CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations ('Cu*Ag') acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of 'Cu*Ag', and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis.

The effects of embryo culture media on the birthweight of singletons via fresh or frozen-thawed embryo transfer: a large-scale retrospective study.

BACKGROUND: Embryo culture media used for IVF treatment might affect fetal growth and thus birthweight of the newborns. METHODS: A retrospective study was conducted in South China using data from 2370 singleton neonates born after IVF/ICSI between 2009 and 2012. Two culture media, i.e., either Vitrolife or SAGE were used as embryo culture media during the study period. Neonates' birthweights were compared between the two embryo culture media groups. RESULTS: Among the 2370 singletons, 1755 cases came from fresh cleavage embryo transfer while 615 were from frozen-thawed cleavage embryo transfer. Within the fresh embryo transfer newborns, no statistical difference was observed in either birthweight (mean ± SD: 3196.0 ± 468.9 versus 3168.4 ± 462.0g, p > 0.05) or adjusted birthweight controlled for gestational age and gender (z-score mean ± SD: 0.11 ± 1.02 versus 0.11 ± 0.99 g, P > 0.05) between the Vitrolife (n = 419) and the SAGE group (n = 1336). Likewise within frozen embryo transfer neotates, no statistical difference of the birthweight (3300.6 ± 441.3 vs.3256.0 ± 466.7 g, P > 0.05) and adjusted birthweight (0.30 ± 0.99 g versus 0.29 ± 0.97 g, P > 0.05) was found between the Vitrolife (n = 202) and the SAGE group (n = 413). The sex ratio [OR1.17, 95 % CI (0.94-1.46)/OR1.1, 95 % CI (0.78-1.54)], rate of small for gestational age [OR1.14, 95 % CI (0.82-1.59)/OR1.06, 95 % CI (0.56-2.02)] and large for gestational age [OR1.07, 95 % CI (0.64-1.76)/OR0.98, 95 % CI (0.47-2.02)] in fresh and frozen-thawed subgourps are all comparable respectively between the two culture media. No group differences were found in the rate of low birthweight and macosomia. Multiple linear regression analysis demonstrated that maternal weight, gestational age, frozen-thawed embryo transfer and infant gender were significantly related to neonatal birthweight (P < 0.001). CONCLUSIONS: It appears that embryos cultured in SAGE or Vitrolife media after fresh or frozen-thawed cleavage embryo transfer did not affect neonate's birthweight.

[Toxicity studies in vivo and in vitro for sheep skin acellular dermal matrix].

OBJECTIVE: To detect the toxic reaction degree for sheep acellular dermal matrix (ADM) in vivo or vitro by using hemolytic, pyrogen and cell-cytotoxic reaction experiments, respectively.
 Methods: Leach liquor of cross-linked and non-cross-linked sheep ADMs were set for cross-linked group and non-cross-linked group, respectively, with a positive control group (10 mL sterile water for injection in test tube) and a negative control group (10 mL 0.9% sodium chloride solution in test tube). The supernatants were obtained from each group and were measured for the absorbance. The hemolysis degree was calculated; 16 New-Zealand rabbits were selected and then divided into 4 groups, A, B, C and D group. The leach liquor of cross-linked and non-cross-linked sheep ADMs were injected into bodies of the 6 New-Zealand rabbits in the A and B groups, and then the body temperatures were measured in every half hour after injection, 6 times in total. The value of highest temperature among 6 measurements minus the normal temperature was the fever degree for the body temperature. Based on these fever degree, the criterion of biological pyrogen reaction for sheep ADM pyrogen experiment was evaluated; the mice fibroblasts were collected during logarithmic phase and were cultured in the nutrient medium containing sheep ADM leach liquor with different density. The absorbance was measured to evaluate relative growth rate for fibroblast.
 Results: The hemolysis degree for the group A and B are less than 5%. The summary of fever degree for New-Zealand rabbits were lower than 1.8 ℃. MTT experiment showed that the toxicity of 10%-90% or 100% leach liquor nutrient medium with sheep ADM for the mice fibroblast is at level 1 or level 2. There was no significant difference between leach liquor of cross-linked and non-cross-linked sheep ADMs (P>0.05). The effects on relative growth rate for mice fibroblasts were minor. 
 Conclusion: The hemolytic and pyrogen reactions for the sheep ADMs embedded in New-Zealand rabbit were within the evaluation criterion, and the effects on vitality and growth rate for the fibroblast were not significant.

