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To describe the importance of molecular and cellular analyses in intracytoplasmic sperm injection (ICSI) the authors review the literature on biological challenges in ICSI and associated techniques. Several matters can be proposed in molecular and cellular challenges in ICSI for safety and efficacy: (1) a reliable and convenient animal model for understanding the molecular and cellular basis of human ICSI must be established, and molecular and cellular analysis of the first cell cycle of human fertilization should be better understood; (2) a proper assay for human sperm function that contributes to the indication for ICSI should be developed; and (3) de novo and transmitted genetic security in ICSI should be examined. [ABSTRACT FROM AUTHOR]

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle. [ABSTRACT FROM AUTHOR]

The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff. [ABSTRACT FROM AUTHOR]

Notwithstanding the thousands of seemingly healthy children born after intracytoplasmic sperm injection (ICSI), it is not yet possible to conclude absolutely that the ICSI procedure might induce some altered development or that the ICSI protocol might not be improved even further. To address this in a clinically relevant system, the developmental potential of rhesus monkey embryos produced by ICSI is reported. Oocytes collected by laparoscopy from gonadotrophin-stimulated fertile females were fertilized by ICSI using spermatozoa obtained from fertile males by electro-ejaculation. Neither sperm immobilization prior to injection nor an additional chemical stimulus were necessary to achieve oocyte activation and pronuclear formation. Survival and activation of the injected oocytes were judged by the extrusion of the second polar body. Successful fertilization was confirmed by the presence of two pronuclei within 12 h post-ICSI. Some oocytes were fixed and processed for the detection of microtubules and chromatin. Fluorescent labelling revealed that by 12 h post-ICSI the male and female pronuclei were closely apposed and eccentrically positioned within a larger microtubule aster. ICSI resulted in a 76.6 ± 14.9% fertilization rate. First cleavage was completed within 24 h post-ICSI. Two-cell ICSI embryos were co-cultured in CMRL medium on a buffalo rat liver cell monolayer until the hatched blastocyst stage. Oocytes collected laparoscopically from stimulated monkeys can be fertilized by ICSI and will complete preimplantation embryo development in vitro demonstrating that the rhesus monkey is an excellent preclinical model for examining and understanding many aspects of human ICSI. [ABSTRACT FROM PUBLISHER]

The disassembly and reorganization of sperm-derived structures are landmarks for the onset of embryonic development. Since complete information on these events is not yet available, we examined the disassembly of the sperm axoneme, the formation of the sperm aster, and the decondensation and development of the male and female pronuclei in inseminated Rhesus monkey oocytes conceived by in-vitro fertilization (IVF) or by intracytoplasmic sperm injection. During IVF, the spermatozoa lose their acrosomes after contacting the zona pellucida, and the plasma membrane and nuclear envelope disappear after fusion with the oolemma. Subsequently, a sperm aster of microtubules forms around the proximal centriole, which is bound to the sperm connecting piece. This process is then followed by the formation of both pronuclei, which single sperm centriole later duplicates and the bipolar mitotic apparatus is observed. Following sperm injection, the spermatozoa have both an intact plasma membrane and acrosome. Although the microtubules form the sperm aster in a fashion identical to that seen during IVF, the presence of an intact acrosome appears to be associated with a heterogeneity in the decondensation of sperm chromatin. While this may indicate an abnormal pattern of chromatin decondensation during the formation of the male pronucleus following sperm injection, the male pronucleus eventually fully decondenses, as during IVF. Sperm mitochondria are displaced as the sperm centriole is exposed. Annulate lamellae and a previously undescribed organelle which seems to contain annulate lamellae precursors, as well as maternal mitochondria, are found in association with the developing pronuclear envelopes. This information increases understanding of fertilization in primates, and may also be of significance for use in assisted human reproduction as well as in the preservation of endangered mammalian species. In addition, these results demonstrates the similarities between fertilization in Rhesus monkeys and humans, providing additional evidence for the use of this non-human primate as a model system in which to investigate the cellular and molecular biological basis of human reproduction. [ABSTRACT FROM AUTHOR]

Authors discuss the possible genetic and cell biological risks to offspring conceived by ICSI in relation to the lack of fundamental research using relevant animal models. [ABSTRACT FROM AUTHOR]

