The PRAMOS Study: PRostaglandin Analogues Monotherapy-Awareness Survey on Ocular Surface Involvement.

INTRODUCTION: Even though the local tolerance of prostaglandin (PG) analogues has improved drastically since the introduction of preservative-free (PF) eye drops, prescription patterns still vary widely among practitioners and between countries and could have an impact on the ocular surface of treated patients and, in consequence, their adherence. The aim of this study is to explore the prescribing patterns of PG analogues monotherapy in France and to evaluate their impact on ocular surface status. METHODS: This was a national multicenter cross-sectional observational study that was conducted by 18 glaucoma experts in France. Patients over 18 years of age and receiving monotherapy with topical PG analogues for the treatment of ocular hypertension and/or glaucoma, with no history of prior glaucoma surgery, were consecutively selected from the glaucoma outpatient clinics of participating physicians and underwent an ocular surface examination. RESULTS: A total of 344 eyes of 344 patients were enrolled between November 2022 and November 2023. Prescribed PG monotherapy was PF in 271 (78.7%) patients. Clinical history and ocular surface evaluation indicated that 79.4% of the study population (n = 273) presented with at least one symptom or clinical sign of dry eye and that three patients out of four had an unstable tear film. Subgroup analysis comparing preserved and PF PG analogues showed a higher prevalence of conjunctival hyperemia and corneal staining in the preserved group. Multivariate analysis identified conjunctival hyperemia as consistently associated with preservative use (odds ratio = 7.654; p = 0.003 for moderate conjunctival hyperemia). CONCLUSIONS: This study highlights the growing trend toward PF PG analogue prescriptions by specialists in France. However, ocular surface issues remain prevalent, impacting patient adherence and treatment efficacy. Comprehensive ocular surface examinations are crucial in glaucoma management to enhance long-term tolerance, compliance, and overall treatment success.

Comparison of In Vitro Corneal Permeation and In Vivo Ocular Bioavailability in Rabbits of Three Marketed Latanoprost Formulations.

BACKGROUND AND OBJECTIVE: All latanoprost formulations currently available for the treatment of glaucoma or ocular hypertension contain the same concentration of latanoprost (0.005%) but differ in excipients, which may affect corneal drug permeability or stability. This study aimed at comparing corneal penetration of three marketed latanoprost solutions with different excipient formulations in in vitro and in vivo drug permeability studies. METHODS: Three latanoprost formulations were tested under good laboratory practice conditions: a formulation containing benzalkonium chloride (BAK) but no surfactant (Preserved latanoprost); the same formulation except preservative-free (PF) without BAK or surfactant (SF) (PF SF latanoprost); and a different formulation without BAK but containing a non-ionic surfactant (MGHS 40 at 5%) combined with thickening agents (Carbomer 974P, Macrogol 4000) (PF latanoprost). Corneal permeation of latanoprost acid (LAT) was first determined in vitro using a reconstructed human corneal epithelium tissue. Then, in vivo pharmacokinetic studies were performed on pigmented rabbits, for which LAT concentration was measured in the aqueous humour (AH) and iris-ciliary body (ICB). RESULTS: In vitro, the cumulative transport of LAT was linear between 1 h and 4 h for preserved latanoprost and PF SF latanoprost, and LAT concentrations matched exactly at each timepoint. By contrast, the permeation of PF latanoprost was linear between 2 h and 12 h and was significantly lower than that of preserved latanoprost and PF SF latanoprost at 4 and 8 h (p < 0.001). In rabbits, the concentrations of LAT in AH and ICB were not statistically different between preserved latanoprost and PF SF latanoprost at each timepoint, except at 1 h in ICB (p = 0.005). By comparison, the LAT concentration of PF latanoprost was statistically (p < 0.05) lower than that of preserved latanoprost and PF SF latanoprost in AH and ICB from 0.5 to 3 h. CONCLUSION: BAK did not influence the corneal penetration of latanoprost in in vitro and in vivo studies. The formulation containing a non-ionic surfactant resulted in lower and slower ocular penetration compared with preserved or PF SF formulations. This raises questions about the relevance of BAK and some surfactants in enhancing corneal penetration of ocular formulations.

Altered DNA methylation associated with an abnormal liver phenotype in a cattle model with a high incidence of perinatal pathologies.

