Restricted dispersal in a sea of gene flow.

How far do marine larvae disperse in the ocean? Decades of population genetic studies have revealed generally low levels of genetic structure at large spatial scales (hundreds of kilometres). Yet this result, typically based on discrete sampling designs, does not necessarily imply extensive dispersal. Here, we adopt a continuous sampling strategy along 950 km of coast in the northwestern Mediterranean Sea to address this question in four species. In line with expectations, we observe weak genetic structure at a large spatial scale. Nevertheless, our continuous sampling strategy uncovers a pattern of isolation by distance at small spatial scales (few tens of kilometres) in two species. Individual-based simulations indicate that this signal is an expected signature of restricted dispersal. At the other extreme of the connectivity spectrum, two pairs of individuals that are closely related genetically were found more than 290 km apart, indicating long-distance dispersal. Such a combination of restricted dispersal with rare long-distance dispersal events is supported by a high-resolution biophysical model of larval dispersal in the study area, and we posit that it may be common in marine species. Our results bridge population genetic studies with direct dispersal studies and have implications for the design of marine reserve networks.

[Percutaneous treatment of the pulmonary valve]. / Traitement percutané de la valve pulmonaire.

Right ventricular outflow tract diseases are historically outcomes of surgical reconstruction for heart defects in neonates or children (Tetralogy of Fallot, Ross surgery). This kind of surgery performed during childhood lead often to dysfunctional right ventricular outflow tract (stenosis, leak) in older infants or young adults. In this case, reintervention on the right ventricular outflow tract would be associated with a high surgical risk. Development of the first percutaneous valves in the year 2000 paved the way for the pulmonary revalvulation. This percutaneous procedure has emerged as a credible alternative to the surgery for multioperated high risk patients with congenital cardiopathies. Two valves are currently available on the French market (Melody®, Sapien®); they cover all therapy indications, except the example of very wide outflow tracts which remain a surgical issue. Medical teams in charge of these young patients have to be enough trained in order to limit risks during the procedure. To this end, several teams promote the percutaneous pulmonary revalvulation over surgical revalvulation, this latter becoming limited only to cases in which percutaneous treatment is not feasible.

Malaria control in India: A national perspective in a regional and global fight to eliminate malaria.

Since the declaration of the vision of malaria eradication in 2007, the overall burden of malaria has been reduced substantially in many countries in the endemic world. This progress has, however, recently slowed worldwide and even an increase of morbidity and mortality has been observed in some regions. That reality has led to reflection on the strategy for malaria elimination, noting that focusing only on low transmission sites has competed with the efforts in countries that still have foci with high malaria burdens. This opinion piece outlines the collaboration of the ICMR-National Institute of Malaria Research (ICMR-NIMR) and other partner Institutions in India with the WorldWide Antimalarial Resistance Network (WWARN), one part of a global effort to manage the spread of Plasmodium falciparum parasites associated with antimalarial resistance.

In-vitro validation of 4D flow MRI measurements with an experimental pulsatile flow model.

PURPOSE: The purpose of this study was to assess the precision of four-dimensional (4D) phase-contrast magnetic resonance imaging (PCMRI) to measure mean flow and peak velocity (Vmax) in a pulsatile flow phantom and to test its sensitivity to spatial resolution and Venc. MATERIAL AND METHODS: The pulsatile flow phantom consisted of a straight tube connected to the systemic circulation of an experimental mock circulatory system. Four-dimensional-PCMR images were acquired using different spatial resolutions (minimum pixel size: 1.5×1.5×1.5mm3) and velocity encoding sensitivities (up to three times Vmax). Mean flow and Vmax calculated from 4D-PCMRI were compared respectively to the reference phantom flow parameters and to Vmax obtained from two-dimensional (2D)-PCMRI. RESULTS: 4D-PCI measured mean flow with a precision of -0.04% to+5.46%, but slightly underestimated Vmax when compared to 2D-PCMRI (differences ranging from -1.71% to -3.85%). 4D PCMRI mean flow measurement was influenced by spatial resolution (P<0.001) with better results obtained with smaller voxel size. There was no effect of Venc on mean flow measurement. Regarding Vmax, neither spatial resolution nor Venc did influence the precision of the measurement. CONCLUSION: Using an experimental pulsatile flow model 4D-PCMRI is accurate to measure mean flow and Vmax with better results obtained with higher spatial resolution. We also show that Venc up to 3 times higher than Vmax may be used with no effect on these measurements.