Aims: Healthcare services worldwide have been significantly impacted by the COVID-19 pandemic. Recent reports have shown a decline in hospitalization for emergency cardiac conditions. The impact of the COVID-19 pandemic on hospitalization and particularly mortality due to acute heart failure has not been thoroughly described., Methods and Results: In this single-centre observational study, we examined referrals to the acute heart failure team over a period of 16 weeks (7 January to 27 April 2020) spanning the ongoing COVID-19 pandemic; 283 patients referred to our acute heart failure services over the study period were included on the basis of typical symptoms, raised BNP, and echocardiogram. There was a substantial but statistically non-significant drop in referrals with 164 referred in the 8 weeks before the first UK death due to COVID-19 on 2 March 2020 (BC), compared with 119 referred after (AC) in the subsequent 8 weeks, representing a 27% reduction overall (P = 0.06). The 30 day case fatality rate was increased from 11% in the BC group compared with 21% in the AC group (risk ratio = 1.9, 95% confidence interval 1.09-3.3). Age, gender, length of stay, left ventricular ejection fraction, and N-terminal pro-brain natriuretic peptide were similar between the groups. Admission creatinine, age, and AC cohort status were found to be univariable predictors of mortality. On multivariate Cox regression analysis, only age (hazard ratio 1.04, P = 0.03) and AC cohort status (hazard ratio 2.1, P = 0.017) remained significant predictors of mortality. On sensitivity analysis, this increased mortality was driven by COVID-19 positive status., Conclusions: There was a reduction in referral of patients with acute heart failure with significant increase in mortality in the 8 weeks following the first reported UK death due to COVID-19. The observation of increased mortality does not appear related to a change in population in terms of demographics, left ventricular ejection fraction, or N-terminal pro-brain natriuretic peptide. The observed increased mortality appears to be related to the coexistence of COVID19 infection with acute heart failure. The study highlights the need for widespread preventative and shielding measures particularly in this group of patients especially in the light of the second wave. Longer follow-up with inclusion of data from other centres and community heart failure services will be needed., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and social interaction and restricted, repetitive patterns of behavior, interests, or activities. Given the lack of specific pharmacological therapy for ASD and the clinical heterogeneity of the disorder, current biomarker research efforts are geared mainly toward identifying markers for determining ASD risk or for assisting with a diagnosis. A wide range of putative biological markers for ASD is currently being investigated. Proteomic analyses indicate that the levels of many proteins in plasma/serum are altered in ASD, suggesting that a panel of proteins may provide a blood biomarker for ASD. Serum samples from 76 boys with ASD and 78 typically developing (TD) boys, 18 months-8 years of age, were analyzed to identify possible early biological markers for ASD. Proteomic analysis of serum was performed using SomaLogic's SOMAScanTM assay 1.3K platform. A total of 1,125 proteins were analyzed. There were 86 downregulated proteins and 52 upregulated proteins in ASD (FDR < 0.05). Combining three different algorithms, we found a panel of 9 proteins that identified ASD with an area under the curve (AUC) = 0.8599±0.0640, with specificity and sensitivity of 0.8217±0.1178 and 0.835±0.1176, respectively. All 9 proteins were significantly different in ASD compared with TD boys, and were significantly correlated with ASD severity as measured by ADOS total scores. Using machine learning methods, a panel of serum proteins was identified that may be useful as a blood biomarker for ASD in boys. Further verification of the protein biomarker panel with independent test sets is warranted., Competing Interests: The authors have declared that no competing interests exist.

This brief report examines the implementation of dietary intervention utilizing the specific carbohydrate diet (SCD) for the management of gastrointestinal issues in a 4 year old boy diagnosed with Autism Spectrum Disorder (ASD) and Fragile X Syndrome (FXS). Data relating to anthropometrics, dietary intake, blood markers, gastrointestinal (GI) symptoms, sleep issues, and behavioral concerns were gathered at baseline and after 4 months of dietary intervention. The dietary intervention was well tolerated. Improvements in nutrient status, GI symptoms, and behavioral domains were reported. The use of the SCD protocol in children with ASD/FXS and GI symptoms warrants further investigation.

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.