Cloning enables the generation of both clinically normal and pathological individuals from the same donor cells, and may therefore be a DNA sequence-independent driver of phenotypic variability. We took advantage of cattle clones with identical genotypes but different developmental abilities to investigate the role of epigenetic factors in perinatal mortality, a complex trait with increasing prevalence in dairy cattle. We studied livers from pathological clones dying during the perinatal period, clinically normal adult clones with the same genotypes as perinatal clones and conventional age-matched controls. The livers from deceased perinatal clones displayed histological lesions, modifications to quantitative histomorphometric and metabolic parameters such as glycogen storage and fatty acid composition, and an absence of birth-induced maturation. In a genome-wide epigenetic analysis, we identified DNA methylation patterns underlying these phenotypic alterations and targeting genes relevant to liver metabolism, including the type 2 diabetes gene TCF7L2. The adult clones were devoid of major phenotypic and epigenetic abnormalities in the liver, ruling out the effects of genotype on the phenotype observed. These results thus provide the first demonstration of a genome-wide association between DNA methylation and perinatal mortality in cattle, and highlight epigenetics as a driving force for phenotypic variability in farmed animals.

Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium.

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

Multicenter clinical trial of high-intensity focused ultrasound treatment in glaucoma patients without previous filtering surgery.

PURPOSE: To evaluate the efficacy and safety of the ultrasonic circular cyclocoagulation procedure in patients with open-angle glaucoma naïve of previous filtering surgery. METHODS: Prospective non-comparative interventional clinical study conducted in five French University Hospitals. Thirty eyes of 30 patients with open-angle glaucoma, intra-ocular pressure (IOP) > 21 mmHg and with no previous filtering glaucoma surgeries were sonicated with a probe comprising six piezoelectric transducers. The six transducers were activated with a 6-s exposure time. Complete ophthalmic examinations were performed before the procedure and at 1 day, 1 week, 1, 2, 3, 6 and 12 months after the procedure. Primary outcomes were qualified surgical success (defined as IOP reduction from baseline ≥20% and IOP > 5 mmHg with possible re-intervention and without hypotensive medication adjunction) and complete surgical success (defined as IOP reduction from baseline ≥20%, IOP > 5 mmHg and IOP < 21 mmHg with possible re-intervention and without hypotensive medication adjunction) at the last follow-up visit and vision-threatening complications. Secondary outcomes were mean IOP at each follow-up visit compared with baseline, medication use, complications and re-interventions. RESULTS: Intra-ocular pressure was significantly reduced (p < 0.05) from a mean pre-operative value of 28.2 ± 7.2 mmHg (n = 3.6 hypotensive medications) to 19.6 ± 7.9 mmHg at 12 months (n = 3.1 hypotensive medications and n = 1.1 procedures) (mean IOP reduction of 30%). Qualified success was achieved in 63% of eyes (19/30) (mean IOP reduction of 37% in these eyes) and complete success in 46.7% of eyes (14/30) (mean IOP reduction of 37% in these eyes) at the last follow-up. No major intra- or post-operative complications occurred. CONCLUSIONS: The UC(3) procedure seems to be an effective and well-tolerated method to reduce IOP in patients with open-angle glaucoma without previous filtering surgery.

Progression of visual field in patients with primary open-angle glaucoma - ProgF study 1.

PURPOSE: To evaluate the visual field rate of progression of patients with treated ocular hypertension (OHT) and primary open-angle glaucoma (POAG) in clinical practice, using the mean deviation (MD) and the visual field index (VFI). METHODS: Non-interventional cohort study. From a large multicentre database representative of the French population, 441 eyes of 228 patients with treated OHT or POAG followed up at least 6 years with Humphrey 24.2 Sita-Standard visual field examination at least twice a year were identified. From initial data, eyes were classified in five groups: 121 with OHT, 188 with early glaucoma (MD greater than -6 dB), 45 with moderate glaucoma (MD -6 to -12 dB), 41 with advanced glaucoma (MD -12 to -18 dB) and 46 with severe glaucoma (MD less than -18 dB). Rate of progression during the follow-up period was calculated using the trend analysis of the Guided Progression Analysis software. RESULTS: The mean duration of follow-up was 8.4 ± 2.7 years and the mean number of visual field, 18.4 ± 3.5. In eyes with OHT, rate of progression was -0.09 dB/year (-0.17%VFI/year). In eyes with POAG, rate of progression was -0.32 dB/year (-0.83%VFI/year) in eyes with early glaucoma, -0.52 dB/year (-1.81%VFI/year) in moderate glaucoma, -0.54 dB/year (-2.35%VFI/year) in advanced glaucoma and -0.45 dB/year (-1.97%VFI/year) in severe glaucoma. In eyes with POAG, a significant progression (p < 0.05) was detected in 159 of 320 eyes (49.7%) with trend analysis and 117 of 320 eyes (36.6%, likely progression) or 183 of 320 eyes (57.2%, possible and likely progression) with event analysis. CONCLUSIONS: Primary open-angle glaucoma is a progressive disease in the majority of patients despite cautioned treatment and follow-up. The rate of progression varies greatly among subjects.

