A simplified checklist for the visual inspection of finished pharmaceutical products: a way to empower frontline health workers in the fight against poor-quality medicines.

BACKGROUND: Substandard and falsified medicines, mainly prevalent in low and middle-income countries (LMICs), cause avoidable morbidity and mortality, and put at stake the performance of health systems. They may be prevented by an adequate implementation of pharmaceutical Quality Assurance (QA) guidelines, but unfortunately, most guidelines address upstream stakeholders and specialized staff in the supply chain. A multi-layered approach is needed, in order to empower the health workers at the point-of-care to proactively contribute to the fight against poor-quality medicines.Visual inspection is a simple technique, suitable for field screening. The findings of a survey conducted in the Democratic Republic of the Congo (DRC) suggested that it might be a fairly good (yet partial) predictor of poor-quality, when compared to full laboratory tests. METHODS AND RESULTS: Starting from the 68-questions checklist originally used in the survey in the DRC, we developed a simplified checklist, specifically designed to guide health workers at the point of care to rapidly identify suspect poor-quality medicines. We selected those medicines' attributes the assessment of which does not require technical expertise, or access to regulatory information. Attributes were categorized according to a 3-level risk scale, to guide decision-making on suspect poor-quality medicines, based on an informed risk assessment.The simplified checklist contains 26 binary questions (YES/NO), grouped into four themes: packaging, identification, traceability, and physical appearance. Each non-conformity corresponds to a level of risk for patients. The user is guided towards three possible actions: A) reasonably safe for dispensing; B) dispense with explanation; C) quarantine and make a risk-benefit evaluation before dispensing. CONCLUSION: The simplified checklist should now be implemented in real-life setting in LMICs. If proven useful in guiding health workers at the point-of-care to take rapid, transparent, patient-centred actions when facing a suspect poor-quality medicine, it could be further extended to address specific formulations. Digitalization for linkage with pharmacovigilance programs could also be considered.

The insulin sensitizing effect of topiramate involves KATP channel activation in the central nervous system.

BACKGROUND AND PURPOSE: Topiramate improves insulin sensitivity, in addition to its antiepileptic action. However, the underlying mechanism is unknown. Therefore, the present study was aimed at investigating the mechanism of the insulin-sensitizing effect of topiramate both in vivo and in vitro. EXPERIMENTAL APPROACH: Male C57Bl/6J mice were fed a run-in high-fat diet for 6 weeks, before receiving topiramate or vehicle mixed in high-fat diet for an additional 6 weeks. Insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamp. The extent to which the insulin sensitizing effects of topiramate were mediated through the CNS were determined by concomitant i.c.v. infusion of vehicle or tolbutamide, an inhibitor of ATP-sensitive potassium channels in neurons. The direct effects of topiramate on insulin signalling and glucose uptake were assessed in vivo and in cultured muscle cells. KEY RESULTS: In hyperinsulinaemic-euglycaemic clamp conditions, therapeutic plasma concentrations of topiramate (∼4 µg·mL(-1) ) improved insulin sensitivity (glucose infusion rate + 58%). Using 2-deoxy-D-[(3) H]glucose, we established that topiramate improved the insulin-mediated glucose uptake by heart (+92%), muscle (+116%) and adipose tissue (+586%). Upon i.c.v. tolbutamide, the insulin-sensitizing effect of topiramate was completely abrogated. Topiramate did not directly affect glucose uptake or insulin signalling neither in vivo nor in cultured muscle cells. CONCLUSION AND IMPLICATIONS: In conclusion, topiramate stimulates insulin-mediated glucose uptake in vivo through the CNS. These observations illustrate the possibility of pharmacological modulation of peripheral insulin resistance through a target in the CNS.

The history of Belgian assisted reproduction technology cycle registration and control: a case study in reducing the incidence of multiple pregnancy.

STUDY QUESTION: What is the effect of a legal limitation of the number of embryos that can be transferred in an assisted reproductive technology (ART) cycle on the multiple delivery rate? SUMMARY ANSWER: The Belgian national register shows that the introduction of reimbursement of ART laboratory costs in July 2003, and the imposition of a legal limitation of the number of embryos transferred in the same year, were associated with a >50% reduction of the multiple pregnancy rate from 27 to 11% between 2003 and the last assessment in 2010, without any reduction of the pregnancy rate per cycle. WHAT IS KNOWN ALREADY: Individual Belgian IVF centres have published their results since the implementation of the law, and these show a decrease in the multiple pregnancy rate on a centre by centre basis. However, the overall national picture remains unpublished. STUDY DESIGN, SIZE, DURATION: Cohort study from 1990 to 2010 of all ART cycles in Belgium (2685 cycles in 1990 evolving to 19 110 cycles in 2010), with a retrospective analysis from 1990 to 2000 and prospective online data collection since 2001. PARTICIPANTS/MATERIALS, SETTING, METHODS: Registration evolved from paper written reports per centre to a compulsory online registration of all ART cycles. From 2001 up to mid-2009, data were collected from Excel spread sheets or MS Access files into an MS Access database. Since mid-2009, data collection is done via a remote and secured web-based system (www.belrap.be) where centres can upload their data and get immediate feedback about missing data, errors and inconsistencies. MAIN RESULTS AND THE ROLE OF CHANCE: National Belgian registration data show that reimbursement of IVF laboratory costs in July 2003, coupled to a legal limitation in the number of embryos transferred in utero, were associated with a 50% reduction of the multiple pregnancy rate from 27 to 11% without reduction of the pregnancy rate per cycle, and with an increase in the number of fresh and frozen ART cycles due to improved access to treatment. LIMITATIONS, REASONS FOR CAUTION: There is potential underreporting of complications of ART treatment, pregnancy outcome and neonatal health. WIDER IMPLICATIONS OF THE FINDINGS: Over the 20 years of registration, the pregnancy rate has remained constant, despite the reduction in the number of embryos transferred, optimization of laboratory procedures and stimulation protocols, introduction of quality systems and implementation of the EU Tissue Directive over the period 2004-2010. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study. None of the authors has any conflict of interest to declare.

Pharmacological modulation of dopamine receptor D2-mediated transmission alters the metabolic phenotype of diet induced obese and diet resistant C57Bl6 mice.

High fat feeding induces a variety of obese and lean phenotypes in inbred rodents. Compared to Diet Resistant (DR) rodents, Diet Induced Obese (DIO) rodents are insulin resistant and have a reduced dopamine receptor D2 (DRD2) mediated tone. We hypothesized that this differing dopaminergic tone contributes to the distinct metabolic profiles of these animals. C57Bl6 mice were classified as DIO or DR based on their weight gain during 10 weeks of high fat feeding. Subsequently DIO mice were treated with the DRD2 agonist bromocriptine and DR mice with the DRD2 antagonist haloperidol for 2 weeks. Compared to DR mice, the bodyweight of DIO mice was higher and their insulin sensitivity decreased. Haloperidol treatment reduced the voluntary activity and energy expenditure of DR mice and induced insulin resistance in these mice. Conversely, bromocriptine treatment tended to reduce bodyweight and voluntary activity, and reinforce insulin action in DIO mice. These results show that DRD2 activation partly redirects high fat diet induced metabolic anomalies in obesity-prone mice. Conversely, blocking DRD2 induces an adverse metabolic profile in mice that are inherently resistant to the deleterious effects of high fat food. This suggests that dopaminergic neurotransmission is involved in the control of metabolic phenotype.

Blocking dopamine D2 receptors by haloperidol curtails the beneficial impact of calorie restriction on the metabolic phenotype of high-fat diet induced obese mice.

Calorie restriction is the most effective way of expanding life-span and decreasing morbidity. It improves insulin sensitivity and delays the age-related loss of dopamine receptor D(2) (DRD2) expression in the brain. Conversely, high-fat feeding is associated with obesity, insulin resistance and a reduced number of DRD2 binding sites. We hypothesised that the metabolic benefit of calorie restriction involves the preservation of appropriate DRD2 transmission. The food intake of wild-type C57Bl6 male mice was restricted to 60% of ad lib. intake while they were treated with the DRD2 antagonist haloperidol or vehicle using s.c. implanted pellets. Mice with ad lib. access to food receiving vehicle treatment served as controls. All mice received high-fat food throughout the experiment. After 10 weeks, an i.p. glucose tolerance test was performed and, after 12 weeks, a hyperinsulinaemic euglycaemic clamp. Hypothalamic DRD2 binding was also determined after 12 weeks of treatment. Calorie-restricted (CR) vehicle mice were glucose tolerant and insulin sensitive compared to ad lib. (AL) fed vehicle mice. CR mice treated with haloperidol were slightly heavier than vehicle treated CR mice. Haloperidol completely abolished the beneficial impact of calorie restriction on glucose tolerance and partly reduced the insulin sensitivity observed in CR vehicle mice. The metabolic differences between AL and CR vehicle mice were not accompanied by alterations in hypothalamic DRD2 binding. In conclusion, blocking DRD2 curtails the metabolic effects of calorie restriction. Although this suggests that the dopaminergic system could be involved in the metabolic benefits of calorie restriction, restricting access to high-fat food does not increase (hypothalamic) DRD2 binding capacity, which argues against this inference.

Relationship between hyaluronic acid binding assay and outcome in ART: a pilot study.

The sperm-hyaluronan binding assay (HBA) is a diagnostic kit for assessing sperm maturity, function and fertility. The aim of this prospective cohort pilot study was to evaluate the relationship between HBA and WHO sperm parameters (motility, concentration and detailed morphology) and possible influence of sperm processing on hyaluronic acid binding. A cohort of 68 patients undergoing a first combo in vitro fertilisation/intracytoplasmic sperm injection treatment after failure of three or more intrauterine insemination cycles were included in the study. Outcome measures studied were fertilisation rate, embryo quality, ongoing pregnancy rate and cumulative pregnancy rate. HBA outcome improved after sperm preparation and culture, but was not correlated to detailed sperm morphology, concentration or motility. HBA did not provide additional information for identifying patients with poor or absent fertilisation, although the latter had more immature sperm cells and cells with cytoplasmic retention present in their semen. HBA outcome in the neat sample was significantly correlated with embryo quality, with miscarriage rates and ongoing pregnancy rates in the fresh cycles, but not with the cumulative ongoing pregnancy rate. No threshold value for HBA and outcome in combo IVF/ICSI treatment could be established. The clinical value for HBA in addition to routine semen analysis for this patient population seems limited.

Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics.

BACKGROUND: Gene expression of cumulus cells (CC) could predict oocyte developmental quality. Knowledge of the genes involved in determining oocyte quality is scanty. The aim was to correlate clinical and biological characteristics during ovarian stimulation with the expression of 10 selected genes in CC. METHODS: Sixty-three ICSI patients were stimulated with GnRH-agonist plus highly purified hMG (n = 35) or recombinant FSH (n = 28). Thirteen variables were analyzed: Age, BMI, duration of stimulation, serum concentrations of progesterone, 17beta-estradiol, FSH and LH on day of hCG, Ovarian Response, Oocyte Maturity, 2 pronuclei and three embryo morphology related variables: > or =7 cells, Low Fragmentation, Good Quality Embryos score. Expression of HAS2, VCAN, SDC4, ALCAM, GREM1, PTGS1, PTGS2, DUSP16, SPROUTY4 and RPS6KA2 was analyzed in pooled CC using quantitative PCR, and the relationship to the 13 variables was evaluated by multivariable analysis. RESULTS: All 10 genes are expressed at oocyte retrieval, with PTGS1, SPROUTY4, DUSP16 and RPS6KA2 described in human ovary for the first time. The three variables that correlated most often with differential expression were Age, BMI and serum FSH level. Significant correlation was found with Oocyte Maturity (VCAN, P < 0.005), Low Fragmentation (RPS6KA2, P < 0.05), Embryos with > or =7 cells (ALCAM and GREM1, P < 0.05). The expression of the other genes was also correlated to oocyte developmental quality but to a less extent. SDC4, VCAN, GREM1, SPROUTY4 and RPS6KA2 showed gonadotrophin preparation-dependent expression and/or interactions (all P < 0.05). CONCLUSION: The expression of ovulation related genes in CC is associated with patient and treatment characteristics, oocyte developmental potential and differs with the type of gonadotrophin used.

Growth hormone and ghrelin secretion are associated with clinical severity in Huntington's disease.

BACKGROUND AND PURPOSE: Huntington's disease (HD) is a fatal hereditary neurodegenerative disorder caused by an increased CAG repeat size in the huntingtin gene. Apart from neurological impairment, the disease is also accompanied by progressive weight loss, abnormalities in glucose homeostasis and a higher prevalence of diabetes mellitus, which may partly be caused by disturbed growth hormone (GH) and ghrelin secretion. Therefore, we aimed to perform a detailed analysis of GH and ghrelin secretion in HD patients in relation to clinical signs and symptoms. METHODS: In nine early-stage, medication-free HD patients and nine age-, gender- and body mass index-matched controls, we measured serum GH levels every 10 min for 24 h and assessed ghrelin response to food intake. Multi-parameter auto-deconvolution and approximate entropy analysis were applied to quantify basal, pulsatile, and total GH secretion rates as well as the regularity of GH secretion. RESULTS: We found no significant differences in GH and ghrelin secretion characteristics between HD patients and controls (total GH secretion: 137 +/- 36 vs. 181 +/- 43 mU/l/24 h, respectively; P = 0.439). However, in HD patients, both GH secretion and its irregularity as well as the degree of postprandial ghrelin suppression significantly increased with worsening motor and functional impairment (all P < 0.05). Moreover, postprandial ghrelin suppression also increased with decreasing body weight and higher CAG repeat number (both P < 0.05). CONCLUSIONS: These findings suggest changes in the regulation of GH and ghrelin secretion dynamics in early stage HD patients that could become more prominent in the later stages of the disease.

Low-dose HCG may improve pregnancy rates and lower OHSS in antagonist cycles: a meta-analysis.

Human chorionic gonadotrophin (HCG) may substitute FSH to complete follicular growth in IVF cycles. This may be useful in the prevention of ovarian hyperstimulation syndrome. Relevant studies were identified on Medline. To evaluate outcomes, a meta-analysis of low-dose HCG-supplemented IVF cycles versus non-supplemented ones was performed with data from 435 patients undergoing IVF who were administered low-dose HCG in various agonist and antagonist protocols and from 597 conservatively treated patients who served, as control subjects. Using these published data, a decision analysis evaluated four different management strategies. Effectiveness and economic outcomes were assessed by FSH consumption, clinical pregnancy and incremental cost-effectiveness ratios. Clinical pregnancy and ovarian hyperstimulation were the main outcome measures. Nine trials published in 2002-2007 were included. From the prospective studies, in the gonadotrophin-releasing hormone antagonist group, a trend for significance in clinical pregnancy rate was evident (odds ratio [OR], 1.54; 95% confidence interval [CI], 0.98-2.42). Ovarian hyperstimulation was less significant in the antagonist low-dose HCG protocol compared with the non-supplemented agonist protocol (OR 0.30; 95% CI 0.09-0.96). Less FSH was consumed in the low-dose HCG group but this difference was not statistically significant. Low-dose HCG supplementation may improve pregnancy rates in antagonist protocols. Overall, low-dose HCG-supplemented protocols are a cost-effective strategy.

Fluorescence microscopy and flow cytometry in measuring activated caspases in human spermatozoa.

Staining of spermatozoa with the fluorescein-Val-Ala-Asp-fluoromethylketone has already been performed on ejaculated sperm samples, using fluorescence microscopy (FM) or flow cytometry (FCM) in order to score activated caspases. This assay may help in assessing apoptosis and its role in male fertility. The present study compares the above two techniques in order to adopt the most accurate method for detection in human frozen-thawed testicular, epididymal and ejaculated spermatozoa. The analyses were carried out on frozen/thawed testicular (n = 14), epididymal (n = 8) and ejaculated (n = 10) sperm samples. Activated caspases were detected in living spermatozoa using fluorescein-labelled inhibitors of poly-caspases (FLICA). For the measurements by FM, the same-observer and different-observer reliability were assessed in testicular and epididymal sperm samples. The inter-method (FM and FCM) reliability was assessed both in epididymal and ejaculated sperm samples. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements from the same sample were tested by Wilcoxon test for matched pairs. For the same-observer and the different-observer data, the ICC were 0.980 and 0.986. In testicular suspensions, the presence of different types of germinal and somatic cells hampers the differentiation of stained spermatozoa by FCM. For the inter-method reliability, the ICC was 0.903. A lower proportion of the viable spermatozoa stained with FLICA was observed by using FM (-6.60 +/- 7.38 %, mean +/- SD; p = 0.003) compared with FCM. To measure the proportion of spermatozoa with activated caspases by this test, FM is a highly accurate and reliable method whatever the sperm origin (ejaculate, epididymis, and testis). FCM cannot be used for testicular samples but seems to be more appropriate for analysis of epididymal and ejaculated sperm samples. The systematic lower proportion by FM in measuring the proportion of stained viable spermatozoa with FLICA involves that only the data measured by the same method (FM or FCM) may be compared.

Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer.

BACKGROUND: Single-embryo transfer is a well-accepted strategy to avoid multiple pregnancies in an assisted reproductive technology (ART) programme. Besides the morphological quality and embryo kinetics up to the blastocyst stage, preimplantation genetic screening (PGS) of aneuploidy has been advocated as an adjuvant approach to select the embryo. METHODS: Couples with a female partner younger than 36 were randomly assigned to undergo transfer of a single blastocyst in a cycle with or without PGS using FISH for the chromosomes X, Y, 13, 16, 18, 21, 22. RESULTS: After the enrolment of 120 of the projected 447 patients in each group, study recruitment was terminated prematurely on the basis of futility. The observed live birth delivery rates after ART were 30.8 versus 30.8% per randomized patient, 34.6 versus 34.6% per cycle initiated, 37.8 versus 37.0% per aspirated cycle and 41.6 versus 43.5% per embryo transfer for the control versus the PGS group, respectively, with absolute between-group differences (95% CI; P value) of 0% (-11.7 to 11.7; P = 1.00), 0% (-12.7 to 12.7; P = 1.00), -0.8% (-14.2 to 12.7; P = 0.91) and 2.1% (-12.7 to 16.7; P = 0.79), respectively. Even in this younger age group, only 61% of the embryos had a normal diploid status. CONCLUSIONS: The absence of a beneficial treatment effect in this randomized clinical trial provides no arguments in favour of PGS to improve live birth delivery rate following single-embryo transfer in women under the age 36. Clinical Trials.gov: NCT00670059.

Neonatal outcome of 937 children born after transfer of cryopreserved embryos obtained by ICSI and IVF and comparison with outcome data of fresh ICSI and IVF cycles.

BACKGROUND: To evaluate the safety of cryopreservation in combination with IVF and ICSI, prenatal diagnosis and neonatal outcome were investigated in children conceived from frozen-thawed ICSI embryos (cryo ICSI) and frozen-thawed IVF embryos (cryo IVF). Data were also compared with earlier published results from fresh ICSI and IVF embryos. METHODS: Questionnaire data and results of physical examination at 2 months of 547 cryo ICSI children and 390 cryo IVF children were compared, and these were also compared with those of infants born after transfer of fresh embryos. RESULTS: Birth characteristics were comparable for cryo ICSI and cryo IVF infants. Cryo singletons showed a trend towards higher mean birthweight compared with fresh singletons, in ICSI and IVF, reaching significance when all cryo (ICSI plus IVF) singletons were considered. Low birthweight rate according to multiplicity was comparable between the fresh and the cryo groups, in ICSI and IVF. Non-statistically significantly increased rates of de novo chromosomal anomalies (3.2%) were found in cryo ICSI fetuses/children compared with the fresh ICSI group (1.7%) (OR 1.96; 95% CI 0.92-4.14). Major malformations were more frequently observed in cryo ICSI live borns (6.4%) than in cryo IVF live borns (3.1%) (OR 2.15; 95% CI 1.10-4.20) and fresh ICSI live borns (3.4%) (OR 1.96; 95% CI 1.31-2.91). CONCLUSIONS: In cryo ICSI compared with cryo IVF, prenatal and neonatal outcome results were comparable, except for a higher major malformation rate in the cryo ICSI group. In the total cryo group compared with the total fresh group, we found a higher mean birthweight in singletons and a higher major malformation rate in live borns.

Randomized sibling-oocyte study using recombinant human hyaluronidase versus bovine-derived Sigma hyaluronidase in ICSI patients.

BACKGROUND: The enzyme hyaluronidase from bovine origin is commonly used for oocyte-cumulus cell removal in ICSI. A recombinant human hyaluronidase (rHuPH20) has been introduced as a quality-controlled and safe alternative. METHODS: In order to validate its effectiveness, a non-inferiority trial was started on sibling cumulus-oocyte complexes (135 ICSI patients). Oocyte denudation involved enzyme incubation under Pasteur pipetting, followed by further mechanical stripping. Primary end-points were oocyte intactness after ICSI and fertilization rate. Secondary end-points were embryo development and positive hCG. RESULTS: Oocyte intactness after ICSI was 89.6% and 92.9% with rHuPH20 and bovine hyaluronidase, respectively [absolute difference -3.3% (-7.4 to 10.7)]. The fertilization rate was 73.9% after rHuPH20 and 77.1% after bovine hyaluronidase treatment [absolute difference -3.2% (-8.3 to 1.8)]. Embryo development was similar in both treatment groups up till Day 5. Positive hCGs were equally distributed over mixed transfers 21/45 (46.7%) and transfers of only embryos from rHuPH20 treatment 17/35 (48.6%) or transfers of only embryos from bovine hyaluronidase treatment 22/48 (45.8%). CONCLUSIONS: Our results indicate that rHuPH20 is not inferior to bovine hyaluronidase for oocyte denudation, with regard to oocyte survival and fertilization. rHuPH20 treatment of human oocytes is compatible with good embryo development, with positive hCG results and with live birth.

CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation.

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.

Real and expected delivery rates of patients with myotonic dystrophy undergoing intracytoplasmic sperm injection and preimplantation genetic diagnosis.

BACKGROUND: This study aimed to analyse the reproductive outcome of a large cohort of myotonic dystrophy type 1 (DM1) patients undergoing ICSI and PGD. The secondary outcome parameter of this study was ovarian response as a way to express gonadal function in female DM1 patients. METHODS: Prospective cohort study. Real and expected cumulative delivery rates are descriptive. The reproductive outcome per cycle was compared with that of a control group of patients with X-linked recessive disorders. The comparative analysis of ovarian stimulation parameters in the study group versus the control group was carried out using both bivariate (crude) and multivariate (linear regression) analysis. RESULTS: Between 1995 and 2005, 205 cycles of ICSI and PGD were carried out for DM1 in 78 couples. The real cumulative delivery rate (max 6 cycles) overall was 46%. The expected overall cumulative delivery rate was 72%. Multivariate analysis did not show a significant difference in total dose of gonadotrophins used for ovarian stimulation between Group A (in which the female partner was affected) and a control group. CONCLUSIONS: This study shows that ICSI and PGD for DM1 offer good reproductive outcome, both in cumulative terms and per treatment cycle. There is no evidence of impaired gonadal function in female DM1 patients.

Back muscle as a promising site for ovarian tissue transplantation, an animal model.

BACKGROUND: The aim of this study was to evaluate the optimal transplantation site for ovarian tissue fragments in murine hosts. We compared the transplantation to the back muscle (B) versus the kidney capsule (K) in a mouse allograft model. METHODS: Hemi-ovaries from 12-day-old mice were allografted into B and K of bilaterally ovariectomized same strain recipients which had undergone gonadotrophin stimulation (n = 15). Graft survival after 27 days, angiogenesis and follicle development were scored and compared to age-matched control ovaries (38-day old, n = 5). The ability of oocytes to be fertilized was studied after IVF, ICSI and embryos were transferred to recipient mothers. Anti-mouse CD 31+ antibody was used to evaluate neo-vascularization in grafts. RESULTS: Primordial follicle survival was higher (P < 0.01) and vascular support was better (P < 0.01) in B- than in K-grafts. From 34 oocytes retrieved from B-grafts (15 metaphase I, of which 14 matured in vitro, and 19 collected at metaphase II), 18 morulae were obtained. Transfer of 12 embryos obtained by ICSI led to three live offspring, and transfer of six IVF embryos to another recipient mother yielded four offspring, one of which was born dead and one showed placental anomalies. CONCLUSIONS: The back muscle is a promising site for ovarian allografts in mice. This is the first report of live offspring obtained after back muscle grafting using both IVF and ICSI.

Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes.

BACKGROUND: Somatic cell nuclear transfer (SCNT) involves the transfer of somatic cell nuclei into enucleated oocytes. Because human in vivo matured oocytes are scarcely available, we investigated whether in vitro matured (IVM) germinal vesicle (GV) oocytes could also support preimplantation development of human cloned embryos. METHODS: Three groups were used for SCNT: in vitro matured GV oocytes (IVM oocytes), 'in vivo' matured oocytes (in vivo oocytes) and 'failed fertilized' oocytes after routine-ICSI (FF oocytes). After removal of the chromosome-spindle complex, cumulus cell nuclei were injected, and oocytes were artificially activated and cultured. RESULTS: In total 61, 54 and 45 metaphase II oocytes were used for SCNT in the three groups, respectively. Survival and pronuclear rates were 59, 78 and 58% and 61, 64 and 50%, respectively. Of the 22 activated IVM oocytes, 13 cleaved to the 2-cell stage, whereby 2 morulae were formed. For the in vivo oocytes, 17 of 27 activated oocytes cleaved to the 2-cell stage and 1 morula was observed. Cleavage to the 2-cell stage in the group of FF oocytes was compromised. CONCLUSIONS: To our knowledge, this is the first report describing development of cloned human embryos using IVM oocytes and non-autologous transfer using a conventional method of SCNT.

Co-culture of pre-antral ovarian follicles does not increase the yield of in-vitro-matured oocytes in the mouse.

