Embryo Arrest Research Articles

Loss of function of membrane estrogen receptor ERα alters expansion of trophoblast cells and impacts mouse fertility

17β-estradiol following binding to estrogen receptor alpha (ERα) exerts a critical role in the control of reproduction, acting through both nuclear and membrane-initiated signaling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss-of-function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do get pregnant, allowing to investigate the role of ERα during pregnancy for the first time. Total neonatal deaths of the mice offspring resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibit partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality results from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells (SpA-TGCs) into the utero-placental unit, associated with the imbalance expression of angiogenic factors, together controlling the trophoblast-mediated spiral arterial remodeling. Hence, maternal loss of membrane ERα function clearly alters the activity of invasive trophoblast cells during placentogenesis. This novel function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.

Endosperm cellularization failure induces a dehydration-stress response leading to embryo arrest.

The endosperm is a nutritive tissue supporting embryo growth in flowering plants. Most commonly, the endosperm initially develops as a coenocyte (multinucleate cell) and then cellularizes. This process of cellularization is frequently disrupted in hybrid seeds generated by crosses between different flowering plant species or plants that differ in ploidy, resulting in embryo arrest and seed lethality. The reason for embryo arrest upon cellularization failure remains unclear. In this study, we show that triploid Arabidopsis thaliana embryos surrounded by uncellularized endosperm mount an osmotic stress response that is connected to increased levels of abscisic acid (ABA) and enhanced ABA responses. Impairing ABA biosynthesis and signaling aggravated triploid seed abortion, while increasing endogenous ABA levels as well as the exogenous application of ABA-induced endosperm cellularization and suppressed embryo growth arrest. Taking these results together, we propose that endosperm cellularization is required to establish dehydration tolerance in the developing embryo, ensuring its survival during seed maturation.

Loss of function of the maternal membrane oestrogen receptor ERα alters expansion of trophoblast cells and impacts mouse fertility.

The binding of 17β-oestradiol to oestrogen receptor alpha (ERα) plays a crucial role in the control of reproduction, acting through both nuclear and membrane-initiated signalling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss of function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do become pregnant, allowing the investigation of the role of ERα during pregnancy for the first time. All neonatal deaths of the mutant offspring mice resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibited partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality resulted from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells into the utero-placental unit, which is associated with an imbalance in expression of angiogenic factors. Together, these processes control the trophoblast-mediated spiral arterial remodelling. Hence, loss of membrane ERα within maternal tissues clearly alters the activity of invasive trophoblast cells during placentogenesis. This previously unreported function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.

Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM.

With improvements in the survival rate of patients with cancer, fertility maintenance has become a major concern in terms of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that are used clinically or are undergoing trials. The HuR small-molecule inhibitor MS-444 has been used in many cancer treatment studies, but its reproductive toxicity in females is unknown. Here, we reported that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization, resulting in the developmental arrest of 2-cell stage embryos in mouse. Combining analysis of low-input RNA-seq for MS-444-treated 2-cell embryos and mapping binding sites of RNA-binding protein, Agbl2 was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed that Agbl2 knockdown reduced the proportion of embryos successfully developed to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analyses showed that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA and reduced its stability by inhibiting HuR dimerization. In addition, optimized stochastic optical reconstruction microscopy (STORM) imaging showed that MS-444 significantly reduced the HuR dimerization, and HuR mainly existed in cluster form in 2-cell stage embryos. In conclusion, this study provides clinical guidance for maintaining fertility during the treatment of cancer with MS-444 in women of reproductive age. And also, our research provides guidance for the application of STORM in nanometer scale studies of embryonic cells. HuR inhibitor MS-444 arrested embryonic development at 2-cell stage. Low-input RNA-seq revealed that Agbl2 was the target gene of MS-444. MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization and reduced the stability of Agbl2 mRNA. STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos.

