Growth status of children with autism spectrum disorder: a case-control study.

BACKGROUND: Children with autism spectrum disorder are at risk of a compromised dietary intake and nutritional status that could impact growth over both the short and long term. The limited body of published research addressing this concern has been contradictory and inconclusive to date. METHODS: This case-control study investigated the height, weight, body mass index (BMI) and other anthropometric measurements of children diagnosed with autism spectrum disorder (ASD). Eighty-six children with ASD and 57 healthy controls participated in the study. Caregivers of participants who met the inclusion criteria completed a health history questionnaire, provided information on dietary intake and feeding behaviour, and completed a nutrition physical with a healthcare professional, which provided all of the anthropometric measurements required for the study. RESULTS: Body mass index and BMI Z-scores for females with ASD and corresponding healthy controls were significantly different. Female participants with ASD had significantly lower BMI and BMI Z-scores than control participants. The prevalence of risk for failure-to-thrive status was consistent across ASD subjects and controls. The prevalence of overweight and obesity was consistent across ASD subjects and controls. Children with ASD comprised 60% of the total number of children across BMI categories and mid-arm muscle circumference percentile ranges, which is consistent with the proportion of children in the overall sample. CONCLUSIONS: More research is needed to fully assess physical status and potential growth concerns of children with ASD. A full physical assessment should be a component of primary care for all children with ASD.

Intracytoplasmic sperm injection: stiletto conception or a stab in the dark.

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.

Fertilization imaged in 2-, 3- and four dimensions: molecular insights for treating infertility.

Fertilization in humans follows a complex series of events including binding of the sperm to the oocyte plasma membrane, oocyte activation, the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei and the restoration of the sperm centrosome. This duplicates and separates, forming two mitotic spindle poles upon which the parental genomes can intermix to complete fertilization. The use of intracytoplasmic sperm injection (ICSI) has been highly effective as a treatment for severe male infertility and thousands of ICSI babies have been born world-wide. Working with rhesus monkey gametes, we have developed a preclinical animal model for understanding the cell biological basis of ICSI. Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear structures normally removed at the oolemma during in vitro fertilization. These unusual modifications raise concerns that the ICSI procedure itself might lead to the observed increase in chromosome anomalies reported for

ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

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.

SNAREs in mammalian sperm: possible implications for fertilization.

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.

TransgenICSI reviewed: foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey.

This brief review considers the status of transgenesis by intracytoplasmic sperm injection (ICSI) with nonhuman primates. GFP expressing rhesus macaques embryos (mean = 34.6%; N = 81) were produced by ICSI using rhodamine-tagged DNA encoding the green fluorescence protein (GFP) gene bound on sperm. Rhodamine signal was lost at the egg surface during in vitro fertilization (IVF) but could be traced by dynamic imaging during ICSI within the egg cytoplasm. GFP gene was expressed as early as the 4-cell stage in ICSI embryos but not in embryos produced by in vitro fertilization (IVF). The percentage of GFP expressing blastomeres increased during embryogenesis to the blastocyst stage. Three offspring resulted from seven embryo transfers-a set of anatomically normal twins (a male and a female) stillborn 35 days premature, and a healthy male born at term. Although transgene was not detected in the offspring, the successful production of live primates using DNA bound sperm by ICSI suggests an alternative route to creating transgenic animals. It also raises concern regarding transmission of infectious material during ICSI.

Cellular and molecular events after in vitro fertilization and intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has heralded an era of tremendous improvements in treating male infertility leading to the births of thousands of babies. However, recent concerns over possible long-term effects of ICSI on offspring has prompted the development of a preclinical, nonhuman primate model to assess the safety of ICSI. Fluorescent imaging of rhesus macaque IVF zygotes revealed that this species shares many similarities with humans in terms of cytoskeletal and chromatin dynamics during fertilization. However, rhesus monkey zygotes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region. Despite these differences, normal rhesus monkey ICSI embryos have been produced and have lead to several births after transfer. The irregularities described in this paper raise concerns that the ICSI procedure may result in chromatin damage during DNA decondensation and further highlight the need for devising improved pre-clinical assessment prior to global acceptance of this, and other, novel methods of assisted reproduction.

