General anesthesia with propofol during oocyte retrieval and in vitro fertilization outcomes: retrospective cohort study.

General anesthesia is frequently administered during oocyte retrieval. Its effects on the outcomes of IVF cycles are uncertain. This study investigated whether administration of general anesthesia (specifically propofol) during oocyte retrieval affects IVF outcomes. A total of 245 women undergoing IVF cycles were included in this retrospective cohort study. IVF outcomes of 129 women who underwent oocyte retrieval under propofol anesthesia and 116 without anesthesia were compared. Data were adjusted for age, BMI, estradiol on triggering day and total gonadotropin dose. The primary outcomes were fertilization, pregnancy and live birth rates. A secondary outcome was the efficiency of follicle retrieval associated with the use of anesthesia. Fertilization rate was lower in retrievals under anesthesia compared to without (53.4% ± 34.8 vs. 63.7% ± 33.6, respectively; p = 0.02). There was no significant difference in the ratio of expected to retrieved oocytes between retrievals with and without anesthesia (0.8 ± 0.4 vs. 0.8 ± 0.8, respectively, p = 0.96). The differences in pregnancy and live birth rates between the groups were not statistically significant. General anesthesia administered during oocyte retrieval may have adverse effects on the fertilization potential of oocytes. This impact on the developmental potential of oocytes may lead to negative IVF outcomes and should be investigated further.

Standardization of Post-Vitrification Human Blastocyst Expansion as a Tool for Implantation Prediction.

The increased use of vitrified blastocysts has encouraged the development of various criteria for selecting the embryo most likely to implant. Post-thaw assessment methods and timetables vary among investigators. We investigated the predictive value of well-defined measurements of human blastocyst re-expansion, following a fixed incubation period. Post-thaw measurements were taken exactly at 0 and 120 ± 15 min. Minimum and maximum cross-sectional axes were measured. Three groups were defined: Group 1: embryos that continued to shrink by 10 µm or more; group 2: embryos that ranged from -9 to +9 µm; and group 3: re-expansion of 10 µm or more. Patient and morphokinetic data were collected and integrated into the analysis. A total of 115 cases were included. The clinical pregnancy rate for group 1 was 18.9%; group 2, 27%; and group 3, 51.2% (p = 0.007). Pre-thaw morphologic grading and morphokinetic scores of the study groups did not reveal differences. p-values were 0.17 for the pre-thaw morphologic score, 0.54 for KID3, and 0.37 for KID5. The patients' demographic and clinical data were similar. The clinical pregnancy rate correlated with the degree of thawed blastocyst re-expansion measured 2 h after incubation. This standardized measure is suggested as a tool to predict the potential of treatment success before embryo transfer.

The Wave complex controls epidermal morphogenesis and proliferation by suppressing Wnt-Sox9 signaling.

Development of the skin epidermis requires tight spatiotemporal control over the activity of several signaling pathways; however, the mechanisms that orchestrate these events remain poorly understood. Here, we identify a key role for the Wave complex proteins ABI1 and Wave2 in regulating signals that control epidermal shape and growth. In utero RNAi-mediated silencing of Abi1 or Wasf2 induced cellular hyperproliferation and defects in architecture of the interfollicular epidermis (IFE) and delayed hair follicle growth. Unexpectedly, SOX9, a hair follicle growth regulator, was aberrantly expressed throughout the IFE of the mutant embryos, and its forced overexpression mimicked the Wave complex loss-of-function phenotype. Moreover, Wnt signaling, which regulates SOX9+ cell specification, was up-regulated in Wave complex loss-of-function IFE. Importantly, we show that the Wave complex regulates filamentous actin content and that a decrease in actin levels is sufficient to elevate Wnt/ß-catenin signaling. Our results identify a novel role for Wave complex- and actin-regulated signaling via Wnt and SOX9 in skin development.

Pax6 regulation of Sox9 in the mouse retinal pigmented epithelium controls its timely differentiation and choroid vasculature development.