Objective: This study sought to assess the effectiveness of ultrasound simulation as a component of high-fidelity trauma simulation, in training diagnostic capabilities of resident and attending physicians participating in simulated trauma scenarios., Methods: Twelve residents and 20 attending physicians participated in 114 trauma simulations. Participants generated a ranked differential diagnosis list after a physical exam and subsequently after a simulated extended focused assessment with sonography for trauma (E-FAST) ultrasound scan. We compared reports to determine whether the addition of ultrasound improved diagnostic performance., Results: The primary diagnosis accuracy improved significantly with the addition of simulated ultrasound (p<0.0001). Median diagnostic ranking scores also improved (p<0.0001). Further, participants reported a higher confidence in their diagnoses (p<0.0001) and narrowed their differential diagnosis list (p<0.0001)., Conclusion: We demonstrated that a low-cost ultrasound simulator can be successfully integrated into trauma simulations, resulting in an associated improvement in measures of diagnostic accuracy, confidence, and precision for participating resident and attending physicians.

Although thimerosal, an ethylmercury-based preservative, has been removed from most pediatric vaccines in the United States, some multidose vaccines, such as influenza vaccines, still contain thimerosal. Considering that a growing number of studies indicate involvement of the gut microbiome in infant immune development and vaccine responses, it is important to elucidate the impact of pediatric vaccines, including thimerosal-containing vaccines, on gut microbial structure and function. Here, a non-human primate model was utilized to assess how two vaccine schedules affect the gut microbiome in infants (5-9 days old) and juveniles (77-88 weeks old) through 16S ribosomal RNA sequencing and metabolomics analyses of the fecal samples. Two treatment groups (n = 12/group) followed either the vaccine schedule that was in place during the 1990s (intensive exposure to thimerosal) or an expanded schedule administered in 2008 (prenatal and postnatal exposure to thimerosal mainly via influenza vaccines), and were compared with a control group (n = 16) that received saline injections. The primary impact on gut microbial structure and function was age. Although a few statistically significant impacts of the two common pediatric vaccine schedules were observed when confounding factors were considered, the magnitude of the differences was small, and appeared to be positive with vaccination.

This study investigated categories of autism spectrum disorder (ASD) symptoms measured by the Autism Diagnostic Observation Schedule-Second Edition and their association with maternal stress. Social affect and restricted and repetitive behaviors were compared with levels of maternal stress, measured by the Parenting Stress Index, in 102 children with ASD ages 2-12 years of age. Results indicated that social affect and restricted and repetitive behaviors were associated with the mother's stress regarding acceptability of the child's condition. Additionally, restricted and repetitive behaviors were significantly related to stress involving the child's hyperactivity and impulsivity. These findings highlight specific areas of stress experienced by mothers of children with ASD that are related to the child's symptoms, providing information for caregiver support and intervention.

This study compared bone mineral density (BMD) of the spine obtained by dual-energy X-ray absorptiometry (DEXA), nutritional status, biochemical markers, and gastrointestinal (GI) symptoms in 4-8 year old boys with Autism Spectrum Disorder (ASD) with a group of age-matched, healthy boys without ASD. Boys with ASD had significantly lower spine BMD compared to controls but this was not correlated with any biochemical markers, dietary intake of calcium and vitamin D, elimination diet status, or GI symptomology. Reduced BMD in 4-8 year old boys with ASD appears to involve factors other than nutrient intake and GI status, and requires further study.