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm.

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.

Clonal propagation of primate offspring by embryo splitting.

Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis.

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.

Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births.

Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound spermatozoa, results in the production of transgene expressing embryos in rhesus macaques (Macaca mulatta, mean = 34.6%; n = 81). Rhodamine-tagged DNA encoding the green fluorescent protein (GFP) gene binds avidly to spermatozoa. The rhodamine signal, while lost at the egg surface during in-vitro fertilization (IVF), is traced by dynamic imaging during ICSI and remains as a brilliant marker on the microinjected spermatozoa within the oocyte cytoplasm. The transgene is expressed in preimplantation embryos produced by ICSI, but not IVF, as early as the 4-cell stage with the number of expressing cells and the percentage of expressing embryos increasing during embryogenesis to the blastocyst stage. The three offspring that resulted from seven embryo transfers (a set of anatomically normal twins, one male and one female, stillborn 35 days premature, and a healthy male born at term) demonstrate that primate spermatozoa with exogenously bound DNA retain their full reproductive capacity in ICSI, but raise the concern that, theoretically, ICSI could transmit infectious material as well.

Cytoskeletal aspects of assisted fertilization.

During mammalian fertilization, the zygotic centrosome organizes a large sperm aster, critical for uniting the male and female pronuclei prior to first mitosis. Fluorescent imaging of inseminated human oocytes has shown that centrosomal defects may result in abnormal microtubule nucleation preventing genomic union, suggesting a novel cause of fertilization failure. Working with rhesus monkey gametes, we have developed a preclinical model for understanding the cell biological basis of intracytoplasmic sperm injection (ICSI). Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear theca, structures normally removed at the oolemma during IVF; this is turn causes a delay in the onset of DNA synthesis. These unusual modifications raise concerns that the ICSI procedure itself may result in chromatin damage during DNA decondensation and further highlight the need for a more rigorous assessment of methods of assisted reproduction prior to their global application.

On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.

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.

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Optimization strategies for production of mammalian embryos by nuclear transfer.

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomedical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.

Assembly of nuclear pore complexes and annulate lamellae promotes normal pronuclear development in fertilized mammalian oocytes.

In addition to functional nuclear pore complexes engaged in nucleo-cytoplasmic transport, the cytoplasmic stacks of pore complexes, called annulate lamellae, exist in numerous cell types. Although both annulate lamellae and nuclear pore complexes are present in fertilized mammalian oocytes, their relative roles in the process of fertilization and preimplantation development are not known. Using epifluorescence and electron microscopy, we explored their fate during bovine fertilization. The assembly of annulate lamellae in bovine oocytes was triggered by sperm-oocyte binding and continued concomitantly with the incorporation of the nuclear pores in the nuclear envelopes of the developing male and female pronuclei. This process was also induced by the parthenogenetic activation of metaphase-II-arrested oocytes. Depletion of Ca2+, previously implicated in oocyte activation and in the insertion of pore complexes into the nuclear envelope, prevented the formation of nuclear pore complexes, but not the assembly of annulate lamellae in oocyte cytoplasm. Injection of the nuclear pore antagonist, wheat germ agglutinin, into the cytoplasm of mature oocytes that were subsequently fertilized caused the arrest of pronuclear development, indicating the requirement of nuclear pore complexes for normal pronuclear development. Treatment of the fertilized oocytes with the microtubule inhibitor, nocodazole, prevented gathering of annulate lamellae around the developing pronuclei, insertion of nuclear pores into their nuclear envelopes, and further pronuclear development. The formation of the male pronuclei was reconstituted in Xenopus egg extracts and reflected the behavior of nuclear pores during natural fertilization. These data suggest that nuclear pore complexes are required for normal pronuclear development from its beginning up until pronuclear apposition. Annulate lamellae may be involved in the turnover of nuclear pore complexes during fertilization, which is in turn facilitated by the reorganization of oocyte microtubules and influx of Ca2+ into oocyte cytoplasm.

Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey.

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 large 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.

Cell and molecular biological challenges of ICSI: ART before science?

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.