Position paper of French Interventional Group (GACI) for TAVI in France in 2018.

Aortic stenosis is a frequent disease in the elderly. Its prevalence is 0.4% with a sharp increase after the age of 65, and its outcome is very poor when the patient becomes symptomatic. The interventional procedure known as TAVI (trans-catheter aortic valve implantation), which was developed in France and carried out for the first time in Rouen by Prof. Alain Cribier and his team in 2002, has proven to be a valid alternative to surgical aortic valve replacement. At first, this technique was shown to be efficient in patients with contra-indications to surgical treatment or deemed to be at high surgical risk. Given the very promising outcomes achieved as a result of close heart team collaboration, appropriate patient selection, simplified procedures and reduced complication rates, transfemoral (TF) TAVI is now preferred in symptomatic intermediate risk patients>75 years old according to the latest ESC guidelines. In 2017, in France, TAVI is currently performed in 50 centers with on-site cardiac surgery. The 2016 TAVI outcomes recorded in the French national TAVI registry (France TAVI) are very encouraging and show that for 7133 patients treated (age 83.4±7 years, logistic Euroscore 14%), 87% of whom via the TF approach, cross-over to surgery was very low (0.5%) with a 3.0% in-hospital mortality rate. The substantial increase in TAVI indications and the improvement of its outcomes may in the near future call for a reconsideration of the number of high volume centers authorized to carry out this technique.

[Percutaneous treatment of mitral regurgitation by Mitraclip in the elderly]. / Traitement percutané de l'insuffisance mitrale par Mitraclip chez le sujet âgé.

Mitral regurgitation (MR) is the second most common form of valvular heart disease. It is classified as either primary (degenerative) or secondary (functional). Secondary MR is the consequence of myocardium disease. Primary MR from degenerative valve disease is due to a primary disruption of the mitral valve apparatus from either prolapsed or flail leaflets. It covers all aetiologies in which intrinsic lesions affect one or several components of the mitral valve apparatus. Gold-standard therapeutic management of severe primary MR is surgery usually to repair but sometime to replace the mitral valve. However patients considered to be at high-risk due to their age or the presence of comorbidities - accounting for 50% of all patients - are not eligible for surgery. Catheter-based interventions have been developed to correct MR percutaneously. The only such intervention which has been evaluated in organic MR is the edge-to-edge procedure using the MitraClip® (Abbott Vascular, Menlo Park, CA). MitraClip® offers an alternative to open surgical repair or replacement via a minimally invasive route and it was shown in the EVEREST II study that MitraClip® was safer than surgery even though it was less effective in reducing MR. A substantial number of patients are ineligible for mitral valve surgery because of prohibitive surgical risk. For those patients, MitraClip® may offer an alternative treatment option. Percutaneous edge-to-edge repair is the first percutaneous option accepted in the 2012 ESC guidelines: Percutaneous edge-to-edge procedure may be considered in patients with symptomatic severe primary MR who fulfill the echo criteria of eligibility, are judged inoperable or at high surgical risk by a 'heart team', and have a life expectancy greater than 1 year (recommendation class IIb, level of evidence C). Because of its frailty, MitraClip® in the elderly may be a good alternative to mitral surgery when indicated for primary or secondary MD.

Complex interactions between malaria and malnutrition: a systematic literature review.

BACKGROUND: Despite substantial improvement in the control of malaria and decreased prevalence of malnutrition over the past two decades, both conditions remain heavy burdens that cause hundreds of thousands of deaths in children in resource-poor countries every year. Better understanding of the complex interactions between malaria and malnutrition is crucial for optimally targeting interventions where both conditions co-exist. This systematic review aimed to assess the evidence of the interplay between malaria and malnutrition. METHODS: Database searches were conducted in PubMed, Global Health and Cochrane Libraries and articles published in English, French or Spanish between Jan 1980 and Feb 2018 were accessed and screened. The methodological quality of the included studies was assessed using the Newcastle-Ottawa Scale and the risk of bias across studies was assessed using the GRADE approach. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guideline were followed. RESULTS: Of 2945 articles screened from databases, a total of 33 articles were identified looking at the association between malnutrition and risk of malaria and/or the impact of malnutrition in antimalarial treatment efficacy. Large methodological heterogeneity of studies precluded conducting meaningful aggregated data meta-analysis. Divergent results were reported on the effect of malnutrition on malaria risk. While no consistent association between risk of malaria and acute malnutrition was found, chronic malnutrition was relatively consistently associated with severity of malaria such as high-density parasitemia and anaemia. Furthermore, there is little information on the effect of malnutrition on therapeutic responses to artemisinin combination therapies (ACTs) and their pharmacokinetic properties in malnourished children in published literature. CONCLUSIONS: The evidence on the effect of malnutrition on malaria risk remains inconclusive. Further analyses using individual patient data could provide an important opportunity to better understand the variability observed in publications by standardising both malaria and nutritional metrics. Our findings highlight the need to improve our understanding of the pharmacodynamics and pharmacokinetics of ACTs in malnourished children. Further clarification on malaria-malnutrition interactions would also serve as a basis for designing future trials and provide an opportunity to optimise antimalarial treatment for this large, vulnerable and neglected population. TRIAL REGISTRATION: PROSPERO CRD42017056934 .