Cyclocoagulation of the ciliary bodies by high-intensity focused ultrasound: a 12-month multicenter study.

PURPOSE: To evaluate the safety and efficacy of high-intensity focused ultrasound (HIFU) cyclocoagulation in reducing intraocular pressure (IOP) in patients with refractory glaucoma by using a novel miniaturized delivery device (EyeOP1). METHODS: We conducted a 12-month open-label multicenter prospective study (EyeMUST1 Study). Patients with primary (primary open-angle glaucoma [POAG]) or secondary refractory glaucoma were treated in two groups depending on the duration of each ultrasound shot (group 1: 4 seconds; group 2: 6 seconds). The primary efficacy outcome was based on IOP reduction at 6 and 12 months. RESULTS: Fifty-two patients were enrolled: 36 (69%) had POAG and 16 (31%) had secondary glaucoma. Group 1 (n = 24) and group 2 (n = 28) had similar demographics and baseline characteristics. In group 1, IOP was reduced from a mean preoperative value of 29.7 ± 7.7 mm Hg (n = 3.5 glaucoma medications) to a mean postoperative value of 21.3 ± 6.7 mm Hg (n = 3.5 glaucoma medications) and 20.1 ± 6.7 mm Hg (n = 3.2 glaucoma medications) at 6 and 12 months, respectively. In group 2, IOP was reduced from a mean preoperative value of 29.0 ± 7.4 mm Hg (n = 3.3 glaucoma medications) to a mean postoperative value of 20.2 ± 7.4 mm Hg (n = 3.4 glaucoma medications) and 18.5 ± 6.6 mm Hg (n = 3.5 glaucoma medications) at 6 and 12 months, respectively. At 12 months, the IOP reduction was sustained in both groups (32% IOP reduction in group 1 and 36% IOP reduction in group 2). The overall tolerance of the technique was good, with no serious adverse events. CONCLUSIONS: The new miniaturized HIFU EyeOP1 delivery device seems to be effective in decreasing IOP in patients with refractory glaucoma. The technology offers a good safety profile. (ClinicalTrials.gov number, NCT01338467.).

Reproducibility of macular ganglion cell-inner plexiform layer thickness measurement with cirrus HD-OCT in normal, hypertensive and glaucomatous eyes.

AIM: To evaluate the intraobserver and interobserver reproducibility of macular retinal ganglion cell-inner plexiform layer (GC-IPL) thickness measurement by automated detection on Optical Coherence Tomography (OCT) images in normal, hypertensive (ocular hypertensive (OHT)) and glaucomatous eyes. METHODS: A total of 138 eyes were enrolled in three groups: 69 normal, 35 OHT and 34 primary open-angle glaucoma eyes. All patients underwent a complete ocular examination, 24-2 automated perimetry, biometry and pachymetry. Macular imaging was performed in each eye using the Cirrus HD-OCT 4000 with software V.6.0. (Carl Zeiss Meditec, Dublin, California, USA) three times on the same day by each of two observers, and the GC analysis (GCA) algorithm provided parameters expressed as average, minimum and six sectoral GC-IPL thicknesses. Reproducibility was assessed by intraclass correlation coefficient (ICC), coefficient of variation (CV) and test-retest variability (TRTV) calculated as 1.96 times the SD. RESULTS: Mean GC-IPL thickness was 82.27 ± 7.37 µm, 76.84 ± 7.01 µm and 66.16 ± 11.16 µm in normal, OHT and glaucoma groups, respectively. GC-IPL thickness was significantly lower in glaucomatous eyes than in normal and OHT eyes (p<0.0001 for all parameters). In all groups, ICC ranged from 96.4 to 99.9% and 92.5 to 99.8%, CV ranged from 0.41 to 2.24% and 0.55 to 1.67%, and TRTV ranged from 0.61 to 2.64 µm and 0.83 to 2.22 µm for intraobserver and interobserver reproducibility, respectively. CONCLUSIONS: To the best of our knowledge, this is the first study of GCA algorithm reproducibility in normal, OHT and glaucomatous eyes. The reproducibility of GC-IPL thickness measurements using the Cirrus HD-OCT GCA algorithm was found to be highly satisfactory. GC-IPL thickness may be a promising new OCT parameter for analysis of ganglion cell damage in glaucoma.