In-vitro culture of ovarian follicles offers the possibility of checking whether there are direct interactions between follicles. Gross follicle morphology and oocyte maturation were investigated as endpoints after co-culture of pre-antral mouse ovarian follicles. Pre-antral mouse follicles with a diameter of 95-110 microm (small) or >110-160 microm (large) were cultured. Pairs of different size or large like-sized follicles were cultured either in physical contact or without in 20 microl culture medium droplets under oil for 10 days. Individual culture of small and large follicles served as controls. On day 10 human chorionic gonadotrophin was administered. On day 11, 'in-vitro ovulated' cumulus-oocyte complexes where the cumuli oophori had expanded (termed 'mucified') were collected. The percentage of metaphase II (MII) oocytes was scored as primary endpoint. There were temporary differences between the degree of follicle attachment and diffusion of granulosa cells, but with the observations of antrum formation by day 10, there was no difference between different types of co-cultures and individual culture of large follicles. The MII-to-follicle rate was highest for individual culture of large follicles (54.7%) followed by coculture of large like-sized follicles growing either in contact or not (40.1% and 42.6%), co-culture of different-sized follicles growing in contact or not (28.5% and 29.8%) and small follicles cultured individually (8.1%). In conclusion, mature oocyte yield was not increased by co-culture of mouse pre-antral ovarian follicles.

Ultrasound-guided embryo transfer does not offer any benefit in clinical outcome: a randomized controlled trial.

BACKGROUND: Ultrasound-guided embryo transfer (ET) is widely suggested as a standard clinical practice that improves overall embryo implantation and pregnancy rates. Various studies of this issue suffer from methodological pitfalls, so that a randomized controlled trial, which overcomes these problems, might be valuable. METHODS: Three hundred women aged <40, who underwent fresh ET, were included in this randomized, double-blind controlled trial. The K-J-SPPE echo tip soft catheter was used for the ultrasound-guided ET and the traditional K-Soft catheter for ETs not using ultrasound. One experienced operator performed all ETs. The primary study outcome was overall pregnancy rate (defined as the number of positive hCG results per transfer). RESULTS: No significant differences between groups were found regarding baseline patient and embryological characteristics, except for male factor and unexplained infertility (higher in the blind and ultrasound-guided ET group, respectively, P < 0.05). Overall pregnancy rates were 53.3 and 51.3% in the ultrasound-guided and blind ET group, respectively. Two ectopic pregnancies were reported in each group. Difficulty in cervical negotiation did not differ between the two groups. CONCLUSIONS: In patients undergoing ET by an experienced operator, ultrasound guidance did not provide any benefit in terms of overall clinical pregnancy and embryo implantation rates.

Single embryo transfer in preimplantation genetic diagnosis cycles for women <36 years does not reduce delivery rate.

BACKGROUND: The Belgian legislation imposes single embryo transfer (SET) on women of <36 years in their first treatment cycle to avoid multiple pregnancies. The aim of this study is to assess the impact of this legislation on the outcome of preimplantation genetic diagnosis (PGD) for inherited diseases in young women undergoing SET. METHODS: A retrospective analysis of PGD cycles for monogenic disorders and translocations in women <36 years on their first treatment cycle. Two groups of patients were defined according to the implementation of the Belgian legislation: (i) double embryo transfer (DET), January 2001-June 2003 (ii) SET, July 2003-June 2005. The primary and secondary outcome measures were delivery per embryo transfer and multiple pregnancy rates, respectively. A subgroup analysis for monogenic disorders and translocations was performed. RESULTS: 62 cycles were included in the DET group and 73 cycles in the SET group. The mean age, number of cumulus-oocyte complexes, number of fertilized oocytes, number of biopsied and cryopreserved embryos were comparable between both groups. There was no significant difference in the delivery rates between the DET and the SET groups (33.9% versus 27.4%, respectively). Multiple pregnancies were avoided when SET was performed. When monogenic disorders and chromosomal translocations were separately evaluated, no significant difference in the delivery rate after SET was observed. CONCLUSIONS: The implementation of a SET policy in young women undergoing PGD for monogenic disorders and translocations enables a significant reduction of multiple pregnancies without significantly affecting the delivery rate.