Impact of cirrhosis in patients undergoing laparoscopic liver resection in a nationwide multicentre survey.

BACKGROUND: The aim was to analyse the impact of cirrhosis on short-term outcomes after laparoscopic liver resection (LLR) in a multicentre national cohort study. METHODS: This retrospective study included all patients undergoing LLR in 27 centres between 2000 and 2017. Cirrhosis was defined as F4 fibrosis on pathological examination. Short-term outcomes of patients with and without liver cirrhosis were compared after propensity score matching by centre volume, demographic and tumour characteristics, and extent of resection. RESULTS: Among 3150 patients included, LLR was performed in 774 patients with (24·6 per cent) and 2376 (75·4 per cent) without cirrhosis. Severe complication and mortality rates in patients with cirrhosis were 10·6 and 2·6 per cent respectively. Posthepatectomy liver failure (PHLF) developed in 3·6 per cent of patients with cirrhosis and was the major cause of death (11 of 20 patients). After matching, patients with cirrhosis tended to have higher rates of severe complications (odds ratio (OR) 1·74, 95 per cent c.i. 0·92 to 3·41; P = 0·096) and PHLF (OR 7·13, 0·91 to 323·10; P = 0·068) than those without cirrhosis. They also had a higher risk of death (OR 5·13, 1·08 to 48·61; P = 0·039). Rates of cardiorespiratory complications (P = 0·338), bile leakage (P = 0·286) and reoperation (P = 0·352) were similar in the two groups. Patients with cirrhosis had a longer hospital stay than those without (11 versus 8 days; P = 0·018). Centre expertise was an independent protective factor against PHLF in patients with cirrhosis (OR 0·33, 0·14 to 0·76; P = 0·010). CONCLUSION: Underlying cirrhosis remains an independent risk factor for impaired outcomes in patients undergoing LLR, even in expert centres.

ANTECEDENTES: El objetivo de este estudio fue analizar el impacto de la cirrosis en los resultados a corto plazo después de la resección hepática laparoscópica (laparoscopic liver resection, LLR) en un estudio de cohortes multicéntrico nacional. MÉTODOS: Este estudio retrospectivo incluyó todos los pacientes sometidos a LLR en 27 centros entre 2000 y 2017. La cirrosis se definió como fibrosis F4 en el examen histopatológico. Los resultados a corto plazo de los pacientes con hígado cirrótico (cirrhotic liver CL) (pacientes CL) y los pacientes con hígado no cirrótico (non-cirrhotic liver, NCL) (pacientes NCL) se compararon después de realizar un emparejamiento por puntaje de propension del volumen del centro, las características demográficas y del tumor, y la extensión de la resección. RESULTADOS: Del total de 3.150 pacientes incluidos, se realizó LLR en 774 (24,6%) pacientes CL y en 2.376 (75,4%) pacientes NCL. Las tasas de complicaciones graves y mortalidad en el grupo de pacientes CL fueron del 10,6% y 2,6%, respectivamente. La insuficiencia hepática posterior a la hepatectomía (post-hepatectomy liver failure, PHLF) fue la principal causa de mortalidad (55% de los casos) y se produjo en el 3,6% de los casos en pacientes CL. Después del emparejamiento, los pacientes CL tendieron a tener tasas más altas de complicaciones graves (razón de oportunidades, odds ratio, OR 1,74; i.c. del 95% 0,92-0,41; P = 0,096) y de PHLF (OR 7,13; i.c. del 95% 0,91-323,10; P = 0,068) en comparación con los pacientes NCL. Los pacientes CL estuvieron expuestos a un mayor riesgo de mortalidad (OR 5,13; i.c. del 95% 1,08-48,6; P = 0,039) en comparación con los pacientes NCL. Los pacientes CL presentaron tasas similares de complicaciones cardiorrespiratorias graves (P = 0,338), de fuga biliar (P = 0,286) y de reintervenciones (P = 0,352) que los pacientes NCL. Los pacientes CL tuvieron una estancia hospitalaria más larga (11 versus 8 días; P = 0,018) que los pacientes NCL. La experiencia del centro fue un factor protector independiente de PHLF (OR 0,33; i.c. del 95% 0,14-0,76; P = 0,010) pacientes CL. CONCLUSIÓN: La presencia de cirrosis subyacente sigue siendo un factor de riesgo independiente de peores resultados en pacientes sometidos a resección hepática laparoscópica, incluso en centros con experiencia.

