Successful Live Birth Outcome Following Assisted Activation of Failed Fertilized Oocytes.

Here, we report on a rare case of a live birth following assisted oocyte activation of failed fertilized oocytes. A 34-year-old nulliparous woman presenting at a university-based assisted reproductive technology center with multi-factor infertility underwent an IVF cycle using intracytoplasmic sperm injection (ICSI) of frozen/thawed testicular sperm aspiration (TESA) sample and preimplantation genetic testing for aneuploidy (PGT-A). All oocytes displayed failed fertilization at assessment 18 h post-ICSI. Rescue of this cycle was achieved with the use of an assisted oocyte activation (AOA) protocol, whereby oocytes were subjected to AOA with calcium ionophore at 19 h post-ICSI and assessed for blastocyst development. Blastocyst-stage embryos were biopsied for PGT-A analysis with one of the three embryos reporting as genetically normal. This embryo was transferred in a frozen embryo transfer cycle and resulted in a normal pregnancy and term live birth. In conclusion, application of AOA protocols following failed fertilization outcomes can lead to viable, genetically normal embryos capable of resulting in a live birth.

New Animal Models for Understanding FMRP Functions and FXS Pathology.

Fragile X encompasses a range of genetic conditions, all of which result as a function of changes within the FMR1 gene and abnormal production and/or expression of the FMR1 gene products. Individuals with Fragile X syndrome (FXS), the most common heritable form of intellectual disability, have a full-mutation sequence (>200 CGG repeats) which brings about transcriptional silencing of FMR1 and loss of FMR protein (FMRP). Despite considerable progress in our understanding of FXS, safe, effective, and reliable treatments that either prevent or reduce the severity of the FXS phenotype have not been approved. While current FXS animal models contribute their own unique understanding to the molecular, cellular, physiological, and behavioral deficits associated with FXS, no single animal model is able to fully recreate the FXS phenotype. This review will describe the status and rationale in the development, validation, and utility of three emerging animal model systems for FXS, namely the nonhuman primate (NHP), Mongolian gerbil, and chicken. These developing animal models will provide a sophisticated resource in which the deficits in complex functions of perception, action, and cognition in the human disorder are accurately reflected and aid in the successful translation of novel therapeutics and interventions to the clinic setting.

In Vitro Culture of Embryos from the Cynomolgus Macaque (Macaca fascicularis).

The cynomolgus macaque (Macaca fascicularis) has contributed significantly as an animal model in a variety of biological systems including reproductive biology. The first reported delivery of a cynomolgus infant born through IVF was in 1984, and since then the use of assisted reproductive technologies (ARTs) in the cynomolgus macaque for modeling primate early embryonic development has evolved to include studies on gene editing in primate embryos and embryonic stem cell chimerism. The significance of the cynomolgus as a model of primate reproduction not only lies in the similarity of their reproductive physiology to the human but also in their availability, moderate size, and year-round utility with no seasonal breeding constraints. While many of the ARTs developed and refined in the rhesus also apply to the cynomolgus there are a number of variations that have proven effective in this species. The following chapter provides an overview of the ART methodologies that support the efficient production of M. fascicularis embryos, their cryopreservation and their transfer to recipient females for the establishment of pregnancy.

Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI).

Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP's function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI.

Primate iPS cells as tools for evolutionary analyses.

Induced pluripotent stem cells (iPSCs) are regarded as a central tool to understand human biology in health and disease. Similarly, iPSCs from non-human primates should be a central tool to understand human evolution, in particular for assessing the conservation of regulatory networks in iPSC models. Here, we have generated human, gorilla, bonobo and cynomolgus monkey iPSCs and assess their usefulness in such a framework. We show that these cells are well comparable in their differentiation potential and are generally similar to human, cynomolgus and rhesus monkey embryonic stem cells (ESCs). RNA sequencing reveals that expression differences among clones, individuals and stem cell type are all of very similar magnitude within a species. In contrast, expression differences between closely related primate species are three times larger and most genes show significant expression differences among the analyzed species. However, pseudogenes differ more than twice as much, suggesting that evolution of expression levels in primate stem cells is rapid, but constrained. These patterns in pluripotent stem cells are comparable to those found in other tissues except testis. Hence, primate iPSCs reveal insights into general primate gene expression evolution and should provide a rich source to identify conserved and species-specific gene expression patterns for cellular phenotypes.

SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition.

BACKGROUND: Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. RESULTS: Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. CONCLUSIONS: The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.

Four decades of leading-edge research in the reproductive and developmental sciences: the Infant Primate Research Laboratory at the University of Washington National Primate Research Center.

The Infant Primate Research Laboratory (IPRL) was established in 1970 at the University of Washington as a visionary project of Dr. Gene (Jim) P. Sackett. Supported by a collaboration between the Washington National Primate Research Center and the Center on Human Development and Disability, the IPRL operates under the principle that learning more about the causes of abnormal development in macaque monkeys will provide important insights into the origins and treatment of childhood neurodevelopmental disabilities. Over the past 40 years, a broad range of research projects have been conducted at the IPRL. Some have described the expression of normative behaviors in nursery-reared macaques while others have focused on important biomedical themes in child health and development. This article details the unique scientific history of the IPRL and the contributions produced by research conducted in the laboratory. Past and present investigations have explored the topics of early rearing effects, low-birth-weight, prematurity, birth injury, epilepsy, prenatal neurotoxicant exposure, viral infection (pediatric HIV), diarrheal disease, vaccine safety, and assisted reproductive technologies. Data from these studies have helped advance our understanding of both risk and resiliency in primate development. New directions of research at the IPRL include the production of transgenic primate models using our embryonic stem cell-based technology to better understand and treat heritable forms of human intellectual disabilities such as fragile X.

