Oral dydrogesterone versus micronized vaginal progesterone for luteal phase support: a double-blind crossover study investigating pharmacokinetics and impact on the endometrium.

STUDY QUESTION: How do plasma progesterone (P) and dydrogesterone (D) concentrations together with endometrial histology, transcriptomic signatures, and immune cell composition differ when oral dydrogesterone (O-DYD) or micronized vaginal progesterone (MVP) is used for luteal phase support (LPS)? SUMMARY ANSWER: Although after O-DYD intake, even at steady-state, plasma D and 20αdihydrodydrogesterone (DHD) concentrations spiked in comparison to P concentrations, a similar endometrial signature was observed by histological and transcriptomic analysis of the endometrium. WHAT IS KNOWN ALREADY: O-DYD for LPS has been proven to be noninferior compared to MVP in two phase III randomized controlled trials. Additionally, a combined individual participant data and aggregate data meta-analysis indicated that a higher pregnancy rate and live birth rate may be obtained in women receiving O-DYD versus MVP for LPS in fresh IVF/ICSI cycles. Little data are available on the pharmacokinetic (PK) profiles of O-DYD versus MVP and their potential molecular differences at the level of the reproductive organs, particularly at the endometrial level. STUDY DESIGN, SIZE, DURATION: Thirty oocyte donors were planned to undergo two ovarian stimulation (OS) cycles with dual triggering (1.000 IU hCG + 0.2 mg triptorelin), each followed by 1 week of LPS: O-DYD or MVP, in a randomized, cross-over, double-blind, double-dummy fashion. On both the first and eighth days of LPS, serial blood samples upon first dosing were harvested for plasma D, DHD, and P concentration analyses. On Day 8 of LPS, an endometrial biopsy was collected for histologic examination, transcriptomics, and immune cell analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: All oocyte donors were <35 years old, had regular menstrual cycles, no intrauterine contraceptive device, anti-Müllerian hormone within normal range and a BMI ≤29 kg/m2. OS was performed on a GnRH antagonist protocol followed by dual triggering (1.000 IU hCG + 0.2 mg triptorelin) as soon as ≥3 follicles of 20 mm were present. Following oocyte retrieval, subjects initiated LPS consisting of MVP 200 mg or O-DYD 10 mg, both three times daily. D, DHD, and P plasma levels were measured using liquid chromatography-tandem mass spectrometry. Histological assessment was carried out using the Noyes criteria. Endometrial RNA-sequencing was performed for individual biopsies and differential gene expression was analyzed. Endometrial single-cell suspensions were created followed by flow cytometry for immune cell typing. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 21 women completed the entire study protocol. Subjects and stimulation characteristics were found to be similar between groups. Following the first dose of O-DYD, the average observed maximal plasma concentrations (Cmax) for D and DHD were 2.9 and 77 ng/ml, respectively. The Cmax for D and DHD was reached after 1.5 and 1.6 h (=Tmax), respectively. On the eighth day of LPS, the first administration of that day gave rise to a Cmax of 3.6 and 88 ng/ml for D and DHD, respectively. For both, the observed Tmax was 1.5 h. Following the first dose of MVP, the Cmax for P was 16 ng/ml with a Tmax of 4.2 h. On the eighth day of LPS, the first administration of that day showed a Cmax for P of 21 ng/ml with a Tmax of 7.3 h. All 42 biopsies showed endometrium in the secretory phase. The mean cycle day was 23.9 (±1.2) in the O-DYD group versus 24.0 (±1.3) in the MVP group. RNA-sequencing did not reveal significantly differentially expressed genes between samples of both study groups. The average Euclidean distance between samples following O-DYD was significantly lower than following MVP (respectively 12.1 versus 18.8, Mann-Whitney P = 6.98e-14). Immune cell profiling showed a decrease of CD3 T-cell, γδ T-cell, and B-cell frequencies after MVP treatment compared to O-DYD, while the frequency of natural killer (NK) cells was significantly increased. LIMITATIONS, REASONS FOR CAUTION: The main reason for caution is the small sample size, given the basic research nature of the project. The plasma concentrations are best estimates as this was not a formal PK study. Whole tissue bulk RNA-sequencing has been performed not correcting for bias caused by different tissue compositions across biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study comparing O-DYD/MVP, head-to-head, in a randomized design on a molecular level in IVF/ICSI. Plasma serum concentrations suggest that administration frequency is important, in addition to dose, specifically for O-DYD showing a rapid clearance. The molecular endometrial data are overall comparable and thus support the previously reported noninferior reproductive outcomes for O-DYD as compared to MVP. Further research is needed to explore the smaller intersample distance following O-DYD and the subtle changes detected in endometrial immune cells. STUDY FUNDING/COMPETING INTEREST(S): Not related to this work, C.Bl. has received honoraria for lectures, presentations, manuscript writing, educational events, or scientific advice from Abbott, Ferring, Organon, Cooper Surgical, Gedeon-Richter, IBSA, and Merck. H.T. has received honoraria for lectures, presentations, manuscript writing, educational events, or scientific advice from Abbott, Ferring, Cooper Surgical, Gedeon-Richter, Cook, and Goodlife. S.M. has received honoraria for lectures, presentations, educational events, or scientific advice from Abbott, Cooper Surgical, Gedeon-Richter, IBSA, and Merck and Oxolife. G.G. has received honoraria for lectures, presentations, educational events, or scientific advice from Merck, MSD, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, and OxoLife. S.V.-S. is listed as inventor on two patents (WO2019115755A1 and WO2022073973A1), which are not related to this work. TRIAL REGISTRATION NUMBER: EUDRACT 2018-000105-23.