The mitochondrial DNA content of cumulus cells may help predict embryo implantation.

PURPOSE: The quantification of mtDNA in cumulus granulosa cells (CGCs) surrounding an oocyte has been positively linked with morphological embryonic quality. In the present study, we evaluated the link between the amount of mtDNA in CGCs surrounding an oocyte and the chances for the corresponding embryo of implanting and leading to an ongoing pregnancy. METHODS: This is an observational study, performed on 84 oocyte-cumulus-complexes (OCCs) having led to the replacement of an embryo in the maternal uterus, retrieved from 71 patients undergoing IVF with intracytoplasmic sperm. The OCCs were classified in two groups, one including 26 OCCs having led to an implanted embryo and the other including 58 OCCs having led to a non-implanted embryo. The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. RESULTS: Significantly higher mtDNA copy numbers in CGCs were associated with implanted embryos than with non-implanted embryos (mean 215 [sd 375] and 59 [sd 72], respectively; p < 104). Multivariate analysis, taking into account the women's age, the embryo quality, and the AMH level, suggests an independent relationship between the mtDNA content of CGCs and the potential of embryo implantation. CONCLUSION: During in vitro fertilization (IVF) procedures, the probability of the implantation of the embryo appears to be closely correlated to the mtDNA copy numbers in the CGCs. Our results highlight the interest of mtDNA quantification in GCGs as a biomarker of the potential of embryo implantation.

Clinical response to Nabiximols correlates with the downregulation of immune pathways in multiple sclerosis.

BACKGROUND AND PURPOSE: Nabiximols (Sativex® ) is a cannabinoid-based compound used for the treatment of moderate to severe spasticity in multiple sclerosis (MS). The aim of the study was to investigate the effect of the administration of Nabiximols on blood transcriptome profile of patients with MS and to interpret it in the context of pathways and networks. METHODS: Whole-genome expression profiling was performed in whole blood of 33 subjects with MS at baseline and after 4 weeks of drug treatment. Patients were classified as responders (n = 19) and non-responders (n = 14). Pathway and network analyses on genes modulated by the drug were performed, followed by in vitro stimulation of peripheral blood mononuclear cells with pro-inflammatory agents to support the immunomodulatory properties of the drug. RESULTS: Individual effect size was modest; however, we observed a downregulation of several immune-related pathways after 4 weeks of treatment, which was more pronounced when restricting analyses to responders. Interesting hub molecules functionally related to the immune system emerged from network analysis, including NFKB1, FYN, MAP14 and TP53. The immunomodulatory properties of the drug were confirmed through in vitro assays in peripheral blood mononuclear cells collected from patients with MS. CONCLUSIONS: Our findings support the immunomodulatory activity of cannabinoids in patients with MS. Further studies in more specific cell types are needed to refine these results.

Basal vitamin D levels and disease activity in multiple sclerosis patients treated with fingolimod.

BACKGROUND: Several studies have shown an association between 25-hydroxyvitamin D (25[OH]D) levels and multiple sclerosis (MS) susceptibility and/or level of disease activity in patients treated with first line drugs. AIMS: To investigate whether baseline 25[OH]D values could influence disease activity also during treatment with the second-line drug fingolimod (FTY). PATIENTS AND METHODS: We enrolled 176 MS patients who started FTY at the San Raffaele Hospital (OSR) MS center with available 25[OH]D measurement at the time of treatment start. We then prospectively followed them for 2 years with periodic clinical examinations and MRI scans. RESULTS: We found no linear correlation between baseline 25[OH]D levels and annualized relapse rate (ARR) or time to first relapse. However, we observed that patients with serum 25[OH]D ≥ 100 nmol/l showed a lower number of Gd+ and combined unique activity (CUA) lesions at baseline compared to patients with the lowest 25[OH]D levels (less than 50 nmol/l, p value < 0.05). Moreover, they showed fewer CUA lesions at 2-year follow-up also when accounting for baseline level of disease activity (p value < 0.05). CONCLUSIONS: In patients treated with FTY, those with the highest baseline 25(OH)D levels had a significantly lower number of active lesions at baseline; the same effect, even if weaker, was observed also at 2-year follow-up when adjusting for baseline disease activity. Given Vitamin D supplementation safety profile, also if a causal effect has not yet been shown, most of MS patients could probably benefit from 25[OH]D levels above those currently considered to be sufficient.