The synchronized differentiation of neuronal and vascular tissues is crucial for normal organ development and function, although there is limited information about the mechanisms regulating the coordinated development of these tissues. The choroid vasculature of the eye serves as the main blood supply to the metabolically active photoreceptors, and develops together with the retinal pigmented epithelium (RPE). Here, we describe a novel regulatory relationship between the RPE transcription factors Pax6 and Sox9 that controls the timing of RPE differentiation and the adjacent choroid maturation. We used a novel machine learning algorithm tool to analyze high resolution imaging of the choroid in Pax6 and Sox9 conditional mutant mice. Additional unbiased transcriptomic analyses in mutant mice and RPE cells generated from human embryonic stem cells, as well as chromatin immunoprecipitation and high-throughput analyses, revealed secreted factors that are regulated by Pax6 and Sox9. These factors might be involved in choroid development and in the pathogenesis of the common blinding disease: age-related macular degeneration (AMD).

T-plastin is essential for basement membrane assembly and epidermal morphogenesis.

The establishment of epithelial architecture is a complex process involving cross-talk between cells and the basement membrane. Basement membrane assembly requires integrin activity but the role of the associated actomyosin cytoskeleton is poorly understood. Here, we identify the actin-bundling protein T-plastin (Pls3) as a regulator of basement membrane assembly and epidermal morphogenesis. In utero depletion of Pls3 transcripts in mouse embryos caused basement membrane and polarity defects in the epidermis but had little effect on cell adhesion and differentiation. Loss-of-function experiments demonstrated that the apicobasal polarity defects were secondary to the disruption of the basement membrane. However, the basement membrane itself was profoundly sensitive to subtle perturbations in the actin cytoskeleton. We further show that Pls3 localized to the cell cortex, where it was essential for the localization and activation of myosin II. Inhibition of myosin II motor activity disrupted basement membrane organization. Our results provide insights into the regulation of cortical actomyosin and its importance for basement membrane assembly and skin morphogenesis.

Blastomere biopsy for PGD delays embryo compaction and blastulation: a time-lapse microscopic analysis.

PURPOSE: The purpose of the study was to explore the effect of blastomere biopsy for preimplantation genetic diagnosis (PGD) on the embryos' dynamics, further cleavage, development, and implantation. METHODS: The study group included 366 embryos from all PGD treatments (September 2012 to June 2014) cultured in the EmbryoScope™ time-lapse monitoring system. The control group included all intracytoplasmic sperm injection (ICSI) embryos cultured in EmbryoScope™ until day 5 during the same time period (385 embryos). Time points of key embryonic events were analyzed with an EmbryoViewer™. RESULTS: Most (88 %) of the embryos were biopsied at ≥8 cells. These results summarize the further dynamic development of the largest cohort of biopsied embryos and demonstrate that blastomere biopsy of cleavage-stage embryos significantly delayed compaction and blastulation compared to the control non-biopsied embryos. This delay in preimplanation developmental events also affected postimplantation development as observed when the dynamics of non-implanted embryos (known implantation data (KID) negative) were compared to those of implanted embryos (KID positive). CONCLUSION: Analysis of morphokinetic parameters enabled us to explore how blastomere biopsy interferes with the dynamic sequence of developmental events. Our results show that biopsy delays the compaction and the blastulation of the embryos, leading to a decrease in implantation.

MicroRNAs are essential for differentiation of the retinal pigmented epithelium and maturation of adjacent photoreceptors.

Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.

PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF.

During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of Pax6 in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of Mitf and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.

Pax6 regulates gene expression in the vertebrate lens through miR-204.

During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.

Pax6 dosage requirements in iris and ciliary body differentiation.

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.

Temporal silencing of an androgenic gland-specific insulin-like gene affecting phenotypical gender differences and spermatogenesis.

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis.

Characterization of a vasa-like gene from the pacific white shrimp Litopenaeus vannamei and its expression during oogenesis.