Dual UHPLC-HRMS Metabolomics and Lipidomics and Automated Data Processing Workflow for Comprehensive High-Throughput Gut Phenotyping.

In recent years, feces has surfaced as the matrix of choice for investigating the gut microbiome-health axis because of its non-invasive sampling and the unique reflection it offers of an individual's lifestyle. In cohort studies where the number of samples required is large, but availability is scarce, a clear need exists for high-throughput analyses. Such analyses should combine a wide physicochemical range of molecules with a minimal amount of sample and resources and downstream data processing workflows that are as automated and time efficient as possible. We present a dual fecal extraction and ultra high performance liquid chromatography-high resolution-quadrupole-orbitrap-mass spectrometry (UHPLC-HR-Q-Orbitrap-MS)-based workflow that enables widely targeted and untargeted metabolome and lipidome analysis. A total of 836 in-house standards were analyzed, of which 360 metabolites and 132 lipids were consequently detected in feces. Their targeted profiling was validated successfully with respect to repeatability (78% CV < 20%), reproducibility (82% CV < 20%), and linearity (81% R2 > 0.9), while also enabling holistic untargeted fingerprinting (15,319 features, CV < 30%). To automate targeted processing, we optimized an R-based targeted peak extraction (TaPEx) algorithm relying on a database comprising retention time and mass-to-charge ratio (360 metabolites and 132 lipids), with batch-specific quality control curation. The latter was benchmarked toward vendor-specific targeted and untargeted software and our isotopologue parameter optimization/XCMS-based untargeted pipeline in LifeLines Deep cohort samples (n = 97). TaPEx clearly outperformed the untargeted approaches (81.3 vs 56.7-66.0% compounds detected). Finally, our novel dual fecal metabolomics-lipidomics-TaPEx method was successfully applied to Flemish Gut Flora Project cohort (n = 292) samples, leading to a sample-to-result time reduction of 60%.

Effect of obesity on gastrointestinal transit, pressure and pH using a wireless motility capsule.

BACKGROUND: Despite the increasing prevalence and medical burden of obesity, the understanding of gastrointestinal physiology in obesity is scarce, which hampers drug development. AIM: To investigate the effect of obesity and food intake on gastrointestinal transit, pressure and pH. MATERIAL AND METHODS: An exploratory cross-sectional study using a wireless motility capsule (SmartPill©) was performed in 11 participants with obesity and 11 age- and gender-matched participants with normal weight (group) in fasted and fed state (visit). During the first visit, the capsule was ingested after an overnight fast. During a second visit, the capsule was ingested after a nutritional drink to simulate fed state. Linear mixed models were constructed to compare segmental gastrointestinal transit, pressure and pH between groups (obesity or control) and within every group (fasted or fed). RESULTS: Food intake slowed gastric emptying in both groups (both P < 0.0001), though food-induced gastric contractility was higher in participants with obesity compared to controls (P = 0.02). In the small intestine, a higher contractility (P = 0.001), shorter transit (P = 0.04) and lower median pH (P = 0.002) was observed in participants with obesity compared to controls. No differences were observed for colonic measurements. CONCLUSION: Obesity has a profound impact on gastrointestinal physiology, which should be taken into account for drug development.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Caspase deficiency alters the murine gut microbiome.