Effect of different mini-volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer.

Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

The mitochondrial DNA content of cumulus granulosa cells is linked to embryo quality.

STUDY QUESTION: Could the mitochondrial DNA (mtDNA) content of cumulus granulosa cells (CGCs) be related to oocyte competence? SUMMARY ANSWER: The quality of embryos obtained during IVF procedures appears to be linked to mtDNA copy numbers in the CGCs. WHAT IS KNOWN ALREADY: Oocyte quality is linked to oocyte mtDNA content in the human and other species, and the mtDNA copy number of the oocyte is related to that of the corresponding CGCs. Moreover, the quantification of CGC mtDNA has recently been proposed as a biomarker of embryo viability. STUDY DESIGN SIZE, DURATION: An observational study was performed on 452 oocyte-cumulus complexes retrieved from 62 patients undergoing ICSI at the ART Center of the University Hospital of Angers, France, from January to May 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. The relationship between CGC mtDNA content and oocyte maturity and fertilizability, on one hand, and embryo quality, on the other, was investigated using univariate and multivariate generalized models with fixed and mixed effects. MAIN RESULTS AND THE ROLE OF CHANCE: No relationship was found between CGC mtDNA content and oocyte maturity or fertilizability. In contrast, there was a significant link between the content of mtDNA in CGCs surrounding an oocyte and the embryo quality, with significantly higher mtDNA copy numbers being associated with good quality embryos compared with fair or poor quality embryos [interquartile range, respectively, 738 (250-1228) and 342 (159-818); P = 0.006]. However, the indication provided by the quantification of CGC mtDNA concerning the eventuality of good embryo quality was seriously subject to patient effect (AUC = 0.806, 95%CI = 0.719-0.869). The quantity of CGC mtDNA was influenced by BMI and smoking. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The quantification of CGC mtDNA may indicate embryo quality. However, since it is affected by patient specificity, it should be used with caution. It remains to be seen whether this marker could directly predict the implantation capacity of the embryo, which is the main objective in IVF practice. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that the quantification of CGC mtDNA may be a novel biomarker of embryo viability. However, patient specificity makes it impossible to establish a general threshold value, valid for all patients. Nevertheless, further studies are needed to determine whether the quantification of CGC mtDNA may, in combination with the morpho-kinetic method, offer an additional criterion for selecting the best embryo for transfer from a given cohort. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centres INSERM and the CNRS. There were no competing interests.

Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing.

STUDY QUESTION: Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? SUMMARY ANSWER: Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. WHAT IS KNOWN ALREADY: Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. STUDY DESIGN, SIZE, DURATION: This was an observational study of 53 immature oocyte-cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. MAIN RESULTS AND THE ROLE OF CHANCE: There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being significantly different (respectively 0.321, SD = 0.547 and 1.075, SD = 1.158) (P < 0.0001). Variants with a potential functional consequence were also overrepresented in CCs compared with oocytes (P = 0.0019). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Limitations may be due to the use of immature oocytes discarded during the assisted reproductive technology procedure, the small size of the sample, and the high-throughput sequencing technology that might not have detected heteroplasmy levels lower than 2%. WIDER IMPLICATIONS OF THE FINDINGS: The alteration of mtDNA integrity in oocytes during ovarian ageing is a recurring question to which our pilot study suggests a reassuring answer. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centers, INSERM and the CNRS. There are nocompeting interests.

Targeted metabolomics reveals reduced levels of polyunsaturated choline plasmalogens and a smaller dimethylarginine/arginine ratio in the follicular fluid of patients with a diminished ovarian reserve.

