Day 3 Embryo Fragmentation Is Associated With Singleton Birthweight Following Fresh Single Blastocyst Transfer：A Retrospective Study

Background Previous studies arguably associated poor embryo morphology with low birthweight in singletons following single embryo transfer. However, the association between specific morphological features on the cleavage stage and birthweight is still less known. The purpose of the study was to investigate whether embryo morphological features at the cleavage stage affect birthweight following blastocyst transfer Methods The single-center, retrospective cohort study included 4226 singletons derived from fresh single cleavage stage embryo transfer (ET, n=1185), fresh single blastocyst transfer (BT, n=787), or frozen-thawed single blastocyst transfer (FBT, n=2254) between 2016 and 2019. The morphological parameters including early cleavage, day 3 fragmentation, symmetry, blastomere number, and blastocyst morphology were associated with neonatal birthweight and z-score in multivariate regression models. Models were adjusted for maternal age, BMI, parity, peak estradiol level, endometrial thickness, insemination protocol, female etiologies, order of transfer, mode of delivery, and year of treatment. Results Adjusted for confounders, fragmentation was the only morphology feature associated with birthweight and z-score, while early cleavage, symmetry, blastomere number and blastocyst morphology were not. Fragmentation increased the birthweight in both ET group (115.4g, 95% CI: 26.6 to 204.2) and BT group (168.8g, 95%CI: 48.8 to 288.8), but not in FBT group (7.47g, 95%CI: -46.4 to 61.3). The associations of birthweight and morphological parameters were confirmed in analyses for z-score. Adjusted odds of large for gestational age and high birthweight were also significantly greater in singletons following the transfer of fragmented embryos in BT group (OR 3, 95% CI: 1.2 to 7.51, OR 3.65, 95% CI: 1.33 to 10, respectively). The presence of fragmentation at the cleavage stage also affected the association between blastocyst morphology and birthweight. Inner cell mass grades were negatively associated with birthweight in blastocysts with day 3 fragmentation but not in blastocysts without. Conclusions Birthweight following blastocyst transfer is positively associated with fragmentation at the cleavage stage. The data did not support the argument that transferring a poor-looking embryo may increase the risks of low birthweight. However, concerns for LGA infants still remain.

Effect of endometrial preparation protocols on the risk of ectopic pregnancy for frozen embryo transfer

Studies have consistently reported a significantly reduced incidence of ectopic pregnancy (EP) for frozen-thawed embryo transfer (ET) cycles compared with fresh cycles. However, only a few studies reported an association between endometrial preparation protocols on EP and results were conflicting. A registry-based retrospective cohort study of 153,354 clinical pregnancies following frozen single ETs between 2014 and 2017 were conducted, of which 792 cases of EP (0.52%) were reported. Blastocyst embryo transfers accounted for 87% of the total sample and were significantly associated with a decreased risk for EP compared with early cleavage ET (0.90% vs. 0.46%, adjusted OR = 0.50, 95% CI, 0.41 to 0.60). Compared with natural cycles, hormone replacement cycles (HRC) demonstrated a similar risk for EP (0.53% vs. 0.47%, adjusted OR = 1.12, 95% CI, 0.89 to 1.42). Subgroup analysis with or without tubal factor infertility and early cleavage/blastocyst ETs demonstrated similar non-significant associations. Endometrial preparation protocols using clomiphene (CC) were associated with a significantly increased risk for EP (1.12%, adjusted OR = 2.34; 95% CI, 1.38 to 3.98). These findings suggest that HRC and natural cycles had a similar risk for EP. Endometrial preparation using CC was associated with an increased risk of EP in frozen embryo transfer cycles.

P–206 Does oocyte vitrifcation affect morphokinetics of subsequent embryo development?