Evaluating drug resistance in visceral leishmaniasis: the challenges.

For decades antimonials were the drugs of choice for the treatment of visceral leishmaniasis (VL), but the recent emergence of resistance has made them redundant as first-line therapy in the endemic VL region in the Indian subcontinent. The application of other drugs has been limited due to adverse effects, perceived high cost, need for parenteral administration and increasing rate of treatment failures. Liposomal amphotericin B (AmB) and miltefosine (MIL) have been positioned as the effective first-line treatments; however, the number of monotherapy MIL-failures has increased after a decade of use. Since no validated molecular resistance markers are yet available, monitoring and surveillance of changes in drug sensitivity and resistance still depends on standard phenotypic in vitro promastigote or amastigote susceptibility assays. Clinical isolates displaying defined MIL- or AmB-resistance are still fairly scarce and fundamental and applied research on resistance mechanisms and dynamics remains largely dependent on laboratory-generated drug resistant strains. This review addresses the various challenges associated with drug susceptibility and -resistance monitoring in VL, with particular emphasis on the choice of strains, susceptibility model selection and standardization of procedures with specific read-out parameters and well-defined threshold criteria. The latter are essential to support surveillance systems and safeguard the limited number of currently available antileishmanial drugs.

Circulating PCSK9 levels in acute coronary syndrome: Results from the PC-SCA-9 prospective study.

BACKGROUND: Serum proprotein convertase subtilisin/kexin type 9 (PCSK9) concentrations have been shown to be positively associated with LDL cholesterol (LDL-C), but the relationship between PCSK9 and coronary atherosclerosis lesions remains unclear. OBJECTIVE: This study aims to investigate the correlation between serum PCSK9 levels and coronary damage severity in patients hospitalized for acute coronary syndrome (ACS). METHODS: In this prospective proof-of-concept study, coronary lesions were assessed using SYNTAX scores. Serum PCSK9 concentrations were measured on admission (Day 0) for ACS by Elisa, and on every day of hospitalization. Spearman's correlations were used to determine the association between PCSK9 levels, SYNTAX score and metabolic parameters. RESULTS: A total of 174 patients (mean age: 59±14 years, 79% male) with ACS (on Day 0, 119 patients were not taking statins, but 55 were) were included. After initiation of high-intensity statin therapy, serum PCSK9 concentrations increased significantly, reaching maximum levels on Day 2 (+31% vs. Day 0), and remained stable up to Day 4 (P<0.001, by mixed model). Serum PCSK9 on Day 0 was associated with LDL-C (rho=0.226, P=0.017) and apolipoprotein B (rho=0.282, P=0.005) in the statin-naïve group only, and with triglycerides and non-HDL-C in all groups. More important, PCSK9 levels on Day 0 were positively associated with SYNTAX scores in the statin-naïve group (rho=0.239, P=0.009), but not in the statin-treated group (P=NS). This association was maintained after adjusting for LDL-C (P=0.014) and major CV risk factors (P=0.008). CONCLUSION: Serum PCSK9 levels are positively associated with severity of coronary artery lesions independently of LDL-C concentrations in patients hospitalized for ACS. This reinforces the potential importance of PCSK9 inhibition in the management of ACS.

1.15 PW-850 J compressed beam demonstration using the PETAL facility.

The Petawatt Aquitaine Laser (PETAL) facility was designed and constructed by the French Commissariat à l'énergie atomique et aux énergies alternatives (CEA) as an additional PW beamline to the Laser MegaJoule (LMJ) facility. PETAL energy is limited to 1 kJ at the beginning due to the damage threshold of the final optics. In this paper, we present the commissioning of the PW PETAL beamline. The first kJ shots in the amplifier section with a large spectrum front end, the alignment of the synthetic aperture compression stage and the initial demonstration of the 1.15 PW @ 850 J operations in the compression stage are detailed. Issues encountered relating to damage to optics are also addressed.