Background: Autism spectrum disorder (ASD) affects approximately 1 in 68 children in the USA. An ASD blood biomarker may enable early diagnosis and/or identification of new therapeutic targets. Serum samples from ASD and typically developing (TD) boys (n = 30/group) were screened for differences in 110 proteins using a multiplex immunoassay., Results: Eleven proteins were found that together could confirm ASD with modest accuracy using multiple training and test sets. Two of the 11 proteins identified here were further tested using a different detection platform and with a larger sample of ASD and TD boys. The two proteins, thyroid-stimulating hormone (TSH) and interleukin-8 (IL-8), have been previously identified as putative biomarkers for ASD. TSH levels were significantly lower in ASD boys, whereas IL-8 levels were significantly elevated. The diagnostic accuracy for ASD based upon TSH or IL-8 levels alone varied from 74 to 76%, but using both proteins together, the diagnostic accuracy increased to 82%. In addition, TSH levels were negatively correlated with the Autism Diagnostic Observation Schedule subdomain scores., Conclusions: These data suggest that a panel of proteins may be useful as a putative blood biomarker for ASD.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and communication, and restricted, repetitive patterns of behavior. In order to identify individuals with ASD and initiate interventions at the earliest possible age, biomarkers for the disorder are desirable. Research findings have identified widespread changes in the immune system in children with autism, at both systemic and cellular levels. In an attempt to find candidate antibody biomarkers for ASD, highly complex libraries of peptoids (oligo-N-substituted glycines) were screened for compounds that preferentially bind IgG from boys with ASD over typically developing (TD) boys. Unexpectedly, many peptoids were identified that preferentially bound IgG from TD boys. One of these peptoids was studied further and found to bind significantly higher levels (>2-fold) of the IgG1 subtype in serum from TD boys (n = 60) compared to ASD boys (n = 74), as well as compared to older adult males (n = 53). Together these data suggest that ASD boys have reduced levels (>50%) of an IgG1 antibody, which resembles the level found normally with advanced age. In this discovery study, the ASD1 peptoid was 66% accurate in predicting ASD.

A variety of feeding issues and concerns, including food aversion, food selectivity, and complete food refusal, are not uncommon among children with autism spectrum disorder (ASD). Other underlying issues are often comorbid with the concerns for feeding and ASD. These may include food allergies, gastrointestinal issues, oral motor issues, and swallowing disorders. The refusal to consume particular foods coupled with the inability to tolerate, digest, and absorb these foods can compromise an individual's overall nutrition status. Therefore, a child's behavior toward food and feeding activities has great impact on dietary intake, nutritional status, and growth. This case report is the first to document combined medical, behavioral, and nutritional intervention for a toddler with ASD and comorbid feeding disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder. Some anecdotal reports suggest that ASD is related to exposure to ethyl mercury, in the form of the vaccine preservative, thimerosal, and/or receiving the measles, mumps, rubella (MMR) vaccine. Using infant rhesus macaques receiving thimerosal-containing vaccines (TCVs) following the recommended pediatric vaccine schedules from the 1990s and 2008, we examined behavior, and neuropathology in three brain regions found to exhibit neuropathology in postmortem ASD brains. No neuronal cellular or protein changes in the cerebellum, hippocampus, or amygdala were observed in animals following the 1990s or 2008 vaccine schedules. Analysis of social behavior in juvenile animals indicated that there were no significant differences in negative behaviors between animals in the control and experimental groups. These data indicate that administration of TCVs and/or the MMR vaccine to rhesus macaques does not result in neuropathological abnormalities, or aberrant behaviors, like those observed in ASD.

Background: In the 1990s, the mercury-based preservative thimerosal was used in most pediatric vaccines. Although there are currently only two thimerosal-containing vaccines (TCVs) recommended for pediatric use, parental perceptions that vaccines pose safety concerns are affecting vaccination rates, particularly in light of the much expanded and more complex schedule in place today., Objectives: The objective of this study was to examine the safety of pediatric vaccine schedules in a non-human primate model., Methods: We administered vaccines to six groups of infant male rhesus macaques (n = 12-16/group) using a standardized thimerosal dose where appropriate. Study groups included the recommended 1990s Pediatric vaccine schedule, an accelerated 1990s Primate schedule with or without the measles-mumps-rubella (MMR) vaccine, the MMR vaccine only, and the expanded 2008 schedule. We administered saline injections to age-matched control animals (n = 16). Infant development was assessed from birth to 12 months of age by examining the acquisition of neonatal reflexes, the development of object concept permanence (OCP), computerized tests of discrimination learning, and infant social behavior. Data were analyzed using analysis of variance, multilevel modeling, and survival analyses, where appropriate., Results: We observed no group differences in the acquisition of OCP. During discrimination learning, animals receiving TCVs had improved performance on reversal testing, although some of these same animals showed poorer performance in subsequent learning-set testing. Analysis of social and nonsocial behaviors identified few instances of negative behaviors across the entire infancy period. Although some group differences in specific behaviors were reported at 2 months of age, by 12 months all infants, irrespective of vaccination status, had developed the typical repertoire of macaque behaviors., Conclusions: This comprehensive 5-year case-control study, which closely examined the effects of pediatric vaccines on early primate development, provided no consistent evidence of neurodevelopmental deficits or aberrant behavior in vaccinated animals.