Culture media for human pre-implantation embryos in assisted reproductive technology cycles.

BACKGROUND: Many media are commercially available for culturing pre-implantation human embryos in assisted reproductive technology (ART) cycles. It is unknown which culture medium leads to the best success rates after ART. OBJECTIVES: To evaluate the safety and effectiveness of different human pre-implantation embryo culture media in used for in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles. SEARCH METHODS: We searched the Cochrane Menstrual Disorders and Subfertility Group's Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, the National Research Register, the Medical Research Council's Clinical Trials Register and the NHS Center for Reviews and Dissemination databases from January 1985 to March 2015. We also examined the reference lists of all known primary studies, review articles, citation lists of relevant publications and abstracts of major scientific meetings. SELECTION CRITERIA: We included all randomised controlled trials which randomised women, oocytes or embryos and compared any two commercially available culture media for human pre-implantation embryos in an IVF or ICSI programme. DATA COLLECTION AND ANALYSIS: Two review authors independently selected the studies, assessed their risk of bias and extracted data. We sought additional information from the authors if necessary. We assessed the quality of the evidence using Grades of Recommendation, Assessment, Development and Evaluation (GRADE) methods. The primary review outcome was live birth or ongoing pregnancy. MAIN RESULTS: We included 32 studies in this review. Seventeen studies randomised women (total 3666), three randomised cycles (total 1018) and twelve randomised oocytes (over 15,230). It was not possible to pool any of the data because each study compared different culture media.Only seven studies reported live birth or ongoing pregnancy. Four of these studies found no evidence of a difference between the media compared, for either day three or day five embryo transfer. The data from the fifth study did not appear reliable.Six studies reported clinical pregnancy rate. One of these found a difference between the media compared, suggesting that for cleavage-stage embryo transfer, Quinn's Advantage was associated with higher clinical pregnancy rates than G5 (odds ratio (OR) 1.56; 95% confidence interval (CI) 1.12 to 2.16; 692 women). This study was available only as an abstract and the quality of the evidence was low.With regards to adverse effects, three studies reported multiple pregnancies and six studies reported miscarriage. None of them found any evidence of a difference between the culture media used. None of the studies reported on the health of offspring.Most studies (22/32) failed to report their source of funding and none described their methodology in adequate detail. The overall quality of the evidence was rated as very low for nearly all comparisons, the main limitations being imprecision and poor reporting of study methods. AUTHORS' CONCLUSIONS: An optimal embryo culture medium is important for embryonic development and subsequently the success of IVF or ICSI treatment. There has been much controversy about the most appropriate embryo culture medium. Numerous studies have been performed, but no two studies compared the same culture media and none of them found any evidence of a difference between the culture media used. We conclude that there is insufficient evidence to support or refute the use of any specific culture medium. Properly designed and executed randomised trials are necessary.

Hyperplastic obesity and liver steatosis as long-term consequences of suboptimal in vitro culture of mouse embryos.