STUDY QUESTION: Does the metabolomic profile of the follicular fluid (FF) of patients with a diminished ovarian reserve (DOR) differ from that of patients with a normal ovarian reserve (NOR)? SUMMARY ANSWER: The metabolomic signature of the FF reveals a significant decrease in polyunsaturated choline plasmalogens and methyl arginine transferase activity in DOR patients compared to NOR patients. WHAT IS KNOWN ALREADY: The composition of the FF reflects the exchanges between the oocyte and its microenvironment during its acquisition of gametic competence. Studies of the FF have allowed identification of biomarkers and metabolic pathways involved in various pathologies affecting oocyte quality, but no large metabolomic analysis in the context of ovarian ageing and DOR has been undertaken so far. STUDY DESIGN, SIZE, DURATION: This was an observational study of the FF retrieved from 57 women undergoing in vitro fertilization at the University Hospital of Angers, France, from November 2015 to September 2016. The women were classified in two groups: one including 28 DOR patients, and the other including 29 NOR patients, serving as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were enrolled in the morning of oocyte retrieval after ovarian stimulation. Once the oocytes were isolated for fertilization and culture, the FF was pooled and centrifuged for analysis. A targeted quantitative metabolomic analysis was performed using high-performance liquid chromatography coupled with tandem mass spectrometry, and the Biocrates Absolute IDQ p180 kit. The FF levels of 188 metabolites and several sums and ratios of metabolic significance were assessed by multivariate and univariate analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 136 metabolites were accurately quantified and used for calculating 23 sums and ratios. Samples were randomly divided into training and validation sets. The training set, allowed the construction of multivariate statistical models with a projection-supervised method, i.e. orthogonal partial least squares discriminant analysis (OPLS-DA), applied to the full set of metabolites, or the penalized least absolute shrinkage and selection operator with logistic regression (LASSO-LR), applied to the ratios and sums of the metabolites. Both multivariate models showed good predictive performances when applied to the validation set. The final penalized model retained the three most significant variables, i.e. the total dimethylarginine-to-arginine ratio (Total DMA/Arginine), the sum of the polyunsaturated choline plasmalogens (PUFA ae), and the patient's age. The negative coefficients of Total DMA/Arginine and PUFA ae indicated that these FF variables had lower values in DOR patients than in NOR patients. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study presents two limitations. First, with this targeted metabolomics analysis, we have explored only a limited portion of the FF metabolome. Second, although the signature found was highly significant, the mechanism underlying the dysfunction remains undetermined. WIDER IMPLICATIONS OF THE FINDINGS: The understanding of the mechanisms implied in ovarian ageing is essential for providing an adequate response to affected women desiring pregnancy. Our study proposes an incoming signature that may open new paths towards this goal. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the University Hospital of Angers, the University of Angers, and the French national research centers, INSERM and the CNRS. There were no competing interests.

Neuromyelitis optica spectrum disorders: long-term safety and efficacy of rituximab in Caucasian patients.

OBJECTIVE: To assess the long-term benefit-risk profile of repeated courses of rituximab in Caucasian patients affected by neuromyelitis optica (NMO) and related disorders, in everyday clinical practice. METHODS: This is a prospective observational study performed at San Raffaele Hospital, Milan, Italy. From February 2006, we recruited 21 patients affected by NMO and NMO spectrum of disorders (NMOSD) whom underwent at least one cycle of intravenous (i.v.) rituximab and then were followed for at least 2 years. RESULTS: At a mean follow-up time of 48 months, we observed a significant reduction of the annualized relapse rate (ARR), from 2.0 to 0.16 (p < 0.01); and of the median Expanded Disability Status Scale (EDSS), from 5.5 to 4.0 (p < 0.013). There were 12 patients (57%) who remained disease free during the follow-up period. Five patients (24%) reported mild hematological adverse events. Serious infectious adverse events were reported by another four patients: These were all wheelchair bound at the beginning of their rituximab treatment. CONCLUSIONS: A fixed treatment scheme of rituximab, with re-treatment every 6 months, was efficacious for NMO and NMOSD, with a good safety profile; however, to obtain an even better benefit-risk ratio, close monitoring of CD19(+) B cells should be performed before the re-treatment of patients with high-level disability, concomitant leukopenia and hypogammaglobulinemia.

Relationship between diminished ovarian reserve and mitochondrial biogenesis in cumulus cells.