The Vivax Surveyor: Online mapping database for Plasmodium vivax clinical trials.

INTRODUCTION: Recurrent P. vivax infections are associated with significant morbidity and mortality. Although radical cure can reduce recurrent infection, it is confounded by antimalarial resistance and the lack of safe and effective hypnozoitocidal treatment. This study documents the available literature of published clinical trials of P. vivax, providing an up to date, online, open access tool to view and download available information. METHODS: A systematic review was conducted according to PRISMA guidelines to identify prospective P. vivax therapeutic clinical trials with at least 28 days follow-up published between 1st January 1960 and 12th October 2016. Treatment arms and evidence of chloroquine resistance were mapped to trial sites. RESULTS: Since 1960, a total of 1152 antimalarial clinical trials with a minimum 28 days follow-up have been published, of which 230 (20.0%) enrolled patients with P. vivax and were included. Trials were conducted in 38 countries: 168 (73.0%) in the Asia-Pacific, 13 (5.7%) in Africa and 43 (18.7%) in the Americas. The proportion of antimalarial trials assessing P. vivax rose from 10.7% (12/112) in 1991-1995, to 24.9% (56/225) in 2011-2015. Overall, 188 (81.7%) P. vivax trials included a chloroquine treatment arm, either alone or in combination with primaquine, and 107 (46.5%) trials included a chloroquine treatment arm with early primaquine to assess radical cure. There has been a recent increase in treatment arms with artemisinin derivatives. Of the 131 sites in which chloroquine resistance could be quantified, resistance was present in 59 (45.0%) sites in 15 endemic countries. CONCLUSIONS: Over the last 20 years there has been a substantial increase in clinical research on the treatment of P. vivax, which has generated a greater awareness of the global extent of chloroquine resistance. The WWARN open access, online interactive map provides up to date information of areas where drug resistant P. vivax is emerging.

[Pelvi-perineal chronic pain, urgent demands of care and decision-making]. / Douleurs pelvipérinéales chroniques, demandes urgentes de soins et prise de décision.

OBJECTIVE: Some patients suffering from pelvi-perineal chronic pain express urgent, even vital, demands of care. The objective was to compare the profile and the psychological functioning of the patients, who have shown an imperious demand of care, in that of the patients who do not have an imperious demand of care. MATERIAL AND METHOD: From the medical consultations for chronic pelviperineal pain, we realized a comparative study including 26 patients (experimental group) expressing an urgent demand of care (i.e., patients who, during the last 3months, called an emergency service concerning the pains for which they consult in the service) and 28 patients (control group) without an urgent demand of care (i.e., patient who, during the last 3months, did not call an emergency service concerning the pains for which they consult in the service). All the patients were tested through a cognitive task of decision-making (Iowa Gambling Task) and through explicit measures of pain and its main psychological associated factors (anxiety, depression, impulsivity an catastrophism). RESULTS: In the first place, the patients from the experimental group possess decision-making abilities equivalent to the patients of the control group; however, both groups of patients show, in the beginning of the test, a deficit in the decision-making (F(4.208)=3.4116; P=.009). Secondly, the measures to questionnaires reveal that the patients of the control group have less severe scores in the scales of depression (t(52)=-2.068; P<04), catastrophism (amplification : t(52)=-3.069; P<0035; powerlessness: t(52)=-2.866, P<.006) and impulsivity (positive urgency: t(52)=-2.246, P<029; lack of premeditation: t(52)=-2.175, P<035) than the patients of the experimental group. CONCLUSION: The use of explicit measures (questionnaire) and implicit measures (experimental task) allowed to objectify more precisely the differences between the chronic pain patients in urgent demand of care and the other chronic pain patients. This psychological specificity obliges us to approach differently the caring of these patients in particular by proposing adapted cognitivo-behavioral techniques. LEVEL OF EVIDENCE: 3.

[Real-time monitoring of anti-influenza vaccination in the 65 and over population in France based on vaccine sales]. / Suivi en quasi-temps réel de la vaccination contre la grippe chez les 65 ans et plus en France à partir des ventes de vaccins en pharmacie.