Recent NICE guidance has highlighted the importance of appropriate and safe intravenous fluid use. We aimed to improve the quality of out of hours fluid prescription in a Bristol hospital by ensuring that indications and cautions for fluid therapy were clearly documented at the time of initiation. Time-pressured on-call doctors need quick access to information regarding patients' care. A documented "fluid plan" allows doctors to undertake a more informed assessment of the patient's fluid balance, leading to safer prescriptions. Our ideal was for 100% of out of hours intravenous fluid prescriptions to be appropriate. Our process measures included the proportion of patients on intravenous fluids who had a documented fluid plan in the medical notes or on the prescription chart on Friday, prior to the weekend on call period. This was defined as mention of indications and/or cautions to fluid therapy. The introduction of a sticker to prompt fluid plan documentation did marginally improve use of fluid plans. It was notable that 96% of these were followed where plans were documented (n=23). Initiation of IV fluid with an accompanying plan is likely to make subsequent fluid prescriptions safer. Rapid turnover of staff and stationary proved significant barriers to consistent implementation of the sticker. Despite these challenges we demonstrated a "proof of concept", suggesting system modification to include fluid plans is safe and effective.

In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technology was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15-62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae, Bacteroidaceae, and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.

Autism is a heterogeneous behaviorally defined neurodevelopmental disorder. It is defined by the presence of marked social deficits, specific language abnormalities, and stereotyped repetitive patterns of behavior. Because of the variability in the behavioral phenotype of the disorder among patients, the term autism spectrum disorder has been established. In the first part of this review, we provide an overview of neuropathological findings from studies of autism postmortem brains and identify the cerebellum as one of the key brain regions that can play a role in the autism phenotype. We review research findings that indicate possible links between the environment and autism including the role of mercury and immune-related factors. Because both genes and environment can alter the structure of the developing brain in different ways, it is not surprising that there is heterogeneity in the behavioral and neuropathological phenotypes of autism spectrum disorders. Finally, we describe animal models of autism that occur following insertion of different autism-related genes and exposure to environmental factors, highlighting those models which exhibit both autism-like behavior and neuropathology.

This case report describes the benefits of antibiotic and antifungal therapy on behavior in a child with autism undergoing treatment for encopresis. Over the course of treatment, the child exhibited a reduction in aberrant behaviors, increased gastrointestinal function, and improved quality of life.

Human reproduction has benefited significantly by investigating nonhuman primate (NHP) models, especially rhesus macaques. To expand the Old World monkey species available for human reproductive studies, we present protocols in baboons, our closest Old World primate relatives, for assisted reproductive technologies (ART) leading to live born offspring. Baboons complement rhesus by confirming or modifying observations generated in humans often obtained by the study of clinically discarded specimens donated by anonymous infertility patient couples. Here, baboon ART protocols, including oocyte collection, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), preimplantation development to blastocyst stage, and embryo transfer techniques are described. With baboon ART methodologies in place, motility during baboon fertilization was investigated by time-lapse video microscopy (TLVM). The first ART baboons produced by ICSI, a pair of male twins, were delivered naturally at 165 days postgestation. Genetic testing of these twins confirmed their ART parental origins and demonstrated that they are unrelated fraternal twins not identicals. These results have implications for ART outcomes, embryonic stem cell (ESC) derivation, and reproductive sciences.

This longitudinal, case-control pilot study examined amygdala growth in rhesus macaque infants receiving the complete US childhood vaccine schedule (1994-1999). Longitudinal structural and functional neuroimaging was undertaken to examine central effects of the vaccine regimen on the developing brain. Vaccine-exposed and saline-injected control infants underwent MRI and PET imaging at approximately 4 and 6 months of age, representing two specific timeframes within the vaccination schedule. Volumetric analyses showed that exposed animals did not undergo the maturational changes over time in amygdala volume that was observed in unexposed animals. After controlling for left amygdala volume, the binding of the opioid antagonist [(11)C]diprenorphine (DPN) in exposed animals remained relatively constant over time, compared with unexposed animals, in which a significant decrease in [(11)C]DPN binding occurred. These results suggest that maturational changes in amygdala volume and the binding capacity of [(11)C]DPN in the amygdala was significantly altered in infant macaques receiving the vaccine schedule. The macaque infant is a relevant animal model in which to investigate specific environmental exposures and structural/functional neuroimaging during neurodevelopment.