STUDY QUESTION: What part do mitochondria play in cases of diminished ovarian reserve (DOR)? SUMMARY ANSWER: Mitochondrial biogenesis in cumulus cells may be linked with impaired oocyte competence in patients with DOR. WHAT IS KNOWN ALREADY: DOR, one of the causes of infertility even in young women, is characterized by the depletion of the ovarian pool associated with a decline in oocyte competence. Mitochondria, which play a role in oocyte quality, could be involved in the pathogenesis of DOR. The study of cumulus cells offers an interesting non-invasive approach for evaluating oocyte quality and the metabolic processes on which it depends. If mitochondrial dysfunction is involved in DOR, it is likely to have an impact on the functioning of cumulus cells. STUDY DESIGN, SIZE, DURATION: This is an observational study of 74 immature oocyte-cumulus complexes retrieved from 47 women undergoing in vitro fertilization with intracytoplasmic sperm injection at the University Hospital of Angers, France, from March 2013 to March 2014. The women were divided into two groups: one group included 26 women with DOR, and the other, which included 21 women with a normal ovarian reserve (NOR), served as a control group. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: The oocyte mitochondrial content and the average mitochondrial content of the cumulus cells were assessed by mitochondrial (mt)DNA quantification using a quantitative real-time PCR technique. Microfluidic-based quantitative RT-PCR assays were used to quantify the expression of 13 genes involved in mitochondrial functions such as apoptosis and antioxidant activity or in mitochondrial biogenesis. We used orthogonal partial least-squares discriminant analysis (OPLS-DA) to distinguish between the DOR group and the NOR group of patients, and an OPLS model to predict the value of the oocyte mtDNA content that could be used as a critical marker of oocyte quality. MAIN RESULTS AND THE ROLE OF CHANCE: The OPLS-DA model showed a good predictive capability (Q2 = 0.543). Using the variable importance in projection (VIP) metric we found three mitochondrial variables distinguishing the DOR group from the NOR group of patients, i.e. the oocyte mtDNA content (VIP = 0.92), the cumulus cell mtDNA content (VIP = 0.95) and the expression in cumulus cells of peroxisome proliferator-activated receptor γ coactivator 1 alpha (PPARGC-1A) (VIP = 1.10), all of which were lower in the DOR group than in the NOR group of patients. The OPLS model was able to satisfactorily predict the oocyte mtDNA content in only the NOR group of patients (Q2 = 0.506). We found four new variables positively linked to the oocyte mitochondrial mass, i.e. the cumulus cell mtDNA content (VIP = 1.19), and the expression in cumulus cells of three factors of mitochondrial biogenesis: polymerase gamma (POLG) (VIP = 2.13), optic atrophy 1 (OPA1) (VIP = 1.89) and the transcription factor associated with mitochondria (TFAM) (VIP = 1.32). LIMITATIONS, REASONS OF CAUTION: This is a descriptive study. Because of ethical concerns in human clinical practice, this study has been performed only on immature oocytes and corresponding cumulus cells, which are usually discarded during in vitro fertilization procedures. WIDER IMPLICATIONS OF THE FINDINGS: Cumulus cells may govern mitochondrial biogenesis, creating an adequate oocyte mitochondrial pool to promote embryonic development. The alteration of this process in patients with DOR may account for the impairment of oocyte quality. This suggests that some mitochondrial characteristics of cumulus cells may serve as indicators of oocyte competence and that oocyte quality may be improved by products enhancing mitochondrial biogenesis. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from the University Hospital of Angers, France: 'Appel d'offre interne à la recherche 2014'. TRIAL REGISTRATION NUMBER: N/A.

Are zona pellucida genes involved in recurrent oocyte lysis observed during in vitro fertilization?

PURPOSE: Complete oocyte lysis in in vitro fertilization (IVF) is a rare event, but one against which we remain helpless. The recurrence of this phenomenon in some women in each of their IVF attempts, regardless of treatment, together with the results of animal experiments led us to investigate the possible involvement of the genes encoding for the glycoproteins constituting the zona pellucida (ZP). PATIENTS & METHODS: Over the last ten years, during which we treated over 500 women each year, three women suffered recurrent oocyte lysis during their IVF attempts in our Centre for Reproductive Biology. For each of these three cases, we sequenced the four genes and promoter sequences encoding the glycoproteins of the ZP. The sequence variations likely to cause a change in protein expression or structure, were investigated in a control group of 35 women who underwent IVF without oocyte lysis and with normal rates of fertilization. RESULTS & CONCLUSION: We found no mutations in the ZP genes sequenced. Only some polymorphisms present in the control group and in the general population were detected, excluding their specific involvement in the phenotype observed. Thus, although we suspected that complete oocyte lysis was due to a genetic cause, it did not seem possible to directly incriminate the genes encoding the proteins of the ZP in the observed phenotype. Further study of the genes involved in the processing and organization of ZP glycoproteins may allow elucidation of the mechanism underlying recurrent oocyte lysis during in vitro fertilization.