BACKGROUND: The aim of this study was to describe a tool based on vaccine sales to estimate vaccination coverage against seasonal influenza in near real-time in the French population aged 65 and over. METHODS: Vaccine sales data available on sale-day +1 came from a stratified sample of 3004 pharmacies in metropolitan France. Vaccination coverage rates were estimated between 2009 and 2014 and compared with those obtained based on vaccination refund data from the general health insurance scheme. RESULTS: The seasonal vaccination coverage estimates were highly correlated with those obtained from refund data. They were also slightly higher, which can be explained by the inclusion of non-reimbursed vaccines and the consideration of all individuals aged 65 and over. We have developed an online tool that provides estimates of daily vaccination coverage during each vaccination campaign. CONCLUSION: The developed tool provides a reliable and near real-time estimation of vaccination coverage among people aged 65 and over. It can be used to evaluate and adjust public health messages.

Predictors of Health-Related Quality of Life Decline after Transcatheter Aortic Valve Replacement in Older Patients with Severe Aortic Stenosis.

BACKGROUND: Transcatheter aortic-valve implantation (TAVI) has been shown to improve survival and quality of life in patients with severe aortic stenosis. However, one-third of patients have poor outcome as death, functional decline or quality of life (QoL) decline. The aim of this study was to determine cardiac and geriatric predictors of physical and mental QoL decline 6 months after a TAVI procedure in patients aged 75 and older. METHODS: Between January 2013 and June 2014, we did a prospective and multicenter study including patients ≥ 75 years old referred for TAVI. The primary outcome was the measure of QoL, assessed by the Short Form 36 survey (SF-36), before and 6 months after the intervention. Association between QoL decline and baseline characteristics including cardiac and geriatric factors was analysed by logistic regression models. RESULTS: Mean age of the 150 patients studied was 83.7 years old and 56% were men. The primary end point, mean SF-36 physical summary score, significantly improved between baseline and 6-month (33.6 vs. 36.4, p=0.003) whereas mental component score significantly decreased (48.2 vs. 36.4, p-value<0.001). However, patients with presence of depressive symptoms before the intervention had mental QoL improvement at six months (OR 0.04 [0.01-0.19], p-value<0.001) and no significant geriatric predictors were associated with physical QoL decline. CONCLUSION: The mental QoL significantly decreased and patients with preoperative depressive symptoms had mental QoL improvement at six months. Researches are needed to confirm that mental QoL of patients with depressive symptoms can be improved by TAVI.

Diagnostic and medical strategy for renovascular hypertension: report from a monocentric pediatric cohort.

UNLABELLED: Renovascular hypertension accounts for 5-10 % of hypertension cases in children; there is currently no consensus on treatment. Here, we report on our clinical experience with this disease and outline the different pathways in which to investigate it. We report retrospectively on ten children diagnosed with renovascular hypertension at the University Hospital of Nantes from 2001 to 2012. The main findings were obtained by fortuitous screening of children aged 2 months to 14 years old with neurofibromatosis (n = 2) and fibromuscular dysplasia (n = 8). The hypertension was always severe yet asymptomatic. Lesions were complicated in nine out of ten cases and included bilateral, multiple, mid-aortic syndrome and aneurysm. Doppler ultrasound associated with computed tomography allowed for a precise diagnosis in seven out of ten cases. Where ambiguities persisted, they were highlighted by arteriography, the gold standard investigation. Medical treatment was insufficient, leading to invasive procedures in nine out of ten children: 2 nephrectomies, 2 autotransplantations, and 21 repetitive percutaneous transluminal angioplasties. After invasive procedures, blood pressure control improved in four cases and was resolved in three. CONCLUSION: Arteriography remains to be the gold standard technique for renovascular hypertension in children and can be combined with angioplasty when medical treatment is rendered obsolete. The role of computed tomography is controversial. Despite the heterogeneity of the children studied, we present a general medical and therapeutic management pathway for the treatment of this disease.

Population Pharmacokinetics of Lumefantrine in Pregnant and Nonpregnant Women With Uncomplicated Plasmodium falciparum Malaria in Uganda.

