SDF-1 Bound Heparin Nanoparticles Recruit Progenitor Cells for Their Differentiation and Promotion of Angiogenesis after Stroke.

Angiogenesis after stroke is correlated with enhanced tissue repair and functional outcomes. The existing body of research in biomaterials for stroke focuses on hydrogels for the delivery of stem cells, growth factors, or small molecules or drugs. Despite the ability of hydrogels to enhance all these delivery methods, no material has significantly regrown vasculature within the translatable timeline of days to weeks after stroke. Here, two novel biomaterial formulations of granular hydrogels are developed for tissue regeneration after stroke: highly porous microgels (i.e., Cryo microgels) and microgels bound with heparin-norbornene nanoparticles with covalently bound SDF-1α. The combination of these materials results in perfused vessels throughout the stroke core in only 10 days, in addition to increased neural progenitor cell recruitment, maintenance, and increased neuronal differentiation.

SDF-1 Bound Heparin Nanoparticles Recruit Progenitor Cells for Their Differentiation and Promotion of Angiogenesis After Stroke.

Angiogenesis after stroke is correlated with enhanced tissue repair and functional outcomes. The existing body of research in biomaterials for stroke focuses on hydrogels for the delivery of stem cells, growth factors, or small molecules or drugs. Despite the ability of hydrogels to enhance all these delivery methods, no material has significantly regrown vasculature within the translatable timeline of days to weeks after stroke. Here we developed 2 novel biomaterials for tissue regeneration after stroke, a highly porous granular hydrogel termed Cryo microgels, and heparin-norbornene nanoparticles with covalently bound SDF-1α. The combination of these materials resulted in fully revascularized vessels throughout the stroke core in only 10 days, as well as increased neural progenitor cell migration and maintenance and increased neurons.

Void Volume Fraction of Granular Scaffolds.

Void volume fraction (VVF) is a global measurement frequently used to characterize the void space of granular scaffolds, yet there is no gold standard by which to measure VVF in practice. To study the relationship  between VVF and particles of varying size, form, and composition, a library of 3D simulated scaffolds is used. Results reveal that relative to particle count, VVF is a less predictable metric across replicate scaffolds. Simulated scaffolds are used to explores the relationship between microscope magnification and VVF, and recommendations are offered for optimizing the accuracy of approximating VVF using 2D microscope images. Lastly, VVF of hydrogel granular scaffolds is measured while varying four input parameters: image quality, magnification, analysis software, and intensity threshold. Results show that VVF is highly sensitive to these parameters. Overall, random packing produces variation in VVF among granular scaffolds comprising the same particle populations. Furthermore, while VVF is used to compare the porosity of granular materials within a study, VVF is a less reliable metric across studies that use different input parameters. VVF, a global measurement, cannot describe the dimensions of porosity within granular scaffolds, and the work supports the notion that more descriptors are necessary to sufficiently characterize void space.

Stoichiometric Post-Modification of Hydrogel Microparticles Dictates Neural Stem Cell Fate in Microporous Annealed Particle Scaffolds.

Microporous annealed particle (MAP) scaffolds are generated from assembled hydrogel microparticles (microgels). It has been previously demonstrated that MAP scaffold are porous, biocompatible, and recruit neural progenitor cells (NPCs) to the stroke cavity after injection into the stroke core. Here, the goal is to study NPC fate inside MAP scaffolds in vitro. To create plain microgels that can later be converted to contain different types of bioactivities, the inverse electron-demand Diels-Alder reaction between tetrazine and norbornene is utilized, which allows the post-modification of plain microgels stoichiometrically. As a result of adhesive peptide attachment, NPC spreading leads to contractile force generation which can be recorded by tracking microgel displacement. Alternatively, non-adhesive peptide integration results in neurosphere formation that grows within the void space of MAP scaffolds. Although the formed neurospheres do not impose a contractile force on the scaffolds, they are seen to continuously transverse the scaffolds. It is concluded that MAP scaffolds  can be engineered to either promote neurogenesis or enhance stemness depending on the chosen post-modifications of the microgels, which can be key in modulating their phenotypes in various applications in vivo.

Global research and learning agenda for building evidence on contraceptive-induced menstrual changes for research, product development, policies, and programs.