Early compaction at day 3 may be a useful additional criterion for embryo transfer.

PURPOSE: The reduction of the number of embryos transferred while maintaining a satisfactory rate of pregnancy (PR) with in vitro fertilization calls for a refined technique of embryonic selection. This prospective study investigates the significance of early embryonic compaction at day 3 as a marker of the chances of implantation. METHODS: We examined 317 transfers and their outcome involving 509 embryos including 91 compacted embryos. RESULTS: Early compaction seems linked with the ovarian response to stimulation and embryonic quality. The PR is significantly increased when the embryonic cohort contains at least one compacted embryo (44% versus 29.5%, p = 0.01), and when at least one compacted embryo is transferred (44% versus 31%, p < 0.05). The analysis of our single embryo transfers shows that the implantation rates are significantly better for compacted embryos (50% versus 30%, p < 0.05) (OR 2.98; CI 1.02-5.28). CONCLUSION: Thus, early compaction, sometimes observed at day 3, may serve as a useful additional criterion for selecting the embryos transferred.

Molecular characterization of corona radiata cells from patients with diminished ovarian reserve using microarray and microfluidic-based gene expression profiling.

BACKGROUND: Diminished ovarian reserve (DOR) is one of the causes of infertility in young women. In this prospective study, gene expression profiling (GEP) of corona radiata cells (CRC) was performed to identify genes deregulated in DOR patients. METHODS: Microarray-based GEP of CRC isolated from eight women undergoing IVF was performed to identify genes differentially expressed between patients with normal ovarian reserve and DOR patients. Microfluidic-based quantitative RT-PCR assays were used to validate selected transcripts on 40 independent patients. A principal component analysis was used to identify more homogeneous subgroups of DOR patients. In silico analyses focusing on cis-regulation were performed to refine the interactions between patient's biological characteristics and their GEP. RESULTS: Forty-eight transcripts were differentially expressed, including CXXC finger protein 5 (CXXC5), forkhead box C1 (FOXC1) (down-regulated in DOR) as well as connective tissue growth factor (CTGF), follistatin-like 3 (FSTL3), prostaglandin-endoperoxide synthase 2 (PTGS2) and suppressor of cytokine signaling 2 (SOCS2) (up-regulated in DOR). According to these transcripts, two DOR patients' subgroups (DOR Gr1 and Gr2) were identified. In DOR Gr2 patients, C-terminal domain 2 (CITED2), CTGF, growth arrest-specific 1 (GAS1), insulin receptor substrate 2 (IRS2), PTGS2, SOCS2 and Versican (VCAN) were expressed at significantly higher levels and CXXC5, FOXC1, guanylate-binding protein 2 (GBP2) and zinc finger MIZ-domain containing 1 (ZMIZ1) at significantly lower levels. Higher baseline estradiol (E(2)) levels were observed in DOR Gr2 patients (P < 0.006). The in silico analyses suggested that all 11 genes differentially expressed between DOR Gr1 and DOR Gr2 subgroups could be transcriptional targets of estrogen. CONCLUSIONS: Despite small sample size limitations, 12 genes deregulated in the CRC of DOR patients were identified, which could be involved in DOR pathogenesis. A DOR patient's subgroup with high baseline E(2) levels and deregulated estrogen-responsive genes was also identified.

Influence of Diminished Ovarian Reserve on Early Embryo Morphokinetics during In Vitro Fertilization: A Time-Lapse Study.