In the present study, we identify and describe an obese phenotype in mice as a long-term consequence of a suboptimal in vitro culture that resulted from the addition of fetal calf serum (FCS) into the culture medium. Mice produced with FCS displayed a high mortality rate (approximately 55% versus 15% in control mice within 20 mo) and increased sensitivity to the development of obesity in adulthood when fed either a standard or a high-fat diet. These mice developed hyperplastic obesity that was characterized by a significant expansion of the fat pads (approximately 25% and 32% higher body weight in male and female mice over controls, respectively) with unchanged adipocyte size. We observed a sexual dimorphism in the development of obesity in the mice produced with FCS. Whereas the female mice displayed hypertension, hyperleptinemia, and fatty liver, the male mice only displayed glucose intolerance. The mRNA expression of metabolically relevant genes in the adipose tissue was also affected. The males produced with FCS expressed higher mRNA levels of the genes that activate fatty acid oxidation (peroxisome proliferator-activated receptor alpha [Ppara, PPARalpha] and acyl-CoA oxidase 1 [Acox1, ACOX1]) and thermogenesis (uncoupling protein 1 [Ucp1, UCP1]), which may counteract the metabolic phenotype. Conversely, the females produced with FCS generally expressed lower levels of these metabolic genes. In the females, the obese phenotype was associated with inhibition of the lipogenic pathway (peroxisome proliferator-activated receptor gamma [Pparg, PPARgamma] and fatty acid synthase [Fasn, FAS]), indicating a saturation of the storage capacity of the adipose tissue. Overall, our data indicate that the exposure to suboptimal in vitro culture conditions can lead to the sexually dimorphic development of obesity in adulthood.

A hidden pitfall in the preparation of agar media undermines microorganism cultivability.

Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the "great plate count anomaly," that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27(T) and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.

No effect of embryo culture media on birthweight and length of newborns.

STUDY QUESTION: Does the type of media used to culture embryos for IVF influence the birthweight and length of neonates? SUMMARY ANSWER: No significant differences were observed in birthweight and length among the three embryo culture media used for in vitro embryo culture. WHAT IS KNOWN ALREADY: Since the establishment of IVF as an assisted reproductive technology (ART), many different culture systems have been used for the development of human embryos. Some studies have shown that the types of culture media influence the newborn birthweight; however, other studies have shown no effect. To further explore this contradictory issue, we compared the birthweight and length of neonates born after the transfer of embryos cultured in one of three commercially available media. STUDY DESIGN, SIZE AND DURATION: This retrospective analysis of birthweight and length of newborns included 1201 women who delivered singletons and 445 women who delivered twins. The following three commercially available culture media were used: G5™, Global and Quinn's advantage media. Women who underwent IVF-ET cycles between 2008 and 2010 were analyzed. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Patients younger than 40 years of age with a body mass index (BMI) <30 kg/m(2) were analyzed. Only data from singletons and twins born alive after the 20th week of gestation were included in the data analysis. Patients who received preimplantation genetic diagnosis (PGD) and donor oocytes were excluded. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis of 1201 singletons and 445 sets of twins showed no significant association between mean birthweight or mean birth length and the type of embryo culture medium. Inter-twin mean birthweight and length disparities were analyzed, but were not shown to be significantly different. Multiple linear regression analysis showed that maternal weight, maternal height, gestational age and infant gender were significantly related to birthweight, and paternal height, gestational age and newborn complications were significantly associated with birth length. LIMITATIONS AND REASONS FOR CAUTION: The current study showed that birthweight and length of newborns were not associated with the embryo culture medium. More research needs to be performed to analyze the effects of other culture medium formulations and to evaluate the long-term effects of embryo culture medium on the health of children conceived through ART. WIDER IMPLICATIONS OF THESE FINDINGS: Our retrospective study suggests that embryo culture medium does not influence neonatal birthweight and length; however, the effects of culture medium on epigenetic variation of embryos need to be studied further.

Reactive metabolite of gefitinib activates inflammasomes: implications for gefitinib-induced idiosyncratic reaction.