A novel homozygous mutation in the PADI6 gene causes early embryo arrest

BackgroundIt has been proved that mutations in the PADI6 gene can cause early embryo arrest. This study describes a newly discovered mutation in PADI6 that expands the genetic spectrum of early embryo arrest.MethodsPeripheral blood of a patient diagnosed with early embryo arrest was collected for whole-exome sequencing. Sanger sequencing was performed to confirm this mutation. The effects of the variant were investigated in human embryonic kidney 293T (HEK293T) cells using western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence.ResultsA novel homozygous mutation in PADI6 was identified in the proband. The patient carried a frameshift insertion mutation c.558dupA (p.Thr187Asnfs*48), which was located in the protein arginine deiminase middle domain. The variant destroyed PADI6 protein expression and reduced PADI6 mRNA expression in HEK293T cells.ConclusionsThe newly identified mutation in PADI6 accounts for early embryo arrest. It expands the spectrum of genetic causes and phenotypes of infertility in humans. These findings also provide an additional possible diagnostic marker for patients with recurrent in vitro fertilization/intracytoplasmic sperm injection failure.

Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state

Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos.

O-100 Incidence, morphology, ploidy and developmental arrest in direct cleavage embryos: what are they teaching us? Morphokinetic analysis of 15.081 embryos

Abstract Study question What is the impact of direct cleavage (DC) occurrence during embryo/blastocyst development regarding morphology grade, embryo arrest and ploidy status? Summary answer Blastocyst formation and morphology grade are significantly reduced in DC when compared to non-DC embryos; however, there is no difference in blastocyst ploidy status. What is known already Chromosome instability leading to aneuploidy during early cleavage is well known in humans. DC is defined as the abrupt cleavage of one into three or more daughter blastomeres as the result from an irregular mitosis. Three or more mitosis poles can impair early embryo development; however, how some embryos with these irregular divisions might be able to develop into expanded blastocysts is still unknown. Time-lapse system provides an uninterrupted evaluation of embryo morphological and dynamic parameters, allowing our understanding in such events and if those embryos may be associated with a self-correction mechanism to reach blastocyst stage. Study design, size, duration Retrospective cohort study from 15.081 embryos, 3.962 cycles and 3.106 patients that were undergoing in vitro fertilization (IVF) treatment according to medical referral in a single private ART center from December 2018 to November 2021. Only two-pronuclear (2PN) embryos cultured in time-lapse incubators (EmbryoScope, Vitrolife, Sweden) were included in the study. Direct cleavage occurance was annotated when a single blastomere directly cleaved into three or more daughter blastomeres. Participants/materials, setting, methods DC embryos were also classified in DC 1-3: abnormal cleavage occurred in zygote (1-cell) resulting in 3–4 blastomeres and in DC 2+: abnormal cleavage occurred at the 2-cell stage resulting in 5 or 6 blastomeres. Embryo arrest, blastocyst formation rate and morphology grade (Gardner and Schoolcraft, 1999) were annotated. Blastocyst biopsy reports for ploidy status were also considered for analysis. Mann-Whitney, chi-squared and Fisher tests were applied for statistical analysis. p < 0,05 was considered significant. Main results and the role of chance There were 1168 DC embryos (7,7%) from 896 cycles/841 patients; 502 DC1-3 (3,3%) from 391 cycles/366 patients and 666 DC2+ embryos (4,4%) from 475 patients/505 cycles. In DC group, around 21% of patients have the incidence of more than 1 DC embryo per cycle. Maternal age was lower in DC groups: 38,0±3,61 (DC1-3) and 38,60±3,21 (DC2+) years old versus not-DC (39,02±3,01, p < 0,0001). Embryo arrest was higher (DC1-3:85,5% and DC2+:56,8% versus not-DC:35,4%, p < 0,0001) and blastocyst formation rate was lower (14,5% and 43,2% versus 64,6%, p < 0,0001) in both DC groups and also between DC groups (p < 0,0001). There were 4124 biopsied not-DC blastocysts, 24 in DC1-3 and 154 in DC2+. There was no difference in ploidy between not-DC (57,9%, 40,2%, 1,9%, aneuploid, euploid and mosaic respectively) and DC groups (54,2%, 41,7%, 4,2% and 61%, 37% and 1,9% respectively, p = 0.86) and neither between DC-groups (p = 0.69). Inner cell mass morphology grades were distinct in all groups (not-DC A:45,8%, B:45,2% C:9,0%, DC1-3 A:35,6%, B:33,9%, C:30,5% and DC2+ A:23,1%, B:57,0% C:19,9%, p < 0,0001) and between DC-groups (p < 0,0001). Trophoectoderm morphology grades were also higher for not-DC group (not-DC A:30,5%, B:48,9% C:20,6%, DC1-3 A:15,3%, B:47,5%, C:37,3% and DC2+ A:13,6%, B:44,4% C:42,0%, p < 0,0001), however there was no difference between DC-groups (p = 0.80). Limitations, reasons for caution Limited data are available on the implantation potential of embryos with DC, as they are not prioritize for transfer, although our study did not demonstrated a higher aneyploidy rate. Patient parameters, with the exception of maternal age, were not considered in this study. Wider implications of the findings Although the morphology grades are lower and embryo arrest is higher in DC embryos, probably due to unequal cleavage, if they reach blastocyst stage, the ploidy status of these embryos are similar to not-DC embryos, speculative for a self-correction mechanism. Future steps should focus on their performance in pregnancy/live-birth. Trial registration number Not Applicable