Background: Contraceptive-induced menstrual changes (CIMCs) can affect family planning (FP) users' lives in both positive and negative ways, resulting in both opportunities and consequences. Despite this, and despite the important links between FP and menstrual health (MH), neither field adequately addresses CIMCs, including in research, product development, policies, and programs globally. Methods: In November 2020, a convening of both MH and FP experts reviewed the existing evidence on CIMCs and identified significant gaps in key areas. Results: These gaps led to the establishment of a CIMC Task Force in April 2021 and the development of the Global Research and Learning Agenda: Building Evidence on Contraceptive-Induced Menstrual Changes in Research, Product Development, Policies, and Programs Globally (the CIMC RLA) , which includes four research agendas for (1) measurement, (2) contraceptive research and development (R&D) and biomedical research, (3) social-behavioral and user preferences research, and (4) programmatic research. Conclusions: Guided by the CIMC RLA, researchers, product developers, health care providers, program implementers, advocates, policymakers, and funders are urged to conduct research and implement strategies to address the beneficial and negative effects of CIMCs and support the integration of FP and MH. CIMCs need to be addressed to improve the health and well-being of women, girls, and other people who menstruate and use contraceptives globally. Disclaimer : The views expressed in this article are those of the authors. Publication in Gates Open Research does not imply endorsement by the Gates Foundation.

Particle hydrogels decrease cerebral atrophy and attenuate astrocyte and microglia/macrophage reactivity after stroke.

Increasing numbers of individuals live with stroke related disabilities. Following stroke, highly reactive astrocytes and pro-inflammatory microglia can release cytokines and lead to a cytotoxic environment that causes further brain damage and prevents endogenous repair. Paradoxically, these same cells also activate pro-repair mechanisms that contribute to endogenous repair and brain plasticity. Here, we show that the direct injection of a hyaluronic acid based microporous annealed particle (MAP) hydrogel into the stroke core in mice reduces the percent of highly reactive astrocytes, increases the percent of alternatively activated microglia, decreases cerebral atrophy and preserves NF200 axonal bundles. Further, we show that MAP hydrogel promotes reparative astrocyte infiltration into the lesion, which directly coincides with axonal penetration into the lesion. This work shows that the injection of a porous scaffold into the stroke core can lead to clinically relevant decrease in cerebral atrophy and modulates astrocytes and microglia towards a pro-repair phenotype.

Changes in patient activation following cardiac rehabilitation using the Active+me digital healthcare platform during the COVID-19 pandemic: a cohort evaluation.

BACKGROUND: Restrictions on face-to-face contact, due to COVID-19, led to a rapid adoption of technology to remotely deliver cardiac rehabilitation (CR). Some technologies, including Active+me, were used without knowing their benefits. We assessed changes in patient activation measure (PAM) in patients participating in routine CR, using Active+me. We also investigated changes in PAM among low, moderate, and high risk patients, changes in cardiovascular risk factors, and explored patient and healthcare professional experiences of using Active+me. METHODS: Patients received standard CR education and an exercise prescription. Active+me was used to monitor patient health, progress towards goals, and provide additional lifestyle support. Patients accessed Active+me through a smart-device application which synchronised to telemetry enabled scales, blood pressure monitors, pulse oximeter, and activity trackers. Changes in PAM score following CR were calculated. Sub-group analysis was conducted on patients at high, moderate, and low risk of exercise induced cardiovascular events. Qualitative interviews explored the acceptability of Active+me. RESULTS: Forty-six patients were recruited (Age: 60.4 ± 10.9 years; BMI: 27.9 ± 5.0 kg.m2; 78.3% male). PAM scores increased from 65.5 (range: 51.0 to 100.0) to 70.2 (range: 40.7 to 100.0; P = 0.039). PAM scores of high risk patients increased from 61.9 (range: 53.0 to 91.0) to 75.0 (range: 58.1 to 100.0; P = 0.044). The PAM scores of moderate and low risk patients did not change. Resting systolic blood pressure decreased from 125 mmHg (95% CI: 120 to 130 mmHg) to 119 mmHg (95% CI: 115 to 122 mmHg; P = 0.023) and waist circumference measurements decreased from 92.8 cm (95% CI: 82.6 to 102.9 cm) to 85.3 cm (95% CI 79.1 to 96.2 cm; P = 0.026). Self-reported physical activity levels increased from 1557.5 MET-minutes (range: 245.0 to 5355.0 MET-minutes) to 3363.2 MET-minutes (range: 105.0 to 12,360.0 MET-minutes; P < 0.001). Active+me was acceptable to patients and healthcare professionals. CONCLUSION: Participation in standard CR, with Active+me, is associated with increased patient skill, knowledge, and confidence to manage their condition. Active+me may be an appropriate platform to support CR delivery when patients cannot be seen face-to-face. TRIAL REGISTRATION: As this was not a clinical trial, the study was not registered in a trial registry.