Changes in WNT signaling-related gene expression associated with development and cloning in bovine extra-embryonic and endometrial tissues during the peri-implantation period.

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses.

Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development.

To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1ß and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.

Nutritional, lifestyle and environmental factors in ocular hypertension and primary open-angle glaucoma: an exploratory case-control study.

PURPOSE: To evaluate known and potential risk factors, including nutritional, lifestyle and environmental factors, differentiating patients with high-tension primary open-angle glaucoma (POAG) from control subjects with ocular hypertension (OHT). METHODS: In 2006-2007, 111 French ophthalmologists prospectively enrolled 339 cases of POAG and 339 age-matched controls with OHT. After a clinical examination with assessment of ocular risk factors, the ophthalmologist filled, during face-to-face interview, a detailed questionnaire developed by nutritionists and epidemiologist on lifestyle and environmental risk factors, including socio-demographic variables, dietary habits related to omega-3 fatty acids intake, smoking and alcohol drinking and professional exposure to pesticides and other chemicals. Associations of POAG with risk factors were estimated using conditional logistic regression, with adjustment for age, gender and duration of disease. RESULTS: In the final multivariate model, by comparison with OHT, POAG was significantly associated with more frequent use of pesticides during the professional life [OR = 2.65, 95% confidence interval (CI): 1.04-6.78, p = 0.04] and with low consumption of fatty fish (OR = 2.14, 95% CI: 1.10-4.17, p = 0.02) and walnuts (OR = 2.02, 95% CI: 1.18-3.47, p = 0.01). POAG was also associated with higher frequency of heavy smoking (40 pack-years or more, OR = 3.93, 95% CI: 1.12-13.80, p = 0.03) but not with moderate (20-40 pack-years) and light smoking (<20 pack-years). CONCLUSIONS: These exploratory observations suggest a protective effect of omega-3 fatty acids and a deleterious effect of heavy smoking and professional exposure to pesticides in POAG. This will need to be confirmed in future studies.

Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.

Prevalence and risk factors for ocular surface disease among patients treated over the long term for glaucoma or ocular hypertension.

Purpose. To determine the prevalence of ocular surface diseases and identify risk factors in a population of patients receiving antiglaucomatous eyedrops over the long term. Methods. An observational cross-sectional study was designed to investigate ocular surface signs and symptoms using simple clinical tools. An ocular surface disease intensity score was calculated based on 10 questions regarding ocular surface symptoms and signs with a 4-grade scale. Patients were classified into 3 groups (A, B, and C) according to this total score. A multinomial logistic regression was performed in order to identify risk factors for surface disease. Results. In an overall population of 516 patients, 49% belonged to group A, 30% to group B, and 21% to group C. The multivariate analysis showed that the following factors were correlated with the severity of ocular surface disease: patient age, number of daily eyedrops, past topical treatment changes for ocular intolerance (found in the history of 40% of the patients), intraocular pressure (found to be significantly higher in patients with more severe ocular surface disease), and glaucoma severity. Conclusions. Patients treated for primary open-angle glaucoma or ocular hypertension often have ocular surface diseases, more often and more severely in older patients receiving more drugs and presenting with more severe glaucoma. These high prevalence values might therefore have consequences on the burden of the disease in terms of adherence to treatment and quality of life.

Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors.

Transfer of somatic cell nuclei to enucleated eggs and ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, these methods are poorly efficient, and other unknown factors might be required to increase their success rate. Here we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development, and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4, and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M-phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies, and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M-phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergizes with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial, because none of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei.

Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development.

X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore, Xist is upregulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis.

A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.

Identification of noncompliant glaucoma patients using Bayesian networks and the Eye-Drop Satisfaction Questionnaire.

