrospectively analyzed 1,890 cases underwent frozen-thawed blastocyst transfer from March 2018 to December 2020. A total of 13 blastocysts that were cryopreserved for clinical use between February 2011 and September 2018, then scheduled for disposal and with consented for research, were subjected to RNA-seq without distinguishing between conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI). Participants/materials, setting, methods Blastocysts were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic with informed consent under ethical approval. TE and ICM cells were collected from blastocysts by using a micromanipulator and then subjected to RNA-seq. Gene expression analysis and digital karyotyping using RNA-seq were performed simultaneously for TE and ICM cells, respectively. One-way analysis of variance, chi-square test and Tukey's multiple comparison test were used for this study. Main results and the role of chance Blastocysts were classified into three groups to correlate with pregnancy rates based on the diameter of the blastocyst and the time to reach this size: those taking less than 130 h to reach a diameter of > 170 μm (Group 1, n = 676), those taking more than 140 h to reach a diameter of < 180 μm (Group 2, n = 158), and the rest (Group 3, n = 1,056). The pregnancy rates of Groups 1, 2 and 3 were 59.0%, 16.5%, and 34.2%, respectively (p < 0.01). Assessing the differences in overall transcripts correlated between Group 1 (n = 5), Group 2 (n = 4), and Group 3 (n = 4), 26 and 67 differentially expressed genes (DEGs) were identified in ICM and TE cells, respectively. Importantly, downregulated genes in TE of blastocysts with lower expectation of pregnancy included tight junction-related genes, such as CXADR, CLDN10, and ATP1B1, which were implicated in peri-implantation development. Digital karyotyping revealed karyotypic abnormalities and mosaicism in all groups with no common abnormalities observed, suggesting that aneuploidy alone cannot predict the pregnancy expectation. Limitations, reasons for caution Although 93 genes potentially related to implantation have been identified, it is still unclear how these genes are involved in implantation. In vitro implantation models using human embryos and artificial embryos currently under development are expected to contribute to the elucidation of the functions of these genes. Wider implications of the findings Our results provide reliable candidates for genes that could allow for non-invasive selection of high-quality blastocysts for ART and add to the knowledge base of transcriptional events in human peri-implantation development. Trial registration number not applicable

The objective of this study is to clearly assess the surface energy effect of co-spray-dried mannitol (co-SDM) particles with polyethylene glycol (PEG) on the aerosolization performance in a dry powder inhalation (DPI) formulation. From the results of in vitro deposition studies in the model DPI formulation containing salbutamol sulphate (SS), the effect of PEG molecular weight (MW) and formulated amount of PEG in the co-SDM on the aerosolization performance was evaluated. The fine particle fraction (FPF) of SS in the formulation containing coSDM with PEG 20000 was superior to that in the formulation containing spray-dried mannitol (SDM) without PEG. On the other hand, the FPFs of SS were found to be influenced by the PEG ratio and MW of PEG used in the formulation of co-SDM with PEG. The surface energies of co-SDMs with PEG were measured by an IGC technique. It was considered that the lower dispersive component of the surface energy of co-SDM may contribute to a higher FPF of SS when using PEG with a high MW. Moreover, the elevation of the basic (electron donor) energy might result in the decrease of FPF of SS when using PEG with low MWs. The elevation of the basic (electron donor) energy of a co-SDM would relate to both/either the difference in the adhesion force to the SS and/or the difference in the hygroscopicity of each PEG.