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have been approved for non-small cell lung cancer. Although EGFR TKIs are less toxic than traditional cytotoxic therapies, they cause many severe idiosyncratic drug reactions. Reactive metabolites can cause cellular damage with the release of danger-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. We tested the ability of afatinib, dacomitinib, erlotinib, gefitinib, and osimertinib to induce the release of DAMPs that activate inflammasomes. Human hepatocarcinoma functional liver cell-4 (FLC-4) cells were used for bioactivation of drugs, and the detection of inflammasome activation was performed with the human macrophage cell line, THP-1 cells. Gefitinib is known to be oxidized to a reactive iminoquinone metabolite. We found that the supernatant from the incubation of gefitinib with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells with gefitinib, the heat shock protein (HSP) 40, 70 and 90 were significantly increased. In addition, activated THP-1 cells secreted high mobility group box 1 (HMGB1) protein. These results support the hypothesis that the reactive iminoquinone metabolite can cause the release of DAMPs from hepatocytes, which in turn, can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by gefitinib, which in some patients, can cause immune-related adverse events.

ß-Cypermethrin Alleviated the Inhibitory Effect of Medium from RAW 264.7 Cells on 3T3-L1 Cell Maturation into Adipocytes.

Studies have elucidated that pyrethroids induce adipogenesis. It is also known that macrophages can affect the homeostasis of adipose tissue. However, whether and how the ß-cypermethrin (ß-CYP)-mediated inhibition of the macrophages affects adipogenesis remain unknown. To explore the effects of ß-CYP on adipogenesis through modulating the function of macrophages, 3T3-L1 cells, a preadipocyte cell line, were exposed to culture medium from either RAW 264.7 cells, a macrophage cell line (RM), or ß-CYP-treated RAW 264.7 cells (CRM). CRM decreased the inhibitory effects of RM treatment on cell proliferation and adipogenesis, as lipid accumulation, the CEBPA content, and Fasn and Acaca expression in 3T3-L1 cells were higher following CRM treatment than following RM treatment through the higher levels of the demethylated CEBPA promoter in 3T3-L1 cells. However, the medium from ß-CYP- and N-acetyl-L-cysteine-cotreated RAW 264.7 cells (CNRM) partially restored the inhibitory effects of RAW 264.7 cells on 3T3-L1 cells that had been reduced by CRM, indicating that ß-CYP might reduce the cytotoxicity and inhibitory effects of RAW 264.7 cells on the adipogenesis of 3T3-L1 cells through elevating ROS levels in RAW 264.7 cells. Moreover, exposure to ß-CYP downregulated the TNF-α secretion in RAW 264.7 cells. In conclusion, these data demonstrated that ß-CYP affected the function of RAW 264.7 cells, alleviating their inhibitory effects on adipogenesis and CEBPA demethylation in 3T3-L1 cells. ß-CYP might achieve these effects through downregulating the secretion of TNF-α via elevating ROS levels in RAW 264.7 cells. Our experiments provide a new perspective on the obesogenic effect of pyrethroids.

Embryonic stem cells cultured in serum-free medium acquire bovine apolipoprotein B-100 from feeder cell layers and serum replacement medium.

Previous studies have demonstrated that cell populations that are cultured with heterologous animal products can acquire xenoantigens, potentially limiting their clinical utility because of immune responses. Embryonic stem cells (ESCs) are an attractive source of multiple potential cellular therapies and are typically derived and routinely cultured on murine embryonic fibroblast (MEF) feeder cell layers in commercially available serum replacement (SR) medium or fetal calf serum (FCS)-containing medium. Recently, we found that a strong antibody response was generated in human subjects after the second infusion of therapeutic cells cultured in FCS-containing medium. This response was specific for bovine apolipoprotein B-100 (apoB-100), which is the major protein component of low-density lipoproteins (LDL) and which targets its binding to abundant low-density lipoprotein receptors on the cell surface, from which it is internalized. Here, we have shown that ESCs cultured on MEFs in SR medium acquired bovine apoB-100 from MEFs and from the SR medium as well. Our findings also suggest that bovine LDL are used as critical nutrients for ESC propagation.

Albumin influences expression and function of the membrane transporter P-glycoprotein in HK-2 human proximal tubular cells.

BACKGROUND: In proximal tubular cells exposed to albumin genes encoding membrane transporters were found to be up-regulated or down-regulated. P-glyco-protein (Pgp) is an efflux pump which transports a variety of compounds outside the cell. In the kidney, Pgp is located mainly on the luminal side of proximal tubular cells. The aim of this study was to assess whether albumin overload influences the expression and function of Pgp in HK-2 cells. METHODS: Tubular cells were cultured in the presence of albumin (20 mg/mL) for 24 and 72 hours. Pgp expression was evaluated by Western blot (WB). ABCB1 gene expression was assessed by RT-PCR. Pgp-mediated transport was measured by the rhodamine-123 (R-123) test. RESULTS: WB showed decreased protein expression (-7% after 24 hours and -65% after 72 hours, vs. controls). RT-PCR showed that gene expression decreased to 66% after 72 hours of treatment. The fluorescence of HK-22 cells was 2.4-fold higher compared with controls (R-123) test. TNF-alpha restored Pgp expression and function. CONCLUSIONS: Tubular cells exposed to albumin present a decrease in both protein and gene expression of Pgp with impairment in transport function. The overexposure of tubular cells to toxic substrates due to Pgp transport failure represents a novel mechanism of tubular damage linked to proteinuria.

Contribution of culture media to oxidative stress and its effect on human oocytes.

The adverse effects of reactive oxygen species (ROS) on many aspects of reproduction are well documented. However, much less is known regarding the contribution of culture media to the oxidative stress of gametes during assisted reproductive techniques. This study measured the generation of ROS by culture media during IVF procedures and its effects on human oocytes. Commercially supplied culture media generated ROS at various rates, depending on the composition, whereas follicular fluid generated ROS at a much lower level. The incubation of cumulus-oocyte complexes (COC) in culture media induced marked lipid peroxidation compared with levels found in freshly retrieved COC. This plasma membrane damage, measured with the quenching of cis-parinaric acid fluorescence assay, was attenuated by supplementation of the medium with alpha-tocopherol or catalase. Moreover, there was an association between ROS production by culture medium and thiolic content consumption within the oocytes, suggesting that the intracellular reduced glutathione pool was partially depleted during in-vitro manipulation. The results show that culture medium could damage oocytes (and consequently embryo development) depending on their composition, and it is proposed that current IVF protocols could be revised in order to decrease ROS generation.

The effects of gentamicin and penicillin/streptomycin on the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes in manual patch clamp and multi-electrode array system.

INTRODUCTION: Cell culture media usually contains antibiotics including gentamicin or penicillin/streptomycin (PS) to protect cells from bacterial contamination. However, little is known about the effects of antibiotics on action potential and field potential parameters in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: The present study examined the effects of gentamicin (10, 25, and 50µg/ml) and PS (50, 100, and 200U/µg/ml) on electrophysiological activity in spontaneously beating hiPSC-CMs using manual patch clamp and multi-electrode array. We also measured mRNA expression of cardiac ion channels in hiPSC-CMs grown in media with or without gentamicin (25µg/ml) using reverse transcription-polymerase chain reaction. RESULTS: We recorded action potential and field potential of hiPSC-CMs grown in the presence or absence of gentamicin or PS. We also observed action potential parameters in hiPSC-CMs after short-term treatment with these antibiotics. Changes in action potential and field potential parameters were observed in hiPSC-CMs grown in media containing gentamicin or PS. Treatment with PS also affected action potential parameters in hiPSC-CMs. In addition, the mRNA expression of cardiac sodium and potassium ion channels was significantly attenuated in hiPSC-CMs grown in the presence of gentamicin (25µg/ml). DISCUSSION: The present findings suggested that gentamicin should not be used in the culture media of hiPSC-CMs used for the measurement of electrophysiological parameters. Our findings also suggest that 100U/100µg/ml of PS are the maximum appropriate concentrations of these antibiotics for recording action potential waveform, because they did not influence action potential parameters in these cells.

