Early Cleavage Stage Embryos Research Articles

P-173 An exploratory clinical study in patients with recurrent ART-failure due to severe fragmentation during early-cleavage: the effects of artificial removal of zona-pellucida at the pronuclear-stage

Abstract Study question Can the artificial removal of the zona pellucida (ZP) at the pronuclear (PN)-stage prevent severe fragmentation in early-cleavage embryos and improve good-quality blastocyst development rate? Summary answer The artificial removal of the ZP at the 2PN-stage could prevent significantly severe fragmentation in early-cleavage embryos and improved the good-quality blastocyst development rate. What is known already Recent analysis using time-lapse culture demonstrated that fragments during early-cleavage occur at the site of the perivitelline threads between the ZP and the embryo membrane (Reprod Biomed Online 2017;35:640-5,646-56). Based on these findings we conducted a pilot study on embryonic development after ZP-removal using abnormally-fertilized oocytes (J Assist Reprod Gene2020;37:1349-54). We showed that the inter-blastomere adhesion was maintained and fragmentation was significantly lower in the ZP-free embryos than in the ZP-intact embryos. Therefore, we attempted to apply this method to patients who have given full consent and had repeatedly failed to conceive due to severe fragmentation in early cleavage-stage embryos. Study design, size, duration This is a single-centre retrospective exploratory study of 34 patients that were scheduled to undergo ART-treatment in our clinic between February 2020 and January 2021. They had been previously treated in our facilities (January 2016 to January 2020) with less than 10% morphologically good-quality blastocysts due to severe fragmentation. None of these patients became pregnant during their initial treatments. Totally, 173 2PN-zygotes were obtained, with 101 allocated to ZP-free group and 72 to ZP-intact group. Participants/materials, setting, methods Normally-fertilized oocytes were placed in 0.125M sucrose-containing HEPES-medium (approximately 5min) and cultured under: 1) ZP-free, in which the ZP was completely removed from the ooplasm by laser-irradiation and a medium-blowing method with a biopsy-pipette, or 2) ZP-intact. ZP-free and ZP-intact zygotes were cultured in sequential medium. Embryos were either freshly transferred on Day-2(ZP-intact group only, according to patients’ wishes), or Day-5/6, or they were cryopreserved on Day-5/6/7 for future embryo transfer cycles for both groups. Main results and the role of chance Even allocation of the normally fertilised oocytes to the two groups was not possible, due to the patients’ request to include more zygotes in the ZP-free group. Therefore, no specific criteria were set other than the rule that all the normally fertilised oocytes of each patient must be allocated into both groups at the time of allocation. As a result, each patient had 2PN zygotes assigned to both ZP-free and ZP-intact groups. Rates of good-quality embryos, blastocyst development, morphologically good-quality blastocyst development and cryopreservation demonstrated significant improvement (all P < 0.01) in the ZP-free group compared to the ZP-intact group. Pregnancy rates in ZP-intact group were 5.9% per patient and 5.3% per embryo transferred, while in ZP-free group 37.5% per patient and 24.3% per embryo transferred. Our results demonstrate that in difficult-to-treat cases where embryos with good morphology cannot be obtained due to excessive fragmentation at the early-cleavage, embryonic development can be significantly improved by artificial removal of ZP at the 2PN-stage. We believe that the ZP-free method has opened a new door in assisted reproductive medicine and can provide the opportunity to obtain blastocysts with good-morphology from an embryo that would probably not have become a blastocyst otherwise. Limitations, reasons for caution A comparative analysis between the two groups was not possible due to the small number of patients, the inclusion of fresh embryos as well as frozen embryos, and the non-randomized data. The imbalance was attributed to the fact that patients wanted us to allocate more oocytes to the ZP-free group. Wider implications of the findings The development of treatment methods based on new ideas should always take into consideration the possibility of associated risks. In the future, we will examine the criteria for selecting patients for whom this method is useful, as well as verify its safety and effectiveness by conducting a prospective randomized analysis. Trial registration number not applicable

Shape of the first mitotic spindles impacts multinucleation in human embryos

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

Neddylation inhibition affects early embryonic development by disrupting maternal-to-zygotic transition and mitochondrial function in mice