Induction of pluripotent stem cells from a cynomolgus monkey using a polycistronic simian immunodeficiency virus-based vector, differentiation toward functional cardiomyocytes, and generation of stably expressing reporter lines.

Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.

Primate model of metaphase I oocyte in vitro maturation and the effects of a novel glutathione donor on maturation, fertilization, and blastocyst development.

OBJECTIVE: To assess the effect of glutathione ethyl ester (GSH-OEt) on the development of macaque metaphase (MI) oocytes as a model for human MI oocyte in vitro maturation (IVM). DESIGN: Prospective cohort study. SETTING: Nonhuman primate assisted reproductive technology program. ANIMAL(S): Twenty-three Macaca fascicularis females aged 6.5-12.5 years. INTERVENTION(S): Ovarian stimulation and maturation of MI oocytes in [1] human tubal fluid (HTF), [2] mCMRL-1066, [3] mCMRL-1066+GSH-OEt 3 mM, or [4] mCMRL-1066+GSH-OEt 5 mM. Oocytes were assessed for maturation after 4-6 hours (early) and 18-20 hours (late) of culture. Mature oocytes were inseminated or subjected to glutathione (GSH) assay. Zygotes were cultured to the blastocyst stage for total differential cell counts. MAIN OUTCOME MEASURE(S): Oocyte maturation rate, GSH content, pronuclear formation and blastocyst development, and cell number were compared between IVM treatment groups and sibling in vivo matured (IVO) MII oocytes. RESULT(S): Compared with HTF, mCMRL-1066 supported higher rates of normal fertilization and blastocyst development in early but not late maturing MI-MII oocytes. Five micromoles of GSH-OEt significantly increased blastocyst total cell and inner cell mass cell number in early MI-MII oocytes compared with IVO and IVM controls. GSH-OEt significantly increased oocyte GSH content and fertilization in late maturing oocytes but not blastocyst development. CONCLUSION(S): GSH-OEt positively affects the development of early and late maturing IVM oocytes.

Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium.

Therapeutic application of stem cell derivatives requires large quantities of cells produced in defined media that cannot be produced via conventional adherent culture. We have applied human induced pluripotent stem (hiPS) cells expressing eGFP under control of the OCT4 promoter to establish the expansion of undifferentiated human embryonic stem (hES) and hiPS cells in suspension culture. A defined culture medium has been identified that results in up to six-fold increase in cell numbers within four days. Our culture system is based on initial single cell dissociation which is critical for standardized process inoculation. HES / hiPS cells were expanded for up to 17 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers. The ability to expand HES / hiPS cells in a scalable suspension culture represents a critical step towards standardized production in stirred bioreactors.

Expression of two-pore domain potassium channels in nonhuman primate sperm.

OBJECTIVE: Two-pore domain potassium channels (K(2P)) play integral roles in cell signaling pathways by modifying cell membrane resting potential. Here we describe the expression and function of K(2P) channels in nonhuman primate sperm. DESIGN: Experimental animal study, randomized blinded concentration-response experiments. SETTING: University-affiliated primate research center. ANIMAL(S): Male nonhuman primates. INTERVENTION(S): Western blot and immunofluorescent analysis of epididymal sperm samples. Kinematic measures (curvilinear velocity and lateral head displacement) and acrosome status were studied in epididymal sperm samples exposed to K(2P) agonist (docosahexaenoic acid) and antagonist (gadolinium). MAIN OUTCOME MEASURE(S): Semiquantitative protein expression and cellular localization and quantitative changes in specific kinematic parameters and acrosome integrity. RESULT(S): Molecular analysis demonstrated expression and specific regional distribution of TRAAK, TREK-1, and TASK-2 in nonhuman primate sperm. Docosahexaenoic acid produced a concentration-dependent increase in curvilinear velocity (P<.0001) with concomitant concentration-dependent reductions in lateral head displacement (P=.005). Gadolinium reduced velocity measures (P<.01) without significantly affecting lateral head displacement. CONCLUSION(S): The results demonstrated expression and function of K(2P) potassium channels in nonhuman primate sperm for the first time. The unique, discrete distributions of K(2P) channels in nonhuman primate sperm suggest specific roles for this subfamily of ion channels in primate sperm function.

Subacute necrotizing encephalopathy in a pig-tailed macaque (Macaca nemestrina) that resembles mitochondrial encephalopathy in humans.

A male pig-tailed macaque (Macaca nemestrina), approximately 5 years old, was found to be vision-impaired and to have profound behavioral abnormalities, including hyperactivity and self-injurious behavior that was not amenable to amelioration by environmental enrichment. Facial and skeletal dysmorphisms also were noted. Magnetic resonance imaging (MRI) and positron emission tomography (PET) scanning revealed areas of possible infarction in the occipital lobes and megaventriculosis. At necropsy, following euthanasia for humane reasons, severe polio- and leukoencephalomalacia accompanied by megaventriculosis were seen in both occipital lobes and in several sulci of the parietal and frontal lobes. Light microscopic findings included loss of neocortical structure, with necrosis, neuronal loss, astrogliosis, vascular proliferation, mild spongiosis, and demyelination. The extent and severity of lesions were most pronounced in the occipital lobes and were greater in the left than in the right hemisphere. Other lesions included mild bilateral atrophy of the optic nerves, thymic involution, necrotizing dermatitis due to trauma, and a spectrum of spermatozoal abnormalities. The imaging and gross and light microscopic changes found in this animal resemble the mitochondrial encephalopathies of humans; this was corroborated by results of immunohistochemical analysis demonstrating decreased expression of enzymes of the mitochondrial oxidative complex ([OC]-I, -III, and -IV) in brain and muscle, and detection of fibrinogen immunoreactivity in neurons and glial cells. The spermatozoal defects may represent yet another aspect of a mitochondrial defect.