Caspases are aspartate-specific cysteine proteases that have an essential role in apoptosis and inflammation, and contribute to the maintenance of homeostasis in the intestine. These facts, together with the knowledge that caspases are implicated in host-microbe crosstalk, prompted us to investigate the effect of caspase (Casp)1, -3 and -7 deficiency on the composition of the murine gut microbiota. We observed significant changes in the abundance of the Firmicutes and Bacteroidetes phyla, in particular the Lachnospiraceae, Porphyromonodaceae and Prevotellacea families, when comparing Casp-1, -7 and -3 knockout mice with wild-type mice. Our data point toward an intricate relationship between these caspases and the composition of the murine gut microflora.

First Reports of Brown Fruit Rot on Sweet Cherry (Prunus avium) and Plum (P. domestica) and Shoot Blight on Apricot (P. armeniaca), Kwanzan Cherry (P. serrulata), and Sweet Cherry (P. avium) Caused by Monilinia laxa in New York, Rhode Island, and Massachusetts.

In the eastern United States, Monilinia laxa (Aderh. & Ruhl.) Honey has only been reported on tart cherry in New York (NY) (1). As a result of considerable rain in May of 2009 and 2011, an ornamental planting of Kwanzan cherries in Middletown, Rhode Island (RI), a planting of sweet cherry cvs. Ulster, Hedelfingen, Sam, and Lapins in Lanesboro, Massachusetts (MA), and plantings of apricot cvs. Harcot and Hargrande in Albion, Aurora, and Geneva, NY, and Harogem in Lanesboro, MA developed severe shoot blight (>15 to 100% of first-year shoots). Blighted shoots were wilted with the blight encompassing the distal end and often extending into second-year tissue with a distinct sunken margin. Leaves on symptomatic shoots had flushed, but were blighted and light brown. Blossom spurs were often blighted and gummosis was frequently observed at the base. In these same years, sweet cherry cv. Black Gold in Walworth, NY and plum cv. Stanley in Olcott, NY developed severe fruit rot (35 to 70% incidence). Plantings suffering from fruit rot had fruit lesions that began as pale brown, soft lesions with indiscriminant margins that covered 15 to 85% of the fruit surface area. Many blighted spurs, shoot tissues, and infected fruit were sporulating with tan-to-buff colored conidia produced in chains. From each planting with shoot blight, shoot tips were removed for pathogen isolation. Sections of symptomatic shoots (5 cm long) were surface sterilized in 0.6% NaOCl for 1 min and rinsed in sterile dH20. From plantings displaying blighted spurs or fruit rot, isolation was attempted directly from sporulating tissue. Cross sections of sterilized shoot tissue (3 mm thick) or tufts of sporulation from fruit and spurs were placed on potato dextrose agar amended with 50 µg/ml of streptomycin sulfate. After incubation at 24°C for 5 days, colonies with lobed margins, commonly described for M. laxa (4), were obtained. Several colonies resembling M. fructicola were isolated from all locations, but the majority of isolates from spurs and shoots resembled M. laxa. Conidia from both colony morphotypes were lemon shaped, but as expected, those from putative M. laxa isolates were smaller (10.75 × 12.0 µm) compared with those from putative M. fructicola isolates (15.75 × 18.25 µm) (4). Confirmation of M. laxa was further achieved by PCR amplification of the ß-tubulin gene using M. laxa-specific primers as previously described (3). Pathogenicity of M. laxa isolates was proven by inoculating fruit of the stone fruit crop from which they were isolated as previously described (2). Fruit inoculated with M. laxa developed brown, soft sporulating lesions identical to the original observations, while those inoculated with water remained healthy. M. laxa was reisolated from symptomatic shoots and spurs, but not from water-inoculated tissues. The presence of M. laxa has been reported on tart cherries in NY (1), but to our knowledge, this is the first instance of economically devastating shoot blight on apricot in NY and MA, ornamental cherry in RI, and sweet cherry in MA and fruit rot on sweet cherry and plum in NY caused by M. laxa. In wet seasons, stone fruit growers may need to revise their chemical management programs to better prepare for M. laxa epidemics on several stone fruit species. References: (1) K. D. Cox and S. M. Villani. Plant Dis. 94:783, 2010. (2) K. D. Cox and S. M. Villani. Plant Dis. 95:828, 2011. (3) Z. Ma et al. Pest Manag. Sci. 61:449, 2005. J.M. (4) G. C. M. van Leeuwen and H. A. van Kesteren. Can. J. Bot. 76:2042, 1998.

Quantitative assessment of protein function prediction from metagenomics shotgun sequences.