The design of a carrier particle is important for the development of dry powder inhalation (DPI) formulations. Most DPI formulations rely on lactose as a carrier particle; however, lactose has several disadvantages with DPI formulations. In this study we focused on mannitol as an alternative carrier to lactose. Spray drying was reported as challenging to produce α-mannitol, as β-mannitol is obtained in almost all cases. The polymorphic state of a carrier particle is reported as one of the properties which affect the aerosolization performance of DPI formulations. To control the polymorphic state and to evaluate its effect for the aerosolization performance was considered to be important for the development of DPI formulations. The purpose of this study is to control the polymorphic state and to selectively produce αmannitol by spray drying, instead of β-mannitol. The effect of polyethylene glycol (PEG) 4000 on a polymorphic state of mannitol was investigated. Powder X-ray diffraction studies showed that spray drying mannitol without PEG 4000 completely produced β-mannitol. In contrast, mannitol/PEG 4000 (PEG 4000 concentration was above 1%) co-spray dried products were found to be completely α-mannitol. It can be estimated that the molecular mobility of mannitol in the presence of PEG 4000 must have been slower than that of mannitol alone and reduced molecular mobility inhibited the transformation from α-mannitol nuclei into β-mannitol nuclei during the spray drying process. Consequently, controlling the polymorphic state and selectively producing α-mannitol by spray drying was successful by adding above 1% of PEG 4000.

This study aims to produce and test the performance of novel crystalline respirable particles containing two low-dose active ingredients and mannitol. This technique overcomes the usual requirement of blending with lactose carriers in formulating combination inhalation products. Ternary powders were produced by co-spray drying solutions containing an inhaled corticosteroid (ICS), a long-acting beta2-agonist (LABA), and mannitol as a crystalline excipient. Two formulations comprising widely used ICS and LABA were studied: budesonide/formoterol fumarate dihydrate/mannitol (B/F/M-SD) and fluticasone propionate/salmeterol xinafoate/mannitol (F/S/M-SD). Various physicochemical properties of the powders were analyzed. Aerosol performance was evaluated by dispersing each powder from an Aerolizer at 60 and 100 L/min into a Next Generation Impactor. We obtained partially hollow spherical particles (volume median diameters of 2 microm) with drug-enriched surfaces. Both formulations contained alpha-mannitol, and the ICSs were crystalline. The content of each drug component in the powder was found to conform to the theoretical dose. The ternary powders generated high fine particle fractions (>50% of the loaded dose), with concomitant drug deposition on the impactor stages. The aerosol performance of B/F/M-SD was maintained after storage over silica gel at 22 degrees C for 11 weeks. In conclusion, co-spray dried particles of ICS/LABA/M-SD were largely crystalline, stable and showed excellent aerosol performance. They may provide an attractive alternative strategy to develop combination products without lactose blends.

BACKGROUNDLiquid droplets can be spontaneously charged in the absence of applied electric fields by spraying. It has been shown by computational simulation that charges may influence particle deposition in the airways. The electrostatic properties of jet nebulized aerosols and their potential effects on lung deposition have hardly been studied. A modified electrical low pressure impactor (ELPI) was employed to characterize the aerosol charges generated from jet nebulized commercial products.METHODSThe charge and size measurements were conducted at 50% RH and 22 degrees C with a modified ELPI. Ventolin, Bricanyl, and Atrovent were nebulized using PARI LC Plus jet nebulizers coupled to a DeVilbiss Pulmo-Aide compressor. The aerosols were sampled in 30-sec durations. The drug deposits on the impactor stages were assayed chemically using high-performance liquid chromatography (HPLC). The charges of nebulized deionized water, isotonic saline, and the three commercial products diluted with saline were also measured to analyze the contributions of the major nebule ingredients on charging. No mass assays were performed on these runs.RESULTSAll three commercial nebules generated net negative charges. The magnitude of the charges reduced over the period of nebulization. Ventolin and Bricanyl yielded similar charge profiles. Highly variable charges were produced from deionized water. On the other hand, nebulized saline reproducibly generated net positive charges. Diluted commercial nebules showed charge polarity inversion. The charge profiles of diluted salbutamol and terbutaline solutions resembled those of saline, while the charges from diluted ipratropium solutions fluctuated near neutrality.CONCLUSIONSThe charge profiles were shown to be influenced by the concentration and physicochemical properties of the drugs, as well as the history of nebulization. The drugs may have unique isoelectric concentrations in saline at which the nebulized droplets would carry near-zero charges. According to results from computational simulation models in the literature, the numbers of elementary charges per droplet estimated from the data were not high enough to potentially affect lung deposition.