There is great controversy as to whether women with Diminished Ovarian Reserve (DOR) exhibit only a quantitative decrease in ovarian reserve or also impaired oocyte and embryo quality. In this retrospective study, we aimed to evaluate the impact of DOR on embryo morphokinetic parameters with a time-lapse system. 1314 embryos were obtained from 256 couples undergoing IVF or ICSI cycles, with 242 embryos in the DOR group as classified by the Bologna and POSEIDON criteria and 1072 embryos derived from the Normal Ovarian Reserve (NOR) group. For each morphokinetic parameter (t2, t3, t4, t5, t8, tB, ECC2, cc2a, ECC3, s2, s3), a generalized linear mixed model was created to control for female age, BMI, smoking status, method of insemination and correlation between oocytes from a same cohort. No significant association was found between DOR and any of the morphokinetic parameters studied. In a secondary analysis, we evaluated the influence of maternal aging, comparing morphokinetic characteristics between two age groups (<37 and ≥37 years). In the univariate analysis, we found that embryos from older women displayed a slower embryo development (in particular for t3, t4, t5, tB, and ECC2), although without statistical significance in the multivariate analysis. In conclusion, our study did not reveal any substantial impact of ovarian aging on early morphokinetic parameters and suggested potential biases that may be a source of controversy in the literature.

Burden of rare coding variants in an Italian cohort of familial multiple sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system. It is a complex and heterogeneous disease caused by a combination of genetic and environmental factors, and it can cluster in families. OBJECTIVE: to evaluate at gene-level the aggregate contribution of predicted damaging low-frequency and rare variants to MS risk in multiplex families. METHODS: We performed whole exome sequencing (WES) in 28 multiplex MS families with at least 3 MS cases (81 affected and 42 unaffected relatives) and 38 unrelated healthy controls. A gene-based burden test was then performed, focusing on two sets of candidate genes: i) literature-driven selection and ii) data-driven selection. RESULTS: We identified 11 genes enriched with predicted damaging low-frequency and rare variants in MS compared to healthy individuals. Among them, UBR2 and DST were the two genes with the strongest enrichment (p = 5 × 10-4 and 3 × 10-4, respectively); interestingly enough the association signal in UBR2 is driven by rs62414610, which was present in 25% of analysed families. CONCLUSION: Despite limitations, this is one of the first studies evaluating the aggregate contribution of predicted damaging low-frequency and rare variants in MS families using WES data. A replication effort in independent cohorts is warranted to validate our findings and to evaluate the role of identified genes in MS pathogenesis.

Oral desensitization as a useful treatment in 2-year-old children with cow's milk allergy.

BACKGROUND: Limited published evidence shows oral desensitization to be a potential intervention option for cow's milk protein (CMPs) allergy. OBJECTIVE: The aim of this study was to evaluate the safety and efficacy of oral desensitization in 2-year-old children with cow's milk allergy, as a treatment alternative to elimination diet. METHODS: A total of 60 children aged 24-36 months with IgE-mediated allergy to CMPs were included in this multi-center study and were randomized into two groups. Thirty children (group A: treatment group) began oral desensitization immediately, whereas the remaining 30 (group B: control group) were kept on a milk-free diet and followed-up for 1 year. RESULTS: After 1-year follow-up period, 90% of the children in group A had become completely tolerant vs. 23% of the children in group B. In group A, cow's milk skin reactivity and serum-specific IgE to milk and casein decreased significantly from the initial assessment, whereas group B showed no significant change after 1 year of follow-up. Twenty-four patients (80%) developed some reaction during the treatment period: 14 children developed moderate reaction (47%) and 10 mild reaction (33%). The most common manifestations were urticaria-angioedema, followed by cough. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, oral desensitization was found to be effective in a significant percentage of 2-year-old children with cow's milk allergy. Oral desensitization appears to be efficacious as an alternative to elimination diet in the treatment of 2-year-old children with cow's milk allergy. The side-effect profile appears acceptable but requires further study.

Change in the Strategy of Embryo Selection with Time-Lapse System Implementation-Impact on Clinical Pregnancy Rates.