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke.

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

An Outbreak of Paralytic Shellfish Poisoning in Tasmania.

Paralytic shellfish poisoning (PSP) is a rare illness caused by eating shellfish containing paralytic shellfish toxins (PST). Toxins are produced during harmful algal blooms, which occur most years on the east coast of Tasmania. Contaminated seafood looks and tastes normal and toxins are not destroyed by cooking or freezing. Commercial shellfish farms are monitored for harmful algae and shellfish toxins, but wild shellfish are not and pose a potential public health risk. A case of PSP was documented in Tasmania in 2011, and we are aware of anecdotal reports of cases in the 1980s and 1990s. We are not aware of cases elsewhere in Australia but harmful algal blooms have been detected in Victoria, South Australia and New South Wales. Routine monitoring of commercial shellfish in 2015 detected a large bloom of Alexandrium tamarense on the east coast of Tasmania, which can cause PSP. Between 2 and 12 October 2015, four cases of PSP were identified. All were adults who ate wild mussels from the east coast of Tasmania and had onset of numbness or tingling of the face and muscle weakness from 30 minutes to 12 hours later. Two cases were briefly hospitalised, both recovered. Since the outbreak, permanent signage at locations where shellfish are frequently recreationally foraged has been erected. Additional alerts are released during high risk periods based on surveillance of commercial sites by the Tasmanian Shellfish Quality Assurance Program. Several states in Australia are at risk of cases of PSP. We recommend active surveillance and multi-jurisdictional collaboration to manage this risk.

Comparative performance of four immunological test kits for the detection of Paralytic Shellfish Toxins in Tasmanian shellfish.

Blooms of the toxic dinoflagellate Alexandrium tamarense (Group 1) seriously impacted the Tasmanian shellfish industry during 2012 and 2015, necessitating product recalls and intensive paralytic shellfish toxin (PST) product testing. The performance of four commercial PST test kits, Abraxis™, Europroxima™, Scotia™ and Neogen™, was compared with the official AOAC LC-FLD method for contaminated mussels and oysters. Abraxis and Europroxima kits underestimated PST in 35-100% of samples when using standard protocols but quantification improved when concentrated extracts were further diluted (underestimation ≤18%). The Scotia kit (cut off 0.2-0.7 mg STX-diHCl eq/kg) delivered 0% false negatives, but 27% false positives. Neogen produced 5% false negatives and 13% false positives when the cut off was altered to 0.5-0.6 mg STX-diHCl eq/kg, the introduction of a conversion step eliminated false negatives. Based on their sensitivity, ease of use and performance, the Neogen kit proved the most suitable kit for use with Tasmanian mussels and oysters. Once formally validated for regulatory purposes, the Neogen kit could provide shellfish growers with a rapid tool for harvesting decisions at the farm gate. Effective rapid screening preventing compliant samples undergoing testing using the more expensive and time consuming LC-FLD method will result in significant savings in analytical costs.

Standardized network order sets in rural Ontario: a follow-up report on successes and sustainability.

Unifying, implementing and sustaining a large order set project requires strategic placement of key organizational professionals to provide ongoing user education, communication and support. This article will outline the successful strategies implemented by the Grey Bruce Health Network, Evidence-Based Care Program to reduce length of stay, increase patient satisfaction and increase the use of best practices resulting in quality outcomes, safer practice and better allocation of resources by using standardized Order Sets within a network of 11 hospital sites. Audits conducted in 2007 and again in 2008 revealed a reduced length of stay of 0.96 in-patient days when order sets were used on admission and readmission for the same or a related diagnosis within one month decreased from 5.5% without order sets to 3.5% with order sets.