This study examined whether acquisition of neonatal reflexes in newborn rhesus macaques was influenced by receipt of a single neonatal dose of hepatitis B vaccine containing the preservative thimerosal (Th). Hepatitis B vaccine containing a weight-adjusted Th dose was administered to male macaques within 24 h of birth (n = 13). Unexposed animals received saline placebo (n = 4) or no injection (n = 3). Infants were tested daily for acquisition of nine survival, motor, and sensorimotor reflexes. In exposed animals there was a significant delay in the acquisition of root, snout, and suck reflexes, compared with unexposed animals. No neonatal responses were significantly delayed in unexposed animals. Gestational age (GA) and birth weight (BW) were not significantly correlated. Cox regression models were used to evaluate main effects and interactions of exposure with BW and GA as independent predictors and time-invariant covariates. Significant main effects remained for exposure on root and suck when controlling for GA and BW, such that exposed animals were relatively delayed in time-to-criterion. Interaction models indicated there were various interactions between exposure, GA, and BW and that inclusion of the relevant interaction terms significantly improved model fit. This, in turn, indicated that lower BW and/or lower GA exacerbated the adverse effects following vaccine exposure. This primate model provides a possible means of assessing adverse neurodevelopmental outcomes from neonatal Th-containing hepatitis B vaccine exposure, particularly in infants of lower GA or BW. The mechanisms underlying these effects and the requirements for Th requires further study.

This article has been withdrawn at the request of the editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy., (Copyright © 2009. Published by Elsevier B.V. All rights reserved.)

Here we have developed protocols using the baboon as a complementary alternative Old World Primate to rhesus and other macaques which have severe limitations in their availability. Baboons are not limited as research resources, they are evolutionarily closer to humans, and the multiple generations of pedigreed colonies which display complex human disease phenotypes all support their further optimization as an invaluable primate model. Since neither baboon-assisted reproductive technologies nor baboon embryonic stem cells (ESCs) have been reported, here we describe the first derivations and characterization of baboon ESC lines from IVF-generated blastocysts. Two ESCs lines (BabESC-4 and BabESC-15) display ESC morphology, express pluripotency markers (Oct-4, hTert, Nanog, Sox-2, Rex-1, TRA1-60, TRA1-81), and maintain stable euploid female karyotypes with parentage confirmed independently. They have been grown continuously for >430 and 290 days, respectively. Teratomas from both lines have all three germ layers. Availabilities of these BabESCs represent another important resource for stem cell biologists.

Infants classified as "high risk" are born with a greater chance of developing medical complications at birth, and may have cognitive and other developmental complications later in life. Very few reports exist regarding the survival and outcome of such infants in primate colonies. Here we present early growth and developmental data on three high-risk infant rhesus macaques (one female and two males) that were born either with intrauterine growth restriction (IUGR; born below the 1st birth weight percentile for gestational age) or extremely prematurely (at gestational days 128 and 140; mean full-term gestation=164 days). We compared the outcome of these infants with that of healthy controls born at term and found no gross developmental delays in these infants with respect to growth, neonatal reflex and motor skill development, early cognitive development, or social behavior. Neurological and cognitive assessments were compared in terms of both postnatal and gestational age. The survival of these infants was dependent on a 24-hr staffed nursery and a fluid protocol that catered to each high-risk infant's individual needs. When such measures are implemented, infants such as these have a good chance of survival and can serve as excellent models for high-risk human babies and their subsequent development., ((c) 2007 Wiley-Liss, Inc.)