In our previous paper, we reported the inhalation properties of dry powder inhaler (DPI) formulations containing Compound A and mechanofusion-processed lactose carriers. The mechanofusion process with magnesium stearate (Mg-St) on the lactose carrier enhanced the fine particle fraction (FPF) value of the Andersen cascade impactor (ACI) study. The increase of FPF seemed to be associated with the increase of the dispersibility of drug particles. The objectives of this study were (1) to evaluate the applicability of lactose carrier mechanofusion-processed with Mg-St and (2) to examine the mechanism of FPF alteration by the mechanofusion process applied on the lactose carrier with or without additive. The inhalation profiles of DPI formulations containing four different pharmaceutical compounds were evaluated with an ACI. The dispersibility of the formulations was observed by particle size distribution measurement in the air stream and the adhesive force was measured bydirect separation method. It was found that higher FPF was obtained with lactose mechanofusion-processed with Mg-St as compared to control lactose carriers for all four compounds. This suggested that mechanofusion process with Mg-St is widely applicable in DPI formulations. The homogenization of surface adhesiveness was attributed to the increased FPF of the DPI including lactose mechanofusion-processed with Mg-St, as suggested by the combination of several physicochemical characteristics. Combination of different characterization methods would be of help to clarify the whole mechanism which defines the inhalation properties of DPI formulations.

A novel triple neurokinin receptor antagonist (TNRA) could have pharmaceutical efficacy for asthma and/or chronic obstructive pulmonary disease. TNRA is potentially developed as inhalation medicine. The aim of this investigation was to evaluate the applicability of dry powder inhaler (DPI) formulation for TNRA. DPI formulation containing lactose was used for this feasibility study. Mechanofusion process for surface modification was applied on lactose particles to prepare four different DPI formulations. The mixture of TNRA and lactose was administered to rats intratracheally using an insufflator. The deposition pattern and blood concentration profile of TNRA were evaluated. Although there was no significant difference in deposition on deep lungs between the four formulations, DPI formulations containing mechanofusion-processed lactose showed longer T(max) and t(1/2) and higher AUC(0-infinity) and MRT compared to that containing intact lactose. On the other hand, the contact angle measurement showed that the mechanofusion process decreased the polar part of the surface energy of the lactose. Therefore, the prolongation of the wetting of the formulated powder mixture seemed to delay the dissolution of TNRA deposited in respiratory tract. It was concluded that DPI formulation containing mechanofusion-processed lactose could be suitable for inhalation of TNRA.

The effect of lactose carrier surface property on the inhalation profile of dry powder inhaler (DPI) was evaluated using a micronized drug (Compound A) by inverse gas chromatography (IGC). Mechanofusion with magnesium stearate (Mg-St) or sucrose stearate increased the fine particle fraction (FPF), considered to be due to decrease in the interaction between Compound A and the lactose carrier. The effect of Compound A concentration on FPF was smaller in mechanofusion-processed lactose compared to intact lactose, especially when processed with Mg-St. The relationship between the IGC parameters of the lactose and FPF was also investigated. FPF increased as both the dispersive component of the surface energy and acidity similarity between the lactose carriers and Compound A increased. Although further investigation is necessary, it could be suggested that acidity similarity decreases the interaction between Compound A and lactose, thus contributing to the increase in the FPF. In conclusion, (1) mechanofusion with Mg-St or sucrose stearate could be an effective method to improve FPF of a DPI drug formulation; (2) IGC would be a valuable method to investigate the interaction between a drug and the DPI carrier; and (3) a relationship between surface acidity and inhalation profile was suggested.