Putative imprinted gene expression in uniparental bovine embryo models.

Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

Dynamic changes in localization of Chromobox (Cbx) family members during the maternal to embryonic transition.

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.

Alpha-adrenergic mRNA subtype expression in the human nasal turbinate.

PURPOSE: Alpha-adrenergic receptor (AR) agonist drugs (e.g., epinephrine) are commonly used for upper airway procedures, to shrink the mucosa, retard absorption of local anesthetic agents, and improve visualization by limiting hemorrhage. Decongestant therapy often also includes alphaAR agonist agents, however overuse of these drugs (e.g., oxymetazoline) can result in chronic rhinitis and rebound increases in nasal secretion. Since current decongestants stimulate alphaARs non-selectively, characterization of alphaAR subtype distribution in human airway (nasal turbinate) offers an opportunity to refine therapeutic targets while minimizing side-effects. We, therefore, investigated alphaAR subtype expression in human nasal turbinate within epithelial, duct, gland, and vessel cells using in situ hybridization. METHODS: Since sensitive and specific anti-receptor antibodies and highly selective alphaAR subtype ligands are currently unavailable, in situ hybridization was performed on sections of three human nasal turbinate samples to identify distribution of alphaAR subtype mRNA. Subtype specific (35)S-labelled mRNA probes were incubated with nasal turbinate sections, and protected fragments remaining after RNase treatment analyzed by light and darkfield microscopy. RESULTS: In non-vascular tissue alpha(1d) AR mRNA predominates, whereas notably the alpha(2c) is the only alphaAR subtype present in the sinusoids and arteriovenous anastamoses. CONCLUSION: Combined with the current understanding that AR-mediated constriction of nasal sinusoids underpins decongestant therapies that minimize secretions and shrink tissues for airway procedures, these findings suggest that alpha(2c) AR subtypes provide a novel selective target for decongestant therapy in humans.

beta2 adrenoceptor gene therapy ameliorates left ventricular dysfunction following cardiac surgery.

OBJECTIVE: Heart surgery is associated with impairment of the myocardial beta-adrenoceptor (betaAR) system. Effective therapies for post-operative ventricular dysfunction are limited. Prolonged inotrope exposure is associated with further betaAR down-regulation. Left ventricular (LV) dysfunction and myocardial betaAR impairment were assessed following cardiopulmonary bypass (CPB) and cardioplegic arrest in a pig model. Transfer of the human beta2-adrenoceptor transgene (Adeno-beta2AR) during cardioplegic arrest was then tested as a potential therapy. METHODS: Five groups of six neonatal piglets were studied. One group did not undergo surgery (Group A). Adeno-beta2AR or phosphate buffered saline (PBS) were delivered via the aortic root during cardioplegic arrest. Groups B (PBS) and C (Adeno-beta2AR) were assessed at 2 days while Groups D (PBS) and E (Adeno-beta2AR) were assessed at 2 weeks from the time of surgery. An LV micromanometer was inserted under sedation to obtain pressure recordings following surgery. betaAR density was measured subsequently. RESULTS: Following cardiac surgery LV betaAR density was reduced (104+/-5.7 vs 135+/-6.1 fmol/mg membrane protein; P=0.007), and, in response to beta agonist stimulation, LV dP/dtmax was reduced (4337+/-405 vs 5328+/-194 mmHg/s; P<0.05) compared to animals which did not undergo surgery. Adeno-beta2AR therapy during cardiac surgery resulted in elevated LV betaAR density (520+/-250.9 fmol/mg) 2 days post-operatively compared to PBS (104+/-5.7 fmol/mg; P=0.002) and compared to the no surgery group (135+/-6.1 fmol/mg; P=0.002). Elevated LV betaAR density was also present at 2 weeks (315+/-74.1 vs 119+/-7.1 fmol/mg; P=0.002). In addition, Adeno-beta2AR therapy enhanced beta agonist stimulated LV dP/dtmax (5348+/-121 vs 4337+/-405 mmHg/s; P<0.05) and heart rate (209+/-6.9 vs 173+/-11.0 bpm; P<0.05), and reduced LVEDP (2.1+/-0.4 vs 6.4+/-1.8 mmHg; P<0.05) compared to PBS treatment. Interestingly, gene delivery was cardiac-selective and beneficial effects on function persisted for 2 weeks. Moreover, beta2AR gene transfer ameliorated LV dysfunction following surgery such that there were no significant differences between non-operated controls and animals treated with Adeno-beta2AR during CPB and cardioplegic arrest. CONCLUSIONS: Reduced betaAR density and impaired LV function were present following CPB and cardioplegic arrest. Cardiac-selective beta2AR gene transfer during CPB resulted in amelioration of LV dysfunction after cardiac surgery. Such a technique may offer a new approach to post-operative ventricular support.