To assess the potential of protein function prediction in environmental genomics data, we analyzed shotgun sequences from four diverse and complex habitats. Using homology searches as well as customized gene neighborhood methods that incorporate intergenic and evolutionary distances, we inferred specific functions for 76% of the 1.4 million predicted ORFs in these samples (83% when nonspecific functions are considered). Surprisingly, these fractions are only slightly smaller than the corresponding ones in completely sequenced genomes (83% and 86%, respectively, by using the same methodology) and considerably higher than previously thought. For as many as 75,448 ORFs (5% of the total), only neighborhood methods can assign functions, illustrated here by a previously undescribed gene associated with the well characterized heme biosynthesis operon and a potential transcription factor that might regulate a coupling between fatty acid biosynthesis and degradation. Our results further suggest that, although functions can be inferred for most proteins on earth, many functions remain to be discovered in numerous small, rare protein families.

Quantitative phylogenetic assessment of microbial communities in diverse environments.

The taxonomic composition of environmental communities is an important indicator of their ecology and function. We used a set of protein-coding marker genes, extracted from large-scale environmental shotgun sequencing data, to provide a more direct, quantitative, and accurate picture of community composition than that provided by traditional ribosomal RNA-based approaches depending on the polymerase chain reaction. Mapping marker genes from four diverse environmental data sets onto a reference species phylogeny shows that certain communities evolve faster than others. The method also enables determination of preferred habitats for entire microbial clades and provides evidence that such habitat preferences are often remarkably stable over time.

Deletions involving long-range conserved nongenic sequences upstream and downstream of FOXL2 as a novel disease-causing mechanism in blepharophimosis syndrome.

The expression of a gene requires not only a normal coding sequence but also intact regulatory regions, which can be located at large distances from the target genes, as demonstrated for an increasing number of developmental genes. In previous mutation studies of the role of FOXL2 in blepharophimosis syndrome (BPES), we identified intragenic mutations in 70% of our patients. Three translocation breakpoints upstream of FOXL2 in patients with BPES suggested a position effect. Here, we identified novel microdeletions outside of FOXL2 in cases of sporadic and familial BPES. Specifically, four rearrangements, with an overlap of 126 kb, are located 230 kb upstream of FOXL2, telomeric to the reported translocation breakpoints. Moreover, the shortest region of deletion overlap (SRO) contains several conserved nongenic sequences (CNGs) harboring putative transcription-factor binding sites and representing potential long-range cis-regulatory elements. Interestingly, the human region orthologous to the 12-kb sequence deleted in the polled intersex syndrome in goat, which is an animal model for BPES, is contained in this SRO, providing evidence of human-goat conservation of FOXL2 expression and of the mutational mechanism. Surprisingly, in a fifth family with BPES, one rearrangement was found downstream of FOXL2. In addition, we report nine novel rearrangements encompassing FOXL2 that range from partial gene deletions to submicroscopic deletions. Overall, genomic rearrangements encompassing or outside of FOXL2 account for 16% of all molecular defects found in our families with BPES. In summary, this is the first report of extragenic deletions in BPES, providing further evidence of potential long-range cis-regulatory elements regulating FOXL2 expression. It contributes to the enlarging group of developmental diseases caused by defective distant regulation of gene expression. Finally, we demonstrate that CNGs are candidate regions for genomic rearrangements in developmental genes.

Molecular characterization of Arabidopsis PHO80-like proteins, a novel class of CDKA;1-interacting cyclins.

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids ("cyclin box") displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.

The dysphonia severity index: an objective measure of vocal quality based on a multiparameter approach.

The vocal quality of a patient is modeled by means of a Dysphonia Severity Index (DSI), which is designed to establish an objective and quantitative correlate of the perceived vocal quality. The DSI is based on the weighted combination of the following selected set of voice measurements: highest frequency (F(0)-High in Hz), lowest intensity (I-Low in dB), maximum phonation time (MPT in s), and jitter (%). The DSI is derived from a multivariate analysis of 387 subjects with the goal of describing, purely based on objective measures, the perceived voice quality. It is constructed as DSI = 0.13 x MPT + 0.0053 x F(0)-High - 0.26 x I-Low - 1.18 x Jitter (%) + 12.4. The DSI for perceptually normal voices equals +5 and for severely dysphonic voices -5. The more negative the patient's index, the worse is his or her vocal quality. As such, the DSI is especially useful to evaluate therapeutic evolution of dysphonic patients. Additionally, there is a high correlation between the DSI and the Voice Handicap Index score.

Aerodynamic measurements of voice production.