The vasa gene encodes an ATP-dependent RNA helicase belonging to the DEAD-box family that, in many organisms, is specifically expressed in germline cells throughout the life cycle. In this study we first cloned Pacific white shrimp (Litopenaeus vannamei) partial cDNAs of two members of the DEAD-box family, one belonging to the vasa subfamily (Lv-Vasa) and the other to the PL10 subfamily (Lv-PL10). Examination of their spatial expression pattern in adult tissues revealed that Lv-Vasa is restricted to the gonads, whereas Lv-PL10 is found in gonads as well as in somatic tissues. Next, we cloned the full-length shrimp vasa cDNA and found that Lv-Vasa encoded a protein with a DEAD-like helicase domain followed by a helicase superfamily C-terminal domain. In addition, Lv-Vasa encoded N-terminal three repeats of the C2HC-type zinc finger domain, a motif encoded by vasa genes of several crustaceans and several other invertebrate organisms. In situ hybridization of ovarian sections showed that the Lv-Vasa transcript is localized to the cytoplasm of the oocyte throughout oogenesis. The abundance of Lv-Vasa mRNA in mature oocytes suggests a maternal contribution for the developing embryo. It is demonstrated that the vasa homolog from L. vannamei is a gonad specific germline cell marker that could be exploited to enhance our understanding of developmental and reproductive processes in the germline of this economically important shrimp.

Males also have their time of the month! Cyclic disposal of old spermatophores, timed by the molt cycle, in a marine shrimp.

That sexually mature females go through hormonally regulated reproductive cycles is a well-established phenomenon in sexually reproducing organisms. Males, on the other hand, are commonly regarded as being continuously ready to mate. 'Programmed sperm degradation' on a periodic basis or an innate sperm 'expiry date' have never been shown. This manuscript describes a newly discovered molt-dependent mechanism by which old sperm is periodically removed from the reproductive system of male Litopenaeus vannamei shrimp. Firstly, it is shown that the spermatophores of males held in captivity become progressively melanized, a process that eventually renders them impotent. Then, by using melanin specks as a color marker, it is demonstrated that this phenomenon can be delayed and even reversed as long as the males remain sexually active. Lastly and most importantly, it is shown that male shrimp go through reproductive cycles that are strictly associated with their molt cycles, which, in turn, are hormonally regulated. Intact intermolt spermatophores disappeared about 12 h premolt, and a new pair of spermatophores appeared in the ampoules the day after the males had molted. This phenomenon was observed in an almost constant portion of males, both those in an all-male population and those in mixed male/female populations, even during the times that the females of those populations were not vitellogenically active. To the best of our knowledge, this is the first report of males of any animal species exhibiting endogenous reproductive cycles, as do females, and of the finding that spermatozoa have a predetermined expiry date, a feature that may possibly contribute to male fitness.

Complete sequence of Litopenaeus vannamei (Crustacea: Decapoda) vitellogenin cDNA and its expression in endocrinologically induced sub-adult females.

The gene that encodes vitellogenin (Vg), the precursor of the major yolk protein, vitellin, is expressed during vitellogenesis in decapod crustaceans. In this study, we sequenced the full-length cDNA from the Pacific white shrimp Litopenaeus vannamei Vg gene (LvVg). This is the first open thelycum penaeid shrimp Vg cDNA to be sequenced. The transcript encodes a 2587 amino acid polypeptide with up to 85% identity to Vg of different penaeid species. Peptide mass fingerprints (PMFs) of the vitelline polypeptides suggest that the predicted endoprotease cleavage site at amino acids 725-728 does indeed undergo cleavage. Five prominent high-density lipoprotein polypeptides of masses 179, 113, 78, 61, and 42kDa were isolated from vitellogenic ovary, and their PMFs were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry. It is likely that these polypeptides are all products of the LvVg gene. Removal of the X-organ sinus gland complex (XO-SG), which secretes the neurohormones that control the endocrine system regulating molt and reproduction, can induce both these processes. During the course of a number of molt cycles in induced sub-adult females, periodic ovarian growth and resorption were observed. Ovary growth correlated with LvVg expression in both the hepatopancreas and the ovary. Expression in ovaries of induced intermolt-early premolt females was significantly higher compared to all other sub-groups. Expression in ovaries of induced females was significantly higher compared to hepatopancreas at all molt cycle stages. Periodicity of molt and vitellogenesis in endocrinologically induced sub-adult shrimps may serve as a model to study alternate regulation of gene expression during these two processes.