P-184 The effect of oocyte vitrification accounting for both open and closed systems on embryo developmental arrest rate. A systematic review and network meta-analysis

Abstract Study question To investigate the effect of oocyte vitrification on embryo developmental arrest rate accounting for both open and closed systems. Summary answer Open and closed vitrification systems are equally associated with a statistically significant higher embryo developmental arrest rate per MII oocyte vitrified compared to fresh oocytes. What is known already Oocyte cryopreservation has increased in popularity as it enhances women’s reproductive autonomy. Numerous studies have been published evaluating its effectiveness. However, the majority of published evidence commonly include comparisons with the now considered as “outdated” method of slow freezing. Additionally, data principally report on fertilization rates and clinical outcomes. It may be timely and essential to focus strictly on the effect of oocyte vitrification on the developmental potential of the embryo. Further to this, data are lacking on whether employing an open or closed vitrification system may affect the outcome of vitrification. Study design, size, duration A systematic search of the literature was performed in the databases Pubmed/Medline, Embase, and Cochrane Central Library limited to articles published in English up to October 2021. Only studies employing vitrification were included in this meta-analysis. A total of 17 published prospective studies were eligible. The population consists of oocytes that were either vitrified or fresh and subjected to ICSI. A network meta-analysis was performed comparing the type of vitrification system employed and fresh oocytes. Participants/materials, setting, methods The primary outcome measure was developmental arrest rate per MII oocyte vitrified prior to reaching cleavage or blastocyst stage. The secondary outcome measures were fertilization rate per MII oocyte vitrified and developmental arrest rate per 2PN zygote. Further to this, a subgroup analysis was performed according to the stage of developmental arrest. To rank the efficiency between the fresh oocytes and the oocytes vitrified employing the open and closed system, the P-Score was employed. Main results and the role of chance The seventeen studies reporting on the effect of oocyte vitrification on embryo developmental arrest per MII oocyte vitrified, presented with high heterogeneity I2=81%. Vitrified oocytes employing either the open or closed vitrification system presented with a statistically higher embryo developmental arrest rate when compared to fresh oocytes (open-systems:RR:1.16; 95%CI:1.07-1.26; closed-systems:RR:1.19 95%CI:1.06-1.34). No statistically significant difference was observed between the two vitrification systems (open vs closed:RR:0.99;95%CI:0.89-1.10). Subgroup analysis was performed according to the developmental stage of embryo arrest. Similarly to the pooled results, when subgrouping for embryos arresting prior to the cleavage stage, a statistically significant difference on developmental arrest was identified when vitrifying (open-systems:RR:1.44; 95%CI:1.18-1.77; closed-systems:RR:1.51 95%CI:1.12-2.04; 8 studies). However, when subgrouping for embryos arresting prior to the blastocyst stage, no statistically significant difference on developmental arrest was observed when vitrifying (open-systems:RR:1.06; 95%CI:0.98-1.15; closed systems:RR:1.10 95%CI:0.98-1.24; 9 studies). Fertilization rate was significantly lower for vitrified oocytes compared to fresh (open-systems:RR:0.86; 95%CI:0.79-0.93; closed-systems:RR:0.81 95%CI:0.72-0.92), while no statistically significant difference was observed between the two vitrification systems (open vs closed:RR:1.04; 95%CI:0.93-1.16). When comparing developmental arrest rate per 2PN zygote no statistically significant difference was detected between vitrification versus fresh (open-systems:RR:1.01; 95%CI:0.87-1.17; closed-systems:RR:0.98 95%CI:0.78-1.22), or between the two vitrification systems (open vs closed:RR:1.03;95%CI:0.82-1.30). Limitations, reasons for caution The limited number of studies included along with the heterogeneity identified present as limitations of this study. Further studies and especially Randomized Controlled Trials should be conducted in order to evaluate possible effects of oocyte vitrification on embryo development. Wider implications of the findings Oocyte vitrification results to higher developmental arrest rates per oocyte vitrified but not per 2PN zygote. Both vitrification systems perform equally in affecting developmental arrest. The differential expression of miRNAs and cytokinesis-related genes are identified by this systematic review as potential pathways influencing developmental potential following oocyte vitrification. Trial registration number Not applicable