This article reviews measurement techniques, normative results and clinical uses of the aerodynamic voice measures maximum phonation time (MPT), vital capacity (VC) and phonation quotient (PQ). Variations in measurement procedures have proven to yield different results. Simple test techniques have resulted in the following normative data: 22.2 sec for MPT, 4800 ml for VC, 269 ml/sec for PQ in adult men, and 18.4 sec for MPT, 3500 ml for VC and 233 ml/sec for PQ in adult women. None of the three parameters can be used to diagnose voice disorders, but each of them is useful for evaluating their degree and for monitoring their evolution. MPT appears to be the most sensitive measure of the three.

An analysis of spontaneous conversational speech fluency in children with acquired aphasia.

We report on an instrumental analysis of spontaneous conversational speech (SCS) fluency in acquired childhood aphasia (ACA). Tape-recorded SCS samples of 25 children with ACA (clinical judgment: 12 nonfluent and 13 fluent), and of 12 dysarthric and 12 nonaphasic and nondysarthric right hemisphere injured children were analysed in order to: (1) investigate whether a more refined analysis can objectively contribute to the differentiation of patients who were labelled as fluent or nonfluent on the basis of a clinical judgment: (2) verify whether an instrumental analysis of phonation duration does confirm the subjective estimation of verbal rate (i.e. the number of words produced in a unit of time) in groups of children with acquired neurogenic speech/language disorders frequently met in clinical practice. The results are: (1) phonation rate (i.e. the vocalization percentage) seems to represent an adequate variable to distinguish clinically diagnosed nonfluent aphasic children from speech/language impaired children belonging to other clinical groups of acquired neurogenic speech/language disorders; (2) the verbal rate is highly correlated to the phonation rate in all investigated groups except the dysarthric one. We suggest the instrumental method discussed here might contribute to the differential diagnosis between dysarthric and aphasic disturbances in the acute stage of the disease. Concerning the study of ACA, the main issue of the present investigation is that an objective fluency measurement has succeeded in identifying aphasic children who obviously do not fit in with the standard doctrine on ACA, which claims that ACA is invariably nonfluent irrespective of lesion location.

[Preventive cryotherapy of the retina and cataract surgery]. / Cryothérapie préventive de la rétine et chirurgie de la cataracte.

The prevention of aphakic retinal detachment by cryotherapy is controversial. No study up to now has compared results. We performed a retrospective study of two groups of patients: both groups consisted of patients with mature cataract and certain risk factors for retinal detachment (myopia, previous retinal detachment in the eye in study or in the fellow eye etc.) in the first group (n = 299) circumferential cryopexy has been performed prior to cataract extraction while in the second group (n = 149) no prophylaxis has been done. Comparison between the two groups did not reveal any protection afforded by prior cryotherapy: despite the prophylactic cryotherapy, 7 patients (2.3%) in the first group developed a retinal detachment post cataract extraction; two patients (1.3%) in the second group developed a retinal detachment; both were high-risk patients. These results lead us to conclude that prophylactic treatment does not necessarily prevent this complication. The best prophylaxis appears to be careful regularly performed examination of the peripheral retina both pre- and post-operatively.

[Videostroboscopy of the larynx]. / Videostroboscopie van de larynx.

The technique of telescopic videostroboscopy permits simultaneous audio and video recording of a laryngoscopy and a subsequent stroboscopy of the larynx during the same examination. The combination of laryngoscopy and stroboscopy is very favorable not only because stroboscopy is not possible in all pathological cases, but also because each of both methods can reveal facts the other does not. In addition, the possibility of playing back the video tapes as often as desirable is an important diagnostic and pedagogic advantage.

Cry analysis in infants with infectious and congenital disorders of the larynx.

Thirty induced pain cries from the same number of infants with infectious or congenital disorders of the larynx (infectious laryngitis, laryngomalacia, paresis of the recurrent nerve and subglottic stricture) have been analyzed by means of sound spectrography. For each cry 21 phonetic features have been evaluated. This cry material was then compared with 120 pain cries from healthy infants. In the pathological group a significant increase could be noted in the occurrence of the following cry attributes: second pause, abnormal melody types (rising, falling-rising, flat and no melody types), instability of the fundamental frequency, bi-phonation, vibrato, half-voiced voice quality, noise concentration, and inspiratory stridor. Furthermore, a significant decrease could be noticed in the occurrence of voiceless cries, falling and rising-falling melody types, and glottal roll. These findings show that such spectrographic features as very high maximum and minimum pitch, bi-phonation, gliding, and abnormal melody type occur more often in cries of infants with central nervous system involvement. Moreover, such parameters as instability of the fundamental frequency and noise concentration as indicators of neurologic disorders need further consideration. Except features of inspiratory stridor, this study revealed no really clear parameters typical of peripheral diseases of the vocal tract.