Pregnancy alters the pharmacokinetic properties of many antimalarial compounds. The objective of this study was to evaluate the pharmacokinetic properties of lumefantrine in pregnant and nonpregnant women with uncomplicated Plasmodium falciparum malaria in Uganda after a standard fixed oral artemether-lumefantrine treatment. Dense venous (n = 26) and sparse capillary (n = 90) lumefantrine samples were drawn from pregnant patients. A total of 17 nonpregnant women contributed with dense venous lumefantrine samples. Lumefantrine pharmacokinetics was best described by a flexible absorption model with multiphasic disposition. Pregnancy and body temperature had a significant impact on the pharmacokinetic properties of lumefantrine. Simulations from the final model indicated 27% lower day 7 concentrations in pregnant women compared with nonpregnant women and a decreased median time of 0.92 and 0.42 days above previously defined critical concentration cutoff values (280 and 175 ng/ml, respectively). The standard artemether-lumefantrine dose regimen in P. falciparum malaria may need reevaluation in nonimmune pregnant women.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e83; doi:10.1038/psp.2013.59; advance online publication 13 November 2013.

[Superior vena cava syndrome induced by pacemaker probes, 12 years after introduction]. / Un syndrome cave supérieur 12ans après l'implantation d'un pacemaker.

Superior vena cava syndrome is a rare disease, most often found to result from a malignant process, which causes extrinsic compression of the superior vena cava. In recent years, there has been an increase of superior vena cava syndrome related to medical devices (implantable site, pacemaker [PM], central venous line for parenteral nutrition...). We report the case of a 37-year-old patient who developed a superior vena cava syndrome 12 years after implantation of a PM. The diagnosis was established on venography after two negative venous-CT focused on the superior vena cava. The superior vena cava syndrome improved immediately after angioplasty and stenting covering the PM probes at the superior vena cava/brachiocephalic venous trunk junction.

Fracture of the lower cervical spine in patients with ankylosing spondylitis: Retrospective study of 19 cases.

INTRODUCTION: Controversy exists surrounding optimal treatment of cervical spine fractures secondary to ankylosing spondylitis (AS). HYPOTHESIS: The anterior approach is an effective surgical technique for these fractures and can be used to correct the AS-induced cervical-thoracic kyphosis. MATERIALS AND METHODS: This continuous, retrospective series between 1990 and 2010 included 19 patients aged 33 to 84 years who presented with a lower cervical spine fracture in the context of AS. The average follow-up was 45 months. Sixteen of these patients were surgically treated using an anterior approach and anterior fixation. In five patients without any neurological deficit, their cervical-thoracic kyphosis was corrected during the same surgery. Regional kyphosis was measured before the surgery, immediately after the surgery and at the last follow-up. RESULTS: Five deaths occurred; these were all patients with post-traumatic complete quadriplegia. Most the incomplete neurological problems improved (66%). In no cases did the neurological condition worsen. Among the 16 patients operated with the anterior approach, two patients also required an additional procedure with a posterior approach because of a persistent neurological deficit. The fractures in the operated patients who survived (14 patients) had healed within an average 4-month delay (range 3-7 months), without worsening of the kyphosis at final follow-up. In the five cases where the kyphosis was corrected, the correction averaged 26° (range 18-36°); there were no neurological complications. DISCUSSION: Based on these results, we suggest using the anterior approach to perform internal fixation as a treatment for cervical fractures secondary to AS and to correct the cervical-thoracic kyphosis in patients without neurological deficits. LEVEL OF EVIDENCE: Level IV - retrospective study.

Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium.

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v(-1)) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L(-1) BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in in vitro-produced bovine embryo cryopreservation solutions.

Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse.

The present study evaluated: (1) in vivo follicular development in canine ovarian tissue after slow freezing and xenotransplantation; and (2) the use of erythropoietin (EPO) as an angiogenic factor to optimise the transplantation procedure. Frozen-thawed ovarian tissue from five bitches was grafted into severe combined immunodeficient (SCID) mice (n=47) treated with or without EPO (500 IU kg(-1), once daily for 3 days) (Groups A and B, respectively) and analysed after 0, 1, 8 or 16 weeks. Follicle grade, follicle density, follicle morphology and stromal cells density were assessed by histological analysis, whereas vascularisation of the graft was quantified by immunohistochemistry with anti-α-smooth muscle actin antibody. Despite a massive loss of follicles after grafting, secondary follicle density was higher at 8 and 16 weeks than at 1 week regardless of EPO treatment. EPO significantly improved early follicle morphology and stromal cell density after 8 weeks and blood vessel density at 16 weeks after transplantation (P<0.05). Intact secondary follicles with more than three granulosa cells layers were observed 16 weeks after transplantation. The results suggest that canine ovarian tissue can be successfully preserved by our slow-freezing protocol because the tissue showed follicular growth after xenotransplantation. EPO treatment did not lessen the massive loss of follicles after transplantation.