OBJECTIVE: To identify poorly compliant glaucoma patients, using the Eye-Drop Satisfaction Questionnaire (EDSQ). METHODS: This was an observational cross-sectional study with compliance data collected by an electronic monitoring device. Patients with primary open-angle glaucoma or ocular hypertension completed the EDSQ, a six-dimension self-reported questionnaire addressing "treatment concern", "disease concern", "patient-clinician relationship", "positive beliefs", "treatment convenience", and "self-declared compliance". A Bayesian network (BN) was applied to explore compliance associations with EDSQ. RESULTS: Among 169 patients who completed the EDSQ, 113 had valid Travalert® data, of whom 25 (22.1%) demonstrated low compliance. All six EDSQ dimensions were associated directly, or indirectly, with compliance. Two profiles exhibited low compliance, ie, patients aged younger than 77.5 years with a poor patient-physician relationship and self-declared poor compliance and patients aged older than 77.5 years with a poor patient-physician relationship and self-declared good compliance. The third profile showed high compliance, ie, patients aged younger than 77.5 years with a good patient-physician relationship and self-declared good compliance. CONCLUSION: Our results confirm a central role for the patient-physician relationship in the compliance process. Age, self-declared compliance, and patient satisfaction with the patient-physician relationship are all dimensions worth exploring before glaucoma medication is switched or proceeding to laser treatment or surgery.

Measurement of treatment compliance using a medical device for glaucoma patients associated with intraocular pressure control: a survey.

OBJECTIVE: To identify and characterize treatment compliance profiles of glaucoma patients and evaluate the association with intraocular pressure (IOP). METHODS: A computerized device (Travalert((R))) that recorded daily instillation times and eye-drop counts was given for 3 months. Patients were declared compliant when at least 2 drops were instilled per day. Compliance rates were calculated for weekdays and weekends, separately, over 8 consecutive weeks. A principal components analysis (PCA) was followed by an ascendant hierarchical classification (AHC) to identify compliance groups. RESULTS: 140 patients were recruited (mean age 65.5 years; 51.8% female) of whom 83.6% had primary open-angle glaucoma with mean IOP 23.9 mmHg before Travalert((R)) use. 60.7% were treated with DuoTrav((R)) (travoprost timolol fixed combination) and 39.3% with travoprost. The PCA identified two axes (compliance and treatment weeks). The AHC identified 3 compliance groups: 'high' (56.6%, approx. 80% compliance), 'medium' (21.2%, approx. 50% compliance), and 'low' (22.1%, approx. 20% compliance). Demographics and glaucoma parameters did not predict low compliance. Final mean IOP was 16.1 mmHg, but higher in the low compliance group (17.7 mmHg, P = 0.02). CONCLUSIONS: Compliance measurement by a medical device showed compliance rates <80% by 50% (approx.) of patients, significantly impacting IOP control. No demographic or glaucoma variable was associated with low compliance.

Quantification of leukocyte genomic 5-methylcytosine levels reveals epigenetic plasticity in healthy adult cloned cattle.

Successful somatic cell nuclear transfer (SCNT) requires epigenetic reprogramming of a differentiated donor cell nucleus. Incorrect reprogramming of epigenetic markings such as DNA methylation is associated with compromised prenatal development and postnatal abnormalities. Clones that survive into adulthood, in contrast, are assumed to possess a normalized epigenome corresponding to their normal phenotype. To address this point, we used capillary electrophoresis to measure 5-methylcytosine (5mC) levels in leukocyte DNA of 38 healthy female bovine clones that represented five genotypes from the Simmental breed and four genotypes from the Holstein breed. The estimated variance in 5mC level within clone genotypes of both breeds [0.104, 95% confidence interval (CI): 0.070-0.168] was higher than between clone genotypes (0, CI: 0-0.047). We quantified the contribution of SCNT to this unexpected variability by comparing the 19 Simmental clones with 12 female Simmental monozygotic twin pairs of similar age. In Simmental clones, the estimated variability within genotype (0.0636, CI: 0.0358-0.127) was clearly higher than in twin pairs (0.0091, CI: 0.0047-0.0229). In clones, variability within genotype (0.0636) was again higher than between genotypes (0, CI: 0-0.077). Twins, in contrast, showed lower variability within genotypes (0.0091) than between genotypes (0.0136, CI: 0.00250-0.0428). Importantly, the absolute deviations of 5mC values of individual SCNT clones from their genotype means were fivefold increased in comparison to twins. Further comparisons with noncloned controls revealed DNA hypermethylation in most of the clones. The clone-specific variability in DNA methylation and DNA hypermethylation clearly show that healthy adult SCNT clones must be considered as epigenome variants.

Nuclear transfer-derived epiblast stem cells are transcriptionally and epigenetically distinguishable from their fertilized-derived counterparts.

Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized- and nuclear transfer-EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT-EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT-EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast.