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.

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.

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.

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.

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 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.

ConFIRM trial - conversion of in vitro fertilization cycles to intrauterine inseminations in patients with a poor ovarian response to stimulation: a protocol for a multicentric, prospective randomized trial.

BACKGROUND: To date, there is no consensus on the ideal management strategy of patients with poor ovarian response (POR) to controlled ovarian stimulation (COS) for in vitro fertilization (IVF). Currently, these patients are given the choice of: (1) canceling the cycle; (2) proceeding with COS regardless of the poor response, and performing the oocyte retrieval and transfer of embryos when available; or (3) conversion to an intrauterine insemination (IUI). When the decision to proceed with the COS cycle is taken, it is not clear whether IVF or conversion to IUI is the best choice. If live birth rates were comparable between the two strategies, conversion to IUI would be the better option for poor responders, since it is less invasive and is associated with a lower cost. METHODS: We designed a non-inferiority, multicentric, randomized controlled trial that will be conducted in 18 French Reproductive Medicine centers. We defined POR as the presence of only two or four mature follicles ≥ 14 mm on ovulation trigger day. Patients with POR will be randomized into two parallel arms: "IVF" and "conversion to IUI." Our main objective is to compare the efficiency of IVF and conversion to IUI in patients with POR to COS. The primary outcome is the live birth rate, defined as the birth of a living infant after 22 weeks' gestational age, or weighing ≥ 500 g. One of the secondary objectives is to compare the cost-efficiency of both strategies at 12 months. We will need to include 940 patients (470 in each arm), and the duration of the inclusion period is estimated to be 36 months. DISCUSSION: This is the first randomized controlled trial to compare the outcomes of IVF and embryo transfer to conversion to IUI in patients with POR to COS. If our study shows that conversion to IUI is non-inferior to IVF in terms of clinical efficiency and live birth rate, it would confirm IUI as a better alternative for patients, both individually (less invasive and more patient-friendly) and collectively (lower cost). TRIALS REGISTRATION: ClinicalTrials.gov, ID: NCT03362489 . Registered on January 10th, 2018.

Surgical treatment of septate uterus in cases of primary infertility and before assisted reproductive technologies.

Septate uterus is the most common congenital uterine malformation in women with infertility. Several criteria are available for the definition of septate uteri, such as the one proposed by the European Society of Human Reproduction and Embryology (ESHRE)/European Society for Gynecological Endoscopy (ESGE) (ESHRE/ESGE), or by the American Society for Reproductive Medicine (ASRM), with notable differences between the two. Recently, a simplified classification was proposed by the Congenital Uterine Malformations Experts (CUME), where a septum is defined as an internal indentation depth≥10mm. To date, there is no consensus on the management of women with a septate uterus and infertility. We have performed an extensive literature appraisal and reviewed all the available international guidelines in order to propose a management strategy for infertile patients with a uterine septum. Hysteroscopic septum incision seems to improve natural conception rates in the year following surgery. Moreover, it improves in vitro fertilization (IVF) outcomes when performed before the embryo transfer, by improving embryo implantation rates. On the other hand, for patients with an arcuate uterus (indentation<1.5cm according to the ASRM guidelines) and infertility, it seems that assisted reproductive technologies are the most appropriate first line treatment. However, in cases of recurrent implantation failure or recurrent pregnancy loss following IVF, hysteroscopic section could be proposed. Overall, we recommend hysteroscopic septum incision for patients with primary infertility, and for patients undergoing assisted reproductive technologies.

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.

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.

Effectiveness and baseline factors associated to fingolimod response in a real-world study on multiple sclerosis patients.

BACKGROUND: Treatment choice in multiple sclerosis (MS) is crucial for optimizing risk-benefit profile. OBJECTIVE: To assess fingolimod (FTY) effectiveness and identify baseline features associated to disease activity in a large Italian cohort of Relapsing-Remitting (RR) MS patients. METHODS: Three-hundred sixty-seven RRMS patients starting FTY treatment at San Raffaele Hospital (Milan-Italy) underwent clinical and MRI evaluations for 2 years. Treatment response was assessed considering the proportion of patients with no evidence of disease activity (NEDA) and recording the time to first relapse. Primary analyses were performed stratifying for Natalizumab (NTZ) treatment in the year before (NO_NTZ vs NTZ group), to account for post-NTZ reactivation. RESULTS: Almost half of patients were NEDA after 2 years, 53.4% in the NO_NTZ group and 36.2% in the NTZ group. Despite an opposite trend during the first 6-12 months, at 2-year follow-up the two groups were comparable for relapses and number of new/enlarging T2 and Gd-enhancing lesions. Baseline parameters of higher disease activity (ARR, Gd enhancing lesions and age at onset) were associated with increased likelihood of failing NEDA criteria or with shorter time to relapse (p < 0.05). CONCLUSIONS: Our data strengthen FTY effectiveness in everyday clinical practice, even in patients switching from NTZ treatment. Baseline parameters of inflammatory activity are the most important prognostic factors for mid-term disease reactivation also during second-line treatment with FTY, providing hints on how to select therapies towards a more personalized management.

[In vitro fertilization versus conversion to intrauterine insemination in patients with poor response to controlled ovarian hyperstimulation]. / Réponse insuffisante à la stimulation en vue de FIV : maintenir la ponction ou choisir l' insémination ?

In women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF), a poor ovarian response, defined as three of fewer mature follicles, can lead to cancellation of the cycle. However, in women with at least one patent tube and normal semen parameters, conversion to intrauterine insemination (IUI) is considered an option, offering reasonable pregnancy rates at a lower cost and without the complications associated with oocyte retrieval. Studies have shown that in cycles with only one mature follicle, IVF should be canceled. However, in cycles with 2 or 3 mature follicles, patients have the choice between IVF and conversion to IUI. Some studies have shown that IVF is superior to IUI in such cases, whereas other reports failed to find any difference. Most of these studies are retrospective and limited by the presence of several biases and low numbers of cycles, and to this date, there is no consensus on the best approach. We have thus designed a multicenter, randomized non-inferiority study, comparing live birth rates following conversion to IUI or IVF in patients with 2 or 3 mature follicles in COH cycles. Nine hundred and forty patients will be randomized on trigger day to either IVF or conversion to IUI. Our study will also include a medico-economic analysis.

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.

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.

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.

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.

Ovarian ageing: the role of mitochondria in oocytes and follicles.

BACKGROUND: There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. OBJECTIVE AND RATIONAL: Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. SEARCH METHODS: PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. OUTCOMES: There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in the deterioration of oocyte quality in terms of competence and of the risk of transmitting mitochondrial abnormalities to the offspring. In contrast, some mtDNA haplogroups are protective against the decline of ovarian reserve. Quantitatively, mitochondrial biogenesis is crucial during oogenesis for constituting a mitochondrial pool sufficiently large to allow normal early embryonic development and to avoid the untimely activation of mitochondrial biogenesis. Ovarian ageing also seriously affects the dynamic nature of mitochondrial biogenesis in the surrounding granulosa cells that may provide interesting alternative biomarkers of oocyte quality. WIDER IMPLICATIONS: A fuller understanding of the involvement of mitochondria in cases of infertility linked to ovarian ageing would contribute to a better management of the disorder in the future.

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.