Time-lapse systems (TLS) and associated algorithms are interesting tools to improve embryo selection. This study aimed to evaluate how TLS and KIDScore™ algorithm changed our practices of embryo selection, as compared to a conventional morphological evaluation, and improved clinical pregnancy rates (CPR). In the study group (year 2020, n = 303 transfers), embryos were cultured in an EmbryoScope+ time-lapse incubator. A first team observed embryos conventionally once a day, while a second team selected the embryos for transfer based on time-lapse recordings. In the control group (year 2019, n = 279 transfers), embryos were selected using the conventional method, and CPR were recorded. In 2020, disagreement between TLS and the conventional method occurred in 32.1% of transfers, more often for early embryos (34.7%) than for blastocysts (20.5%). Irregular morphokinetic events (direct or reverse cleavage, multinucleation, abnormal pronuclei) were detected in 54.9% of the discordant embryos. When it was available, KIDScore™ was decreased for 73.2% of the deselected embryos. Discordant blastocysts mainly corresponded with a decrease in KIDScore™ (90.9%), whereas discordant Day 3 embryos resulted from a decreased KIDScore™ and/or an irregular morphokinetic event. CPR was significantly improved in the TLS group (2020), as compared to the conventional group (2019) (32.3% vs. 21.9%, p = 0.005), even after multivariate analysis. In conclusion, TLS is useful to highlight some embryo development abnormalities and identify embryos with the highest potential for pregnancy.

Transcriptomic Analysis of Peripheral Monocytes upon Fingolimod Treatment in Relapsing Remitting Multiple Sclerosis Patients.

Fingolimod (FTY), a second-line oral drug approved for relapsing remitting Multiple Sclerosis (RRMS) acts in preventing lymphocyte migration outside lymph nodes; moreover, several lines of evidence suggest that it also inhibits myeloid cell activation. In this study, we investigated the transcriptional changes induced by FTY in monocytes in order to better elucidate its mechanism of action. CD14+ monocytes were collected from 24 RRMS patients sampled at baseline and after 6 months of treatment and RNA profiles were obtained through next-generation sequencing. We conducted pathway and sub-paths analysis, followed by centrality analysis of cell-specific interactomes on differentially expressed genes (DEGs). We investigated also the predictive role of baseline monocyte transcription profile in influencing the response to FTY therapy. We observed a marked down-regulation effect (60 down-regulated vs. 0 up-regulated genes). Most of the down-regulated DEGs resulted related with monocyte activation and migration like IL7R, CCR7 and the Wnt signaling mediators LEF1 and TCF7. The involvement of Wnt signaling was also confirmed by subpaths analyses. Furthermore, pathway and network analyses showed an involvement of processes related to immune function and cell migration. Baseline transcriptional profile of the HLA class II gene HLA-DQA1 and HLA-DPA1 were associated with evidence of disease activity after 2 years of treatment. Our data support the evidence that FTY induces major transcriptional changes in monocytes, mainly regarding genes involved in cell trafficking and immune cell activation. The baseline transcriptional levels of genes associated with antigen presenting function were associated with disease activity after 2 years of FTY treatment.

Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods.

In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo-produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.

The cytokine profile of follicular fluid changes during ovarian ageing.

OBJECTIVE: Ovarian ageing is one of the commonest causes of infertility in patients consulting for assisted reproductive technology. The composition of the follicular fluid (FF), which reflects the exchanges between the oocyte and its microenvironment, has been extensively investigated to determine the metabolic pathways involved in various ovarian disorders. Considering the importance of cytokines in folliculogenesis, we focused on the cytokine profile of the FF during ovarian ageing. MATERIAL AND METHODS: Our cross-sectional study assesses the levels of 27 cytokines and growth factors in the FF of two groups of women undergoing in vitro fertilization. One group included 28 patients with ovarian ageing clinically characterized by a diminished ovarian reserve (DOR), and the other group included 29 patients with a normal ovarian reserve (NOR), serving as controls. RESULTS: With univariate analysis, the cytokine profile was found to differ significantly between the two groups. After adjustment of the p-values, platelet-derived growth factor-BB (PDGF-BB) was the only cytokine with a significantly lower concentration in the DOR group (7.34 ± 16.11 pg/mL) than in the NOR group (24.39 ± 41.38 pg/mL) (p = 0.005), independently of chronological age. CONCLUSION: Thus, PDGF-BB would seem to be implicated in the physiopathology of DOR, potentially in relation to its role in folliculogenesis or in the protection against oxidative stress.