Molecular and Cellular Mechanisms Underlying Preimplantation Embryo Development

Preimplantation embryo development refers to the maturation of a fertilized ovum to a blastocyst. This process is highly regulated and required for proper implantation of the blastocyst into the endometrium. During this phase, several tasks must be accomplished. The differentiated zygotic genome must undergo reprogramming back to totipotency in order to generate all of the different types of tissue making up a human. Next, certain cells begin to differentiate to prepare for implantation which occurs at approximately day 7 post-fertilization. This progression is a result of a careful interplay between maternally persistent RNA transcripts and activation of the zygotic genome. After the embryonic genome activation, blastomere differentiation begins to occur. Cellular polarity has been shown to be the signal transduction that initiates this differentiation. Understanding the molecular and cellular mechanisms regulating preimplantation embryo development is of fundamental importance for reproductive science and has numerous applications in fields such as assisted reproductive technology and stem cell therapy

Two Acinetobacter baumannii Isolates Obtained From a Fatal Necrotizing Fasciitis Infection Display Distinct Genomic and Phenotypic Characteristics in Comparison to Type Strains

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii's virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified

Expression of both ubiquitin and histone proteins analyzed in blastocoel fluid from IVF embryos is correlated with positive implantation

Introduction:Due to an influx of patients seeking infertility treatments like IVF, the need to identify additional molecular markers, particularly proteins, in preimplantation embryos to predict successful implantation is essential. We propose assessing expression of proteins from the ubiquitin-proteasome family as well as histones in blastocoel fluid-conditioned media from IVF-embryos with known implantation outcomes. These data may provide evidence for embryologists to eventually predict implantation potential of an embryo with these assays. Methods:Blastocoel fluid conditioned media samples were collected from day-5-IVF embryos at the times of PGT-A biopsy. Media associated with embryos of poor morphology versus good morphology were used in this study. Total RNA and protein concentration were assessed with an Agilent Bioanalyzer. Blastocoel fluid-conditioned media samples were pooled and loaded onto a NuPAGE protein gel to detect total proteins with Colloidal Blue staining and then for Western Blots. Antibodies to detect ubiquitin and histones were used for the blots. Results:Preliminary results suggest that proteins can be detected in pooled blastocoel fluid conditioned media samples based on Colloidal Blue staining. The initial Western Blot assay detected some expression of ubiquitin. Conclusions:We hypothesize that the euploid grade AA embryos will present with stronger markers of histone and ubiquitin proteins as well as a higher concentration of protein in the blastocoel fluid, providing evidence that the embryos with stronger markers of said proteins will result in a successful implantation

Expression of signaling pathway genes in IVF embryos that result in miscarriage

Introduction: Approximately 10 to 20 percent of pregnancies end in miscarriage. Infertility is one of the leading causes of recurrent miscarriages and affects approximately 14% of reproductive-age couples. Recurrent miscarriages are presumed to be the result of embryonic chromosomal abnormalities such as aneuploidy. In response to infertility, many couples seek IVF, often without reliable guidelines on which embryo has the best potential for leading to a live birth. Currently, this process is very expensive, costing on average $23,474 per cycle, and emotionally taxing, exacerbating their comorbidities of anxiety, depression, and isolation. Additionally, IVF typically leads to only an approximately 25-50% birth rate. As a result, there are many couples who are unable to conceive through IVF the first time. Due to the high cost of the procedure, many couples may not be able to afford to do multiple rounds of IVF. Purpose Statement: This study seeks to identify gene expression signatures from blastocoel fluid-conditioned media collected from preimplantation euploid IVF-embryos that resulted in miscarriage. Methods: Candidate genes were previously identified through whole transcriptome (RNASeq) analysis of blastocoel fluid from euploid IVF-embryos that resulted in successful or unsuccessful implantation. Blastocoel fluid from a new set of euploid IVF-embryos resulting in miscarriage or successful implantation were selected for specific gene expression analysis using RT-qPCR. RNA was first purified from the fluid, then cDNA was synthesized for use in RT-qPCR reactions with gene-specific TaqMan primers. Genes assessed using RT-qPCR were BCL2L12, SHARPIN, CUL2, and DRAP1 with GAPDH as a control. These genes represented pathways involved in apoptosis and protein degradation via ubiquitination. Results: Gene expression analysis of is ongoing and future studies will continue to assess the expression of these genes in additional samples. Initial studies suggest altered expression patterns in some samples associated with miscarriage. Tested samples associated with positive implantation outcomes showed higher relative expression levels of CUL2. Those embryos with negative implantation outcomes showed higher relative expression levels of SHARPIN. Conclusion: If genes could be identified that are associated with miscarriage, this additional embryo quality metric may improve the success of each IVF-embryo transfer potentially reducing the number of miscarriages. By reducing the number of miscarriages, fertility treatments may be accessible to a larger population by decreasing the associated costs of having to complete multiple rounds of treating and the psychological burden that may accompany recurrent miscarriage