Study question Are the morphokinetic profiles, as assessed using time-lapse technology, of human embryos developed from vitrified oocytes different to those from fresh oocytes. Summary answer Vitrification of oocytes does have an effect on early developmental morphokinetic profiles, but this is normalized by the time the embryo has reached blastocyst. What is known already Vitrification of oocytes is now commonplace, but little is known about the effect this may have on subsequent embryo development. Study design, size, duration This was a retrospective data analysis, from 8 fertility clinics in the UK between 2012 and 2019. Embryos from patients in the vitrified group (n = 557) were matched to fresh patient controls (n = 539). The matching was performed based on the following criteria: type of treatment, patient age, cause of infertility and number of embryos. Participants/materials, setting, methods The embryos in each group were compared for mean morphokinetics of key developmental stages in hours post insemination (hpi). Parameters compared included early cleavage divisions (t2-t8), time to start of compaction (tSC), time to morula (tM), time to start blastulation (tSB), time to full blastocyst (tB) and duration of compaction (tB-tSC). Treatment outcomes were compared between the two groups, including percentage of blastocyst formation, clinical pregnancy rate, implantation rate and live birth rate. Main results and the role of chance The results showed a significant delay across all early cleavage divisions as follows for vitrified and fresh oocytes respectively: 2-cell (28.14 vs 26.10 (p < 0.001)), 3 cell (37.56 vs 35.37 (p < 0.001)), 4 cell (40.58 vs 37.54 (p < 0.001)), 5 cell (50.31 vs 47.14 (p < 0.001)), 6 cell (53.99 vs 50.87 (p < 0.001)), 7 cell (57.08 vs 54.48 (p < 0.001)) and 8 cell (61.26 vs 58.91 (p < 0.01)). In addition, tSC was also significantly delayed in the vitrified group (80.65 vs 76.36 (p < 0.001)). However, the compaction stage was significantly shorter in the vitrified oocytes (19.02 vs 22.45 (p < 0.001)). Therefore, there was no difference in the time that embryos derived from fresh and vitrified oocytes reached the blastocyst stage (108.03 vs 107.78 (p > 0.05)). No difference was found in clinical pregnancy, implantation or live birth rates but significantly fewer blastocyst developed from vitrified oocytes compared to fresh (36.09% vs 42.4% (p < 0.05)). Limitations, reasons for caution Although this was a matched analysis, it was a retrospective in nature therefore is subject to confounders. However, it would be problematic to perform a prospective randomized controlled trial to address this study question given the need to randomize patients to elective freezing of oocytes prior to embryo creation. Wider implications of the findings: Vitrification of oocytes may affect early developmental morphokinetic profiles, but any effect is normalized by the time the embryo has reached blastocyst. However, fewer blastocysts may develop following oocyte vitrification. This may have implications for oocyte donation banks and those patients choosing to cryopreserve oocytes. Trial registration number NA

Early embryogenesis and organogenesis in the annelid Owenia fusiformis

Background Annelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis, a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored. Results Owenia fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately 5 h post-fertilization (hpf) at 19 °C. Gastrulation occurs via invagination and completes 4 h later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorso-posterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin-1) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide+ neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after ~ 3 weeks post-fertilization at 15 °C, when a conspicuous juvenile rudiment has formed ventrally. Conclusions Owenia fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more elaborated than previously recognized and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.

Germ plasm anchors at tight junctions in the early zebrafish embryo

The zebrafish germline is specified during early embryogenesis by inherited maternal RNAs and proteins collectively called germ plasm. Only the cells containing germ plasm will become part of the germline, whereas other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is key for germ cell specification and its removal is critical for the development of soma. The molecular mechanism underlying this process in vertebrates is largely unknown. Here we show that germ plasm localization in zebrafish is similar toXenopusand amniotes but distinct fromDrosophila. We identified non muscle myosin II (NMII) and tight junction (TJ) components as interaction candidates of Bucky ball (Buc), which is the germ plasm organizer in zebrafish. Remarkably, we also found that TJ protein ZO1 colocalizes with germ plasm and electron microscopy (EM) of zebrafish embryos uncovered TJ like structures at early cleavage furrows. In addition, injection of the TJ-receptor Claudin-d (Cldn-d) produced extra germ plasm aggregates. Our findings discover for the first time a role of TJs in germ plasm localization.

ATP signaling in the integrative neural center of Aplysia californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica—a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Early Embryogenesis and Organogenesis in the Annelid Owenia Fusiformis

Background : Annelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis , a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored.Results : O. fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately five hours post fertilization (hpf) at 19 ºC. Gastrulation occurs via invagination and completes four hours later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorsoposterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin1 ) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide + neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after ~3 weeks post fertilization at 15 ºC, when a conspicuous juvenile rudiment has formed ventrally.Conclusions : O. fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more complex than previously recognized and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.

Early embryogenesis and organogenesis in the annelid Owenia fusiformis

BackgroundAnnelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis, a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored.ResultsO. fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately five hours post fertilization (hpf) at 19 °C. Gastrulation occurs via invagination and completes four hours later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorsoposterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin1) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide+ neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after ∼3 weeks post fertilization at 15 °C, when a conspicuous juvenile rudiment has formed ventrally.ConclusionsO. fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more complex than previously recognised and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.

ATP Signaling in the Integrative Neural Center of Aplysia Californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica – the prominent neuroscience model. acP2X receptors were successfully expressed in Xenopus oocytes and were displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were quite different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the acP2X receptors than the suramin. acP2X were uniquely expressed within the cerebral F-cluster, which contains multiple secretory peptides (e.g., insulins, interleukins, and potential toxins), ecdysone-type receptors, and a district subset of ion channels. We view F-cluster as the multifunctional integrative center, remarkably different from other neurosecretory cells. acP2X receptors were also found in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering evolution of neurotransmitters.