Comparative analysis of media effects on human induced pluripotent stem cell-derived cardiomyocytes in proarrhythmia risk assessment.

INTRODUCTION: Cardiotoxicity assessment using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) forms a key component of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). A potentially impactful factor on iPSC-CM testing is the presence of serum in the experimental media. Generally, serum-free media is used to most accurately reproduce "free" drug concentration. However, caution is needed; drug solubility and cardiomyocyte electrophysiology could be affected by media formulation, potentially impacting interpretation of drug-induced effects. METHODS: Effects of 25 drugs on properties of spontaneous field potentials in iPSC-CMs were assayed using a high-throughput microelectrode array (MEA) in two media formulations: serum-containing and serum-free. Comparative analysis was conducted on rate-corrected field potential duration (FPDc) and prevalence of arrhythmic events. Further MEA experiments were conducted, varying percentages of serum as well as carbon substrate components. Comparative LC-MS/MS analysis was done on two compounds to evaluate drug concentrations. RESULTS: In serum-free media, 9 drugs prolonged FPDc. In serum-containing, 11 drugs prolonged FPDc. Eighteen drugs induced arrhythmias, 8 of these induced arrhythmias at lower concentrations in serum-containing media. At the highest non-arrhythmic concentrations, 13 of 25 drugs exhibited significant differences in FPDc prolongation/shortening between the media. Increasing fractions of serum in media yielded higher FPDc measurements. LC-MS/MS analysis of moxifloxacin and quinidine showed higher concentrations in serum-containing media. DISCUSSION: The present study highlights media formulation as an important consideration for cardiac safety testing with iPSC-CMs. Results described here suggest that media formulation influences both compound availability and baseline electrophysiological properties. Special attention should be paid to media for future iPSC-CM assays.

A survey of scientists' awareness of and attitudes to the use of human blood products and alternatives in human assisted reproductive technology.

Scientists working in assisted reproduction [members of Scientists in Reproductive Technology (SIRT) Australia, and subscribers of the online forums EmbryoMail and Quartec] were invited to complete an online questionnaire on the use of human blood products in assisted reproductive technologies (ART). A total of 260 started the questionnaire, with 208 (80%) completing it. A total of 62% of respondents had worked in human ART ≥8 years and 68% had post-graduate qualifications. The majority (82%) reported using products of animal or human origin, with 75% knowing why protein was added to culture media and 41% not worried by this. Almost half (49%) of respondents were unaware of regulations surrounding the use of human blood products in health care and 70% were unaware of adverse events involving human blood products in human ART. Most respondents (70%) indicated that they were not concerned about infections such as hepatitis, but agents such as prions were a cause for concern (57%). A total of 57% of respondents were unaware of alternatives, but 77% would use a suitable alternative. Using blood products in human ART is surrounded by a lack of awareness, often independent of respondents' qualifications or experience. A better understanding of these products and possible alternatives is required if informed decisions about their suitability are to be made.

Pseudo-outbreak of Bordetella bronchiseptica infection associated with contaminated rabbit blood used as a broth culture supplement.

A cluster of clinical isolates of Bordetella bronchiseptica was identified by microbiology laboratory personnel. A clinical and molecular epidemiologic study determined that this cluster represented a pseudo-outbreak due to bacterial contamination of rabbit blood used as a broth culture supplement. This pseudo-outbreak highlights the importance of quality assurance programs in the laboratory.

The influence of donor factors on corneal organ culture contamination.