Post-translational modifications (PTMs) are critical for early development in mice because early cleavage-stage embryos are characterized by transcriptional inactivity. Neddylation is an important ubiquitin-like PTM that regulates multiple biophysical processes. However, the exact roles of neddylation in regulating early embryonic development remain largely unknown. In the present study, we found that inhibition of neddylation by specific inhibitor MLN4924 led to severe arrest of early embryonic development. Transcriptomic analysis showed that neddylation inhibition changed the expression of 3959 genes at the 2-cell stage. Importantly, neddylation inhibition blocked zygotic genome activation and maternal mRNA degradation, thus disrupting the maternal-to-zygotic transition. Moreover, inhibition of neddylation induced mitochondrial dysfunction including aberrant mitochondrial distribution, decreased mitochondrial membrane potential, and reduced ATP content. Further analysis showed that inhibition of neddylation resulted in the accumulation of reactive oxygen species and superoxide anion, thereby resulting in oxidative stress and severe DNA damage at the 2-cell stage. Overall, this study demonstrates that neddylation is vital for early embryonic development in mice. Our findings suggest that proper neddylation regulation is essential for the timely inter-stage transition during early embryonic development.

Genetic tools for the study of the mangrove killifish, Kryptolebias marmoratus, an emerging vertebrate model for phenotypic plasticity.

Kryptolebias marmoratus (Kmar), a teleost fish of the order Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic-varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self-fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage-stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild-type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono- and bi-allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity.

Hypoxic in vitro culture reduces histone lactylation and impairs pre-implantation embryonic development in mice

BackgroundDynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated.ResultsWe examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate.ConclusionsWe provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.

Research Paper: Comparison of Embryo Development and Pregnancy Rates in Continuous Single and Sequential Media Cultures of Sibling Embryos

Embryo development and pregnancy rates for In Vitro Fertilization (IVF) in continuous single medium culture or sequential media culture of sibling embryos were evaluated. Patients who underwent retrieval of ≥8 oocytes from June 2013 to March 2014 were enrolled. All embryos were cultured for at least 5 days and the formation rate and quality of embryos were compared between the groups. No significant differences were observed between the 2 culture media systems regarding the formation rate and quality of early cleavage-stage embryos, although blastulation rate and quality were better with single medium. Clinical pregnancy rate and ongoing pregnancy rate per blastocyst transfer did not differ between the groups. These results suggested that a single continuous medium was as good as or better than sequential media to achieve pregnancy in IVF and embryo transfer.

DUX4-Induced Histone Variants H3.X and H3.Y Mark DUX4 Target Genes for Expression

SUMMARYThe DUX4 transcription factor is briefly expressed in the early cleavage-stage embryo, where it induces an early wave of zygotic gene transcription, whereas its mis-expression in skeletal muscle causes the muscular dystrophy facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4 induces the expression of the histone variants H3.X and H3.Y. We have used a myoblast cell line with doxycycline-inducible DUX4 to show that these histone variants are incorporated throughout the body of DUX4-induced genes. Following a brief pulse of DUX4, these histones contribute to greater perdurance and to enhanced re-activation of DUX4 target gene expression. These findings provide a model for H3.X/Y as a chromatin mechanism that facilitates the expression of DUX4 target genes subsequent to a brief pulse of DUX4 expression.

Intrauterine CRH-treated PBMC in repeated implantation failure.

The intrauterine administration of activated autologous peripheral blood monocytes (PBMC) prior to embryo transfer seems to improve reproductive outcomes in women with repeated implantation failure (RIF). We have previously shown that the intrauterine administration of PBMC treated with corticotropin-releasing hormone (CRH) prior to blastocyst transfer (day 5) improves significantly the clinical pregnancy rate of women with RIF. In the present crossover pilot study, we have investigated whether CRH-PBMC treatment could be of benefit in case of fresh early cleavage stage embryo transfer (day 3) in women with RIF. Twenty-six (n=26) women with at least three previous failed IVF attempts and no history of clinical pregnancy in the past were recruited in this study. Ovarian stimulation was performed following either the long or the short protocol. PBMC were collected during the oocyte retrieval, were treated with CRH, and transferred in the uterine cavity 2days later. Good quality cleavage stage embryos were transferred at day 3, following oocyte retrieval. Following the intrauterine administration of CRH-treated autologous PBMC, 15/26 clinical pregnancies occurred (57.69%). Compared to the null result of the same women prior to recruitment, this observation was considered significant (P<10-2 ). Our findings further support the role of the intrauterine administration of CRH-treated PBMC as an effective approach when transferring cleavage stage embryos in women with RIF. Prospective randomized studies are needed to clarify whether such intervention could be of benefit in clinical practice.