Dose dependent effects of cardiac beta2 adrenoceptor gene therapy.

BACKGROUND: Adenoviral-mediated gene transfer during cardiopulmonary bypass (CPB) achieves efficient myocardial transgene expression. The optimal vector dose required to produce not only increased beta adrenoceptor (betaAR) density but, more importantly, enhanced left ventricular (LV) function is unknown. In addition, it is unclear if absent extracardiac expression in preliminary studies represented cardiac specific, as opposed to selective gene delivery, as a consequence of low vector doses. MATERIALS AND METHODS: Adenoviral vector encoding the human beta(2) adrenoceptor (Adeno-beta(2)AR) was delivered to cardioplegic arrested hearts of neonatal piglets during CPB in three doses ranging from 5 x 10(11) total viral particles (tvp) to 2 x 10(12) tvp. Control animals received adenoviral vector encoding beta galactosidase (Adeno-betagal) or PBS (PBS). LV and liver betaAR density and in vivo LV function were assessed 5 days later. RESULTS: Elevated LV betaAR density was present after delivery of Adeno-beta(2)AR at all doses. Piglets which received 5 x 10(11) tvp and 1 x 10(12) tvp Adeno-beta(2)AR demonstrated enhanced LV dP/dt(max) but in those receiving 2 x 10(12) tvp LV dP/dt(max) was unchanged. Moreover, at this higher dose of adenoviral vector the detrimental effects of cardiac inflammation and extracardiac gene overexpression became apparent. CONCLUSIONS: Although the highest increase in cardiac betaAR density occurred after high-dose Adeno-beta(2)AR, LV dP/dt(max) was not enhanced. Moreover, significant extracardiac gene expression was present at this dose, emphasizing the need for careful dose response studies in gene therapy. However, cardiac selective beta(2)AR overexpression does occur following adenoviral vector delivery during CPB and cardioplegic arrest resulting in enhanced LV dP/dt(max).

Analysis of imprinted messenger RNA expression during bovine preimplantation development.

While the expression and epigenetic differences of imprinted genes have been extensively characterized in the mouse and human, little is known about imprinted genes in livestock species. In the current study, eight genes that are imprinted in the human or mouse were investigated in preimplantation bovine embryos. Amplified cDNA was created from three single metaphase II (MII) oocytes or embryos throughout preimplantation development. The imprinted genes Dlk1 and Mest (isoform 1) had no detectable transcripts during preimplantation development. Gnas and Grb10 were expressed in most embryos from the 2-cell to blastocyst stages of development. Mest (isoform 2) was expressed in all oocytes and embryos, except for one blastocyst sample. Ndn and Xist were expressed from the 8-16-cell stage (maternal-to-zygotic transition, MZT) onwards. Sgce was expressed until the MZT, and Nnat in both early (alpha form) and late (beta form) stage embryos. The paternally imprinted genes Gnas, Grb10, and Xist were expressed in both in vitro-fertilized (IVF) and parthenogenetically activated (PA) blastocysts as expected. Of the four maternally imprinted genes expressed in the blastocyst (Mest, Ndn, Nnat, and Sgce), Nnat alone showed differential mRNA expression between IVF and PA blastocysts, suggesting imprinting by this stage of development. In conclusion, seven of the eight genes investigated showed mRNA expression during preimplantation development, indicating a potential role during early development. Also significant is the observation that Nnat is imprinted by the blastocyst stage of development although the other genes are not, indicating a temporal imprinting program.