Assisted reproductive technologies (ART) used in fertility clinics include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), followed by embryo transfer into the biological or a surrogate mother. Over 1,000,000 liveborn offspring--an estimated 1 in 150 United States newborns--have been produced worldwide by ART since 1978. IVF appears to produce healthy children in singleton pregnancies, though concerns remain regarding preterm deliveries, multiple pregnancies, as well as the longer-term consequences of all ART procedures. Clinical studies remain difficult to interpret and subject to confounding variables, as developmental problems may be due to a parent's reproductive conditions rather than, or in addition to, an ART procedure. Also, because of expense and time commitments, the United States ART clinical population is not fully representative of society diversities. This socio-economic skewing might compensate for negative effects, masking small, or modest developmental deficits. Embryo splitting (ES), an ART procedure used only with animals, can produce genetically identical offspring. ES involves dividing four- to eight-cell embryos into separate blastomeres and implanting them into empty zona pellucida, followed by embryo transfer. Although these ART techniques have produced nonhuman primate offspring, there has been no research on behavioral safety. Here, we report the first study of behavioral development by rhesus macaques infants produced through ES, ICSI, and IVF. We assessed neonatal reflexes, self-feeding ability, recognition memory, object concept attainment, simple discrimination learning and reversal, and learning set (LS) acquisition. Although the sample sizes are small, we found no overall ART group delayed development. Surprisingly, the ES and ICSI monkeys appeared to be accelerated in attaining age milestones involving sensory-motor behaviors and a difficult Well Hiding object concept task. We conclude that macaque monkeys may provide an excellent model for the study of early human development by offspring of parents with conditions requiring ART pregnancies, as well as a model for the behavioral study of genetic-environment interactions using identical twins produced by ES., (Copyright (c) 2006 Wiley Periodicals, Inc.)

Although the deliberate creation of human embryos for scientific research is complicated by ethical and practical issues, a detailed understanding of the cellular and molecular events occurring during human fertilization is essential, particularly for understanding infertility. It is clear from cytoskeletal imaging studies of mouse fertilization that this information cannot be extrapolated to humans because of unique differences in centrosomal inheritance. However, the cytoskeletal rearrangements during non-human primate fertilization are very similar to humans, providing a compelling animal model in which to examine sperm--egg interactions. In order to address this key step in primate fertilization and to avoid the complexities in working with fertilized human zygotes, studies are now exploring the molecular foundations of various assisted fertilization techniques in a monkey model. While intracytoplasmic sperm injection with ejaculated or testicular sperm is quite successful in primate models, there are some specific differences when compared with standard IVF that warrant further investigation, particularly in regards to nuclear remodeling, genomic imprinting, Y-chromosome deletions and developmental outcomes. Similarly, primate models have been useful for examining spermatid function during fertilization but these have met with limited success. One area of primate reproductive research that has yet to be mastered is reproductive cloning. Genetically identical primates would provide the ultimate approach for accelerating stem cell-based therapies for a number of neurodegenerative diseases such as Alzheimer's and Parkinson's disease, as well as targeted gene therapies for various metabolic disorders.

Sperm centrosomal function was assessed by immunocytochemical analysis after the injection of human sperm into mature rabbit eggs. Three hours after intracytoplasmic sperm injection (ICSI), an astral microtubule array from the base of the human sperm was observed in the rabbit eggs. This sperm aster expanded in the egg cytoplasm, concomitant with pronuclear formation, and a dense microtubule array was organized at the time of pronuclear centration. Using fertile donor sperm, the sperm aster formation rate at 3 hr after ICSI was 35.0 +/- 1.5%. Using sperm from infertile patients, the average aster formation rate was lower (25.4 +/- 14.8%, P<0.05). Among infertile cases, there was no correlation between sperm aster formation rates and conventional parameters of semen analysis. However, the sperm aster formation rate correlated with the embryonic cleavage rate following human in vitro fertilization (IVF). These data suggest that this assay reflects sperm function during embryonic development after sperm entry and that reproductive success during the first cell cycle requires a functional sperm centrosome. Furthermore, sperm centrosomal function cannot be predicted from conventional parameters of semen analysis. We propose that insufficient centrosomal function could be the cause of certain cases of idiopathic infertility. These assays may lead to the discovery of new types of infertility, which have previously been treated as "unexplained infertility," and may also lead to the treatment of infertility incurable even by ICSI. Consequently, an accurate and relevant assay to help assure couples of the success of fertilization is warranted, perhaps prior to ICSI therapy., (Copyright 2004 Wiley-Liss, Inc.)