Mitochondria and lipid metabolism in mammalian oocytes and early embryos.

Mammalian oocytes and early cleavage-stage embryos are critically dependent on their ˜100,000 mitochondria to develop from ovulation to compacted morula stage. They rely almost solely on oxidative phosphorylation of multiple intracellular substrates- namely pyruvate, fatty acids and glutamine- for production of ATP. Increasing evidence exists for the requirement of both fatty acids and pyruvate for mammalian developmental potential. Fatty acids are stored as neutral lipids in lipid droplets, which are liberated into the cytoplasm as free fatty acids and taken up into mitochondria for metabolism. Different mammalian species exhibit different amounts of stored and free lipid, while the types of lipid present tend to remain constant. It is thought that the amount of lipid contained in the oocytes of mammalian species reflects the extent of β-oxidation, but it is unclear why large differences are seen in lipid content. Maternal high fat diet or obesity causes negative intracellular effects such as the ER stress response, and oxidative mitochondrial and DNA damage. While some mechanisms have been established, it is still unclear exactly how high fat leads to compromised oocyte and embryo quality. It is proposed that healthy mammalian oocyte mitochondria require a balance of pyruvate and fatty acid oxidation in order to maintain a low level of otherwise damaging ROS production. This balance is disrupted in conditions of excess or insufficient substrate.

A time-lapse sibling oocyte study: does embryo culture medium have an impact on morphokinetic parameters?

To evaluate the effects of different culture media on embryonic development and morphokinetic parameters in the same culture condition by sibling oocyte study. Retrospective analysis: randomized, sibling oocyte study in a private assisted reproductive technology clinic. This study was approved by the Ladies Clinic Kyono Ethics Committee. The subjects were 450 fertilized zygotes from 66 cycles from which at least six oocytes were retrieved at our clinic, from Oct. 2017 to Feb. 2018. Following IVF/ICSI, normally fertilized zygotes were divided into two groups and cultured in an EmbryoSlide+TM with two kinds of culture media, global®total® LP (GL) and SAGE 1-StepTM (SA). Both media contain 5mg/ml human serum albumin. Embryos were monitored by EmbryoScope+TM with 6% CO2 and 5% O2. We analyzed embryonic development and the appearance of multinucleation (MN) between the two media. Furthermore, in the 155 blastocysts, morphological events (cell division and interval of cell cleavage) evaluations were performed by tPNf (syngamy), t2 (time to 2 cell), t3, t4, t5, t6, t7, t8, tM (fully compacted morula), tSB (start of blastulation), tB (time of blastulation), cc2 (t3-t2) and s2 (t4-t3). We also compared the appearance of multinucleation (MN), direct cleavage (DC) and cell fusion (CF: a reduction in the number of cells of an embryo during its development). Fisher's exact test and Mann-Whitney U tests were used for statistical analysis. P<0.05 was considered statistically significant. In embryonic development - good-quality day-3 embryo rate , blastocyst formation rate and good-quality blastocyst rate - there were no significant differences between the two media. GL showed a significantly higher appearance of MN at 2 or 3-cell stage than SA in both insemination methods (ICSI: GL, 31.1% (38/122) vs SA, 19.2% (19/99); c-IVF: GL, 25.0% (17/68) vs SA, 8.6% (5/58); P<0.05). In the morphokinetic analysis, there were no differences in time points of cell division (tPNf to tB) or appearance of DC and CF. However, there were significant differences in interval of second embryo cell cycle (cc2) (cc2: GL, 11.3±1.7h vs. SA, 10.0±2.8h; P=0.0019) derived from ICSI embryos. There was no difference in embryonic development between the two culture media, but a significant difference was found in the presence of MN at two-cell stage. Although there was no difference in the presence of MN in the embryos that reached the blastocyst stage, since a significant difference was observed in the interval of cell cleavage (cc 2), it was suggested that culture medium may affect the time of DNA replication of early cleavage stage embryos.

Effects of seawater acidification on the early development of sea urchin Glyptocidaris crenularis

In this study, we evaluated the effects of CO2-induced seawater acidification on fertilization, embryogenesis and early larval development in the sea urchin Glyptocidaris crenularis, that inhabits subtidal coastal areas in northern China. The range in seawater pH used in experiments was based on the projections of the Intergovernmental Panel on Climate Change (IPCC), to the year 2100. A natural seawater treatment (pHnbs =7.98±0.03) and three laboratory-controlled acidified treatments (OA1, ΔpH nbs =-0.3 units; OA2, ΔpHnbs =-0.4 units; OA3, ΔpHnbs=-0.5 units) were used in experiments. Results show that: (1) there was a negative effect of seawater acidification on fertilization and on the percentage of abnormal fertilized eggs; (2) the size of early cleavage stage embryos decreased in a dose-dependent manner with decreasing pH; (3) both the hatching rate of blastulae and the survival rate of four-armed pluteus larvae decreased as pH declined; (4) larval abnormalities including asymmetrical development, changes in the length of skeletal elements, and corr oded spicules were observed in all seawater acidified-treatments compared with the control. These data indicate that seawater acidification has a negative impact on the early development of G. crenularis, and supports the hypothesis that the response of echinoderms to ocean acidification (OA) varies among species. Further research is required to clarify the specific cellular mechanisms involved.

Mitochondrial-related consequences of heat stress exposure during bovine oocyte maturation persist in early embryo development.

Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P < 0.0001). Exposure to 41.0ºC for 12 h increased total and reduced glutathione levels in oocytes at 12 h but reduced them by 24 h (time × temperature P < 0.001). ATP content was higher in heat-stressed oocytes at 24 h (P < 0.0001). Heat-induced increases in ATP content of matured oocytes persisted in early cleavage-stage embryos (8- to 16-cell embryos; P < 0.05) but were no longer apparent in blastocysts (P > 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

Differential effects of follistatin on porcine oocyte competence and cumulus cell gene expression in vitro.

Several oocyte-derived genes/proteins are essential to early embryonic development. The expression and stability of these proteins are influenced by the autocrine/paracrine activity of factors released by oocytes and cumulus cells. This study investigated the paracrine and autocrine activity of follistatin (FS), which is secreted by oocytes and cumulus cells as part of porcine embryogenesis. Immunohistochemical (IHC) localization of follistatin was conducted on 100 randomly selected early- and late-cleaving two-cell embryos. Dissociated cumulus cells were treated with various doses of follistatin for determination of the follistatin gene (FST) mRNA expression levels by quantitative real-time PCR analysis. Microinjection of siRNA induced a downregulation of FST mRNA during embryonic development, thereby decreasing the proportion embryos developing to the blastocyst stage (19.33%). Immunolocalization analysis showed enhanced staining for follistatin in early-cleavage stage embryos. Quantitative real-time PCR indicated a significantly lower FST transcript level in cumulus cells after application of the highest dose of follistatin (100ng/ml). Exogenous follistatin treatment of in vitro maturation embryos resulted in statistically significant dose-dependent changes during development. Application of the highest concentration (100ng/ml) of follistatin decreased the maturation rate of the oocytes. On the other hand, the application of 10ng/ml follistatin resulted in an increase in the number of embryos. The observed differential effect of exogenous follistatin might be due to maternal FST and autocrine/paracrine factors secreted by cumulus cells.

Annelids shed light on the evolution of spiralian development

Spiralian development is characterized by stereotypic cell geometry and spindle orientation in early cleavage stage embryos, as well as conservation of ultimate fates of descendent clones. Diverse taxa such as molluscs, annelids, flatworms, and nemerteans exhibit spiralian development, but it is a mystery how such a conserved developmental program gives rise to such diverse body plans. This review highlights examples of variation during early development among spiralians, emphasizing recent experimental studies in the annelid Capitella teleta Blake, Grassle and Eckelbarger, 2009. Intracellular fate mapping studies in C. teleta reveal that many of its cells’ fates are shared among spiralians, but it also has a novel origin for trunk mesoderm (3c and 3d micromeres). Studies have identified an inductive signal in spiralians that has “organizing activity” and that influences cell fates in the surrounding embryo. Capitella teleta also has an organizing activity; however, surprisingly, it is localized to a different cell, it signals at a different developmental stage, and likely utilizes a distinct molecular signaling pathway compared with that in molluscs. A model is presented to provide a mechanistic explanation of evolutionary changes in the cellular identity of the organizer. Detailed experimental investigations in spiralian embryos demonstrate variation in developmental features that may influence the evolution of novel forms.

Nucleologenesis and Nucleolotransfer in Mammalian Oocytes: A Review

AbstractAn effort to improve development potential of early embryos is one of the main goals of biotechnology in the area of reproductive biology with application in veterinary or human medicine. Recent observations of the function of nucleolus or rather its forms before, during and after the fertilisation or parthenogenetic activation show the key role(s) of nucleolus in the processes of early genome activation. The nucleolus is a subnuclear structure (organelle) mainly involved in regulation of transcription and translation. This organelle has been characterized in detail by immunofluorescence, cell transfection and proteomics. This data was, however, mostly obtained in nucleoli of differentiated eukaryotic cells. Much less is known about the nucleolar structural changes and related functional processes in growing and fully grown mammalian oocytes, zygotes and early cleavage stage embryos, especially in the context of embryonic genome activation. It has been shown, that nucleoli in mammalian oocytes and early embryos have several forms and functions, which vary during the oocyte growth and embryonic development. Certain functions have not been fully described or explained, yet. The method of enucleolation, which allows to remove nucleoli from the oocytes or to exchange nucleoli between oocytes or zygotes, together with their proteomic and structural analyses brought new information about functions of nucleoli in oocytes and early cleavage-stage embryos and allowed to explain some new key roles of nucleoli during oocyte maturation and early embryonic development.

Chapter 9 Slow Freezing and Thawing of Human Cleavage Stage Embryos.

The ability to store human embryos in a viable state at very low temperatures has been critical to the evolution of responsible practice in clinical Assisted Reproductive Technology (ART). It has encouraged a reduction in the frequency of simultaneous multiple embryo transfer and thereby reduced the risks associated with multiple pregnancy while maintaining high cumulative pregnancy rates from single oocyte collection cycles. In this chapter, we describe a simple slow freezing procedure for human early cleavage stage embryos that results in a high proportion of post-thaw embryos surviving and retaining their implantation potential.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.

Intrauterine administration of hCG-activated autologous human peripheral blood mononuclear cells (PBMC) promotes live birth rates in frozen/thawed embryo transfer cycles of patients with repeated implantation failure

Recurrent implantation failure refers to unsuccessful implantation after repeated transfers of morphologically good quality embryos into a normal uterus. Recently, accumulating evidence has suggested that local immune cells at the implantation site have actively contributed to embryo implantation. Our aim was to study the effects of intrauterine administration of hCG-activated autologous human PBMC on clinical pregnancy, implantation rates and live birth rate of patients who received frozen/thawed embryo transfer. We observed patients with one to three failed transplantations cannot benefit from the administration, but the rate of clinical pregnancy (39.58% vs. 14.29%), live birth (33.33% vs. 9.58%) and implantation (22.00% vs. 4.88%) were significantly increased in patients with four or more failures, respectively. For patients with endometrial thickness more than 7mm and less than 8mm on day of embryo transfer, the implantation rate (22.69% vs. 14.21%) and the live birth rate significantly higher in the PBMC-treated group; For patients who had RIF and received frozen/thawed early cleavage stage embryo transfer, the live birth delivery rate (29.63% vs. 13.33%) significant higher in PBMC-treated group. These findings indicate that intrauterine administration of hCG-activated autologous PBMC effectively improves the IVF outcomes for RIF patients, especially for the RIF patients with cleavage stage embryo transfer, patients with thin endometrial thickness also benefit from this approach.

No increased risk of major congenital anomalies or adverse pregnancy or neonatal outcomes following letrozole use in assisted reproductive technology.

Does letrozole use increase the risk of major congenital anomalies and adverse pregnancy and neonatal outcomes in fresh, single-embryo transfer? Letrozole significantly decreases the risk of miscarriage and does not increase the risk of major congenital anomalies or adverse pregnancy or neonatal outcomes compared with natural cycles in patients undergoing ART. Letrozole is the most commonly used aromatase inhibitor for mild ovarian stimulation in ART. However, its safety in terms of pregnancy and neonatal outcomes is unclear. This retrospective cohort study used data from the Japanese national ART registry from 2011 to 2013. A total of 3136 natural cycles and 792 letrozole-induced cycles associated with fresh, single-embryo transfer and resulting in a clinical pregnancy were included in the analysis. The main pregnancy outcomes were miscarriage, ectopic pregnancy and still birth, and the neonatal outcomes were preterm delivery, low birth weight, small/large for gestational age and major congenital anomalies. Terminated pregnancies were included in the analysis of major congenital anomalies. Odds ratios (ORs) and 95% CIs were calculated using multivariate logistic regression analysis adjusted for maternal age and calendar year. The risk of miscarriage was significantly lower in women administered letrozole (adjusted OR [aOR], 0.37, 95% CI, 0.30-0.47, P < 0.001). There was no significant difference in the overall risk of major congenital anomalies between the two groups (natural cycle 1.5%vs letrozole 1.9%, aOR, 1.24, 95% CI, 0.64-2.40, P = 0.52), and no increased risk for any specific organ system. Subgroup analysis demonstrated that the risk of major congenital anomalies was not increased in patients who underwent eitherin vitro fertilization or ICSI, or in those who received early cleavage stage or blastocyst embryo transfer. All other pregnancy and neonatal outcomes were comparable between the two groups. Despite the large sample size, we were only able to rule out the possibility that letrozole might cause large increases in birth-defect risks in ART patients. The results suggest that letrozole stimulation reduces the risk of miscarriage, with no increase in the risk of major congenital anomalies or adverse pregnancy or neonatal outcomes compared with natural cycles in women undergoing ART. Letrozole may thus be a safe option for mild ovarian stimulation. None. Not applicable.