P-703 Developing individualized treatment strategies using detailed subgroup analysis to optimise pregnancy outcomes for women 40 and above

Abstract Study question What are the prognostic factors and treatment strategies for women 40 and above within different ovarian reserve subgroups? Summary answer Women in each ovarian reserve subgroup have different prognostic factors of pregnancy success and specific treatment strategies are needed for each subgroup. What is known already The number of women 40 and above seeking subfertility treatment is increasing and many are willing to undergo treatment despite extremely poor prognosis. POSEIDON criteria suggested the usage of ovarian reserve and female age as a basis for the study of the management of women in different prognostic categories. However, this classification may not be detailed enough to lead to specific recommendations for individualised management in women 40 and above, as there is rapid decline of oocyte quality and quantity. Large observational studies may lack finer detail due to challenges in recruiting women within different ovarian reserve subgroups. Study design, size, duration We carried out a detailed retrospective analysis of 382 IVF/ICSI cycles for 253 women from January 2019 to December 2021 in a special clinical and academic program for reproductive ageing and fertility. Inclusion criteria were all women who had IVF/ICSI cycles aged 40 and over. Analysis was done for all fresh cycles including cancelled cycles, failed fertilization, no eggs collected and embryo arrest. Cycles resulting in freezing of all embryos were excluded in analysis. Participants/materials, setting, methods The participants were divided into three main subroups: Group A, “normal ovarian reserve” (AMH 2.5-29.9pmol/l, n = 271 cycles); Group B, “extremely poor ovarian reserve” (AMH <2.5pmol/l, n = 85 cycles); and Group C “extremely high ovarian reserve” (PCOS or AMH ≥30.0pmol/l, n = 26 cycles). Chi-square and Mann-Whitney U tests were used to compare pregnant and non-pregnant groups for nominal and continuous data respectively. Multi-variate logistic regression was performed when indicated. All descriptive statistics are stated in median and percentages. Main results and the role of chance In Group A, univariate analysis showed that pregnancies were associated with lower female (41 vs 42) and male age (40 vs 43.5), administering long agonist versus antagonist protocol (73% vs 27%), the absence of androgen pretreatment (12% vs 88%), higher oestradiol level (7681pmol/l vs 6114pmol/l), higher number of oocytes retrieved (8 vs 4) and normally fertilized (4 vs 3), and embryo transfer on day 3 compared to 2 or 5 (all p < 0.05). The use of combined FSH/LH vs FSH only for ovarian stimulation given for 12 days at 300IU daily, higher endometrial thickness (11.3mm vs 10.6mm), IVF versus ICSI (65% vs 35%), and addition of subcutaneous progesterone for luteal support versus vaginal progesterone only (51% vs 43%) were trending towards but were not significant (p > 0.05). Multivariate logistic regression analysis revealed lower female and male age, less number of IVF cycles, and higher number of embryos transferred as independent factors contributing to pregnancy. In Group B, significant predictors of pregnancy included younger female age, higher gravidity, previous successful IVF and higher number of eggs fertilized (p < 0.05). In Group C, factors that contributed to positive pregnancy outcome included antagonist cycles versus agonist and usage of FSH and LH versus FSH alone (p > 0.05). Limitations, reasons for caution The retrospective nature of the study design limits control of potential confounding variables. Although there were clear trends, in a few sub-categories there were insufficient numbers to reach statistical significance. Continuing data collection is necessary to achieve adequate power for smaller groups especially Group B and C. Wider implications of the findings This detailed analysis will allow us to better understand the prognostic factors in women 40 and above, especially for women with extremes of ovarian reserve, who may benefit substantially from individualised care. We recommend standardisation of data collection and development of artificial intelligence algorithms to streamline management in the future. Trial registration number N/A