PURPOSE: To evaluate the contamination rate and the corresponding spectrum of microbes and to identify donor risk factors for corneal organ culture contaminations. METHODS: A total of 3306 organ-cultured donor corneas were included in the study. We performed a retrospective database analysis to evaluate donor factors such as gender, age, death-to-explantation interval (DEI), procurement site and cause of death and to determine their influence on donor cornea contaminations. Odds ratios (ORs) were calculated for each factor. RESULTS: The overall contamination rate was 7.8% (n = 259). Younger donor age (OR: 2.2, p = 0.003, chi-squared test), a DEI of more than 24 hr (OR: 1.6, p < 0.001), hospitalization prior to death (OR: 2.2, p < 0.001) and death caused by sepsis (OR: 2.7, p < 0.001) were associated with an increased risk of contamination, whereas donor gender did not have an effect on donor cornea contaminations. The most frequently isolated microbes were Enterococci (19%), Staphylococci (10.8%) and Candida (37.4%). CONCLUSION: This study helps to estimate the contamination risk of a cultured cornea based on specific donor factors. However, donors with risk factors should not be generally excluded from cornea donation. Further studies including antibiograms might clarify whether a change in the antibiotic composition of the culture medium would be useful to deal with the increasing number of multi-resistant microbes.

Integrating insulin into single-step culture medium regulates human embryo development in vitro.

OBJECTIVE: To evaluate the effect of supplementing single-step embryo culture medium with insulin on human embryo development. DESIGN: Comparative study. SETTING: Two private centers. PATIENT(S): The study involved a sibling oocyte split of 5,142 retrieved oocytes from 360 patients. INTERVENTION(S): Sibling oocytes split after intracytoplasmic sperm injection for culture from day 0 through day 5 or 6 in insulin-supplemented or control medium. Women were split to receive their embryos from insulin-supplemented or control medium. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate. RESULT(S): There were significantly higher rates of clinical, ongoing, and twin pregnancies in the insulin-supplemented arm than in the control arm. On day 3, embryo quality and compaction were higher in insulin-supplemented medium. On day 5, insulin supplementation showed higher rates of blastocyst formation, quality, and cryopreservation. CONCLUSION(S): Insulin supplementation of single-step embryo culture medium from day 0 through day 5 or 6 improved clinical pregnancy rate and human embryo development. However, these findings need further confirmation through a multicenter randomized controlled trial that may include other patient populations and different culture media.

Biometry of primate lenses during immersion in preservation media.

PURPOSE: The purpose of this study was to assess the condition of human lenses (obtained from an eye bank) and of fresh monkey lenses, and to determine the effects of maintaining these lenses in various liquid preservation media. METHODS: Freshly excised human and monkey lenses were maintained for 5 h in one of four solutions (Balanced Saline Solution [BSS], Ringer's Solution, Dulbecco's Modified Eagle Medium with Ham's F-12 [DMEM/F-12/F-12], and Tissue Culture Medium 199 [TC-199]) using a custom-designed, temperature-regulated testing cell. A modified optical comparator and digital camera were used to photograph magnified lens profiles and measure lens diameter and thickness. Lens volume was then calculated assuming rotational symmetry about the optical axis. RESULTS: Seven of the 33 human lenses exhibited extensive swelling and separation of the capsule from the lens cell mass prior to the incubation. During incubation, for 12/22 of the remaining human and 27/27 of the monkey lenses, thickness increased by 1.0-1.8%, diameter decreased by 0.7-1.6% and the volume was essentially unchanged. Substantial swelling and capsular separation were observed in 10 of the 22 human lenses, 7/10 for those maintained in salt solutions, and 3/12 for those in tissue culture media. Lens volumes increased by an average of 6.8%, due to an 8.7% increase in the thickness, while the diameter decreased by 0.9%. These changes appeared to be independent of postmortem time and donor age. CONCLUSIONS: Culture media are more effective than simple salt solutions in maintaining lens physical integrity during short-term incubations. Substantial uptake of water, accompanied by separation of the capsule from the lens cell mass, occurs at various stages during storage and experimental manipulations in >60% of human lenses obtained from the eye bank. Data obtained with such lenses will not be representative of the true ex vivo state. It is recommended that lenses be assessed to determine if swelling has taken place before acceptance of data.