Risk factors for abnormal umbilical cord insertions among term singleton art pregnancies

The placenta with velamentous umbilical cord insertion (VCI) is a rare pathologic condition that is supposed to increase the risk of perinatal complications. The reported adverse outcomes include fetal growth restriction, non-reassuring fetal status requiring emergency Cesarean section, fetal exsanguination due to the rupture of vasa previa, and cerebral palsy. We reported on a study, emphasizing a higher incidence of the placenta accompanied by VCI in ART pregnancies when compared with non-ART cases, at the 70th ASRM Annual Meeting. In this research, we examined the ART-conceived cases closely for the purpose of identifying the ART-associated risk factors that might affect the incidence of the placenta with VCI. Retrospective cohort study and comparative analysis. We reviewed the records of 427 consecutive singleton, term labor and delivery ART-conceived cases from January 2011 to April 2016. Information about maternal age, obstetric history, details of ART treatment, mode of delivery, and insertion site of the umbilical cord was all collected from their medical records. Our study population was divided into two categories as follows: cases with VCI (Group A: n=23), and cases without VCI (Group B: n=404). Odds ratios for VCI, 95% confidence intervals (CI), and significance of the odds ratio were calculated according to ART-related variables (i.e., fertilization method (conventional-IVF vs. ICSI), type of embryo (fresh vs. frozen-thawed), embryo transfer timing (day 2-3 early-cleavage stage embryo vs. day 5-6 blastocyst), and fetal sex), using multivariate logistic regression. Maternal basal characteristics and odds ratios were shown in the table below. The incidence of VCI in our study population was 5.4% (23/427). The rate of delivery necessitating emergency C-section or operative vaginal delivery in Group A (14/23, 61%) was much higher than in Group B (138/404, 34%). Blastocyst transfer and female fetus significantly increased the likelihood of developing the placenta with VCI. Fertilization method and type of embryo did not affect the incidence of the placenta with VCI.Tabled 1Baseline characteristics and adjusted odds ratio(aOR) for velamentous umbilical cord insertionGroup A (cases with VCI: n=23)Group B (cases without VCI: n=404)aOR for VCI (95% CI, P value)Maternal age35.9±3.835.5±3.61.03 (0.91 - 1.16, 0.66)Parity (primipara / multipara)20 / 3302 / 1021.00 (reference) / 0.40 (0.09 - 1.23, 0.15)c-IVF / ICSI5 / 18143 / 2611.00 / 1.96 (0.76 - 6.05, 0.19)Fresh / Frozen-thawed7 / 16136 / 2681.00 / 1.25 (0.52 - 3.34, 0.63)Early cleavage / Blastocyst1 / 22117 / 2871.00 / 10.23 (2.09 - 184.78, 0.02*)Male / Female5 / 18200 / 2041.00 / 3.48 (1.35 - 10.71, 0.02*)[Note of the table above: Data are mean±SD or number of subjects. Odds ratios were all adjusted for maternal age and parity. *: P<0.05 was considered statistically significant.] Open table in a new tab [Note of the table above: Data are mean±SD or number of subjects. Odds ratios were all adjusted for maternal age and parity. *: P<0.05 was considered statistically significant.] As VCI often brings about serious obstetric complications, ART pregnancies achieved by blastocyst transfer and/or of female fetus are optimal candidates for checking up on and identifying the umbilical cord insertion site more proactively during routine obstetric ultrasound examinations.