Comparative analysis of viral infection outcomes in human seminal fluid from prior viral epidemics and Sars-CoV-2 may offer trends for viral sexual transmissibility and long-term reproductive health implications

Plain Language Summary This review describes the detection of viruses in seminal fluid and their sexual transmission, focusing on the major viral outbreaks of Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome (SARS)-coronavirus (CoV), and SARS-coronavirus 2 (CoV-2). ZIKV and EBOV were found to be present in semen and to be sexually transmitted, leading the World Health Organization (WHO) to update their guidelines on prevention of the two viruses to include refraining from sexual contact. There are conflicting studies regarding the presence of SARS-CoV in male reproductive tissue, but it has been linked to testicular atrophy and orchitis. To date, two studies have detected SARS-CoV-2 RNA in semen, while seven studies have reported no positive detection. More studies must be completed to accurately determine its risk of sexual transmission to ensure mitigation of further transmission and understand the long-term implications of SARS-CoV-2 on the reproductive health of recovered patients

Patient engagement in fertility research: bench research, ethics, and social justice

Abstract Background Patient and Public Involvement (PPI) in research is increasingly being utilized to better connect patients and researchers. The Patient Engagement Studio (PES) supports PPI in research by working directly with researchers throughout various stages of their projects. Recently, two researchers presented to the PES for assistance with their project, Embryo+™. The purpose of Embryo+™ is to decrease miscarriage rates using RNA sequencing technology that screens for the most viable embryos. To date, no examples of PPI directly in the planning or implementation of bench research concerning in vitro fertilization and embryo transfer have been identified. Main body Embryo+™ researchers met in-person with the PES two times (fall 2019; each meeting had 9 PES members in attendance) for initial feedback and protocol development. After these meetings, PES leadership and Embryo+™ researchers decided that the unique nature of the project merited a PPI evaluation. Subsequent evaluation of engagement efforts occurred by reviewing the PES reports for the Embryo+™ researchers, conducting two recorded web-based discussion meetings with the PES (summer 2020; meeting 1 n = 7; meeting 2 n = 6), and a brief survey (n = 13). The discussion meetings provided an opportunity for the PES members to define engagement themes through consensus via verbal agreement to the studio director’s periodic summaries during the discussions. Combining survey results and PES themes allowed for a broad discussion for meaningful engagement. The Embryo+™ researchers established trust with the patients by changing some of their language in response to patient suggestions, allowing for unintended ethical conversations, and implementing the patient developed protocols. Overall, the patient experts thought this project was very meaningful and valuable, quantified by a mean loyalty score 89.43 (s.d. 10.29). Conclusion Bench science researchers may need additional PPI training prior to engaging with patient groups. PPI in this project was successful in large part due to this training, where the director emphasized the importance of gaining trust with the patients. The researchers applied what they learned and several examples of how to develop trust with patients are discussed. If trust is established, PPI in an ethically charged, basic science research study can be both valuable and successful