P-597 The effects of triple trigger on IVF outcomes and pregnancy rates in poor responder patients

Abstract Study question Are there any positive effects of triple trigger on oocyte morphology, embryo quality and pregnancy rates in poor responder (POR) patients? Summary answer The trigger with human chorionic gonadotropin (hCG), gonadotropin-releasing hormone agonist (GnRHa) and human menopausal gonadotropin (hMG) may improve embryo quality and pregnancy rates in POR. What is known already The ESHRE consensus criteria define a poor ovarian response (POR) as less than four oocytes retrieved. Advanced female age and severe sperm disorder were associated fertilization failure after intracytoplasmic sperm injection (ICSI). Also, oocyte quality is pivotal to determine the fertilization of the oocyte. Hence there is no study to evaluate triple trigger on the embryo quality in POR and severe male infertility are excluded. Study design, size, duration In this prospective randomized study, we analyzed poor responder patients that have history of fertilization failure and cleavage arrest embryo, including 100 patients with only hCG trigger (group 1), 100 patients with dual trigger hCG + GnRHa (Group 2), and 100 patients with triple trigger hCG + GnRHa + hMG (Group 3). Patients who have the POR criteria according to MII oocytes lower than 4 were included in the analysis. Participants/materials, setting, methods Mean age, body mass index (BMI), AMH levels, day-2 FSH and E2, duration of infertility, LH and E2 levels on trigger day, duration of stimulation, total dose of gonadotropins used, number of previous trials, MII oocytes were examined and compared between the groups. Sperm concentration under 5 x 106 mL was not included to exclude severe male factor. Embryo quality, pregnancy rates evaluation was performed for each patient that has been performed GnRH antagonist protocols. Main results and the role of chance Age, Body Mass Index (BMI), AMH levels, day 2 FSH and E2, duration of infertility, duration of stimulation, E2 levels on trigger day, and total dose of gonadotropins usage between the three groups were similar. Number of MII oocytes, fertilization (2PN), and D5 (blastocyst) embryo transfer were significantly higher in Group 3 (Table 1). Pregnancy rates are higher in group 3 however significantly not significant. Limitations, reasons for caution The study is limited by its sample size and a higher sample size might be used in the future studies to corroborate the current findings. hMG trigger dosage may be arranged in another study. Wider implications of the findings Our results showed that the number of MII oocytes 2PN levels, D5 ET were higher in DOR patients triggered with hCG + GnRHa + hMG. Trial registration number 28012022

Application of thromboelastography in diagnosing normal pregnancies and pregnancies with complications.

BackgroundThis observational study aimed to compare the potential application of thromboelastography (TEG) in diagnosing women with normal pregnancy (NP) and women with threatened abortion (TA), missed abortion (MA), embryo arrest (EA), fetal death (FD), history of abnormal pregnancy (HAP), and antiphospholipid antibody syndrome (AA).MethodsAccording to the relevant clinical criteria, patients were divided into groups, and their blood samples were subjected to TEG. Next, the parameters R, K, α‐angle, MA, LY‐30, G, and coagulation index (CI) were analyzed. Partial correlation analysis was used to analyze correlation between groups of data. LSD‐t test and Dunnett's T3 test were used to analyze continuous variables. Ordinal categorical variables were compared using ordinal logistic regression analysis and estimate odds ratio of risk factors. A receiver operating characteristic (ROC) curve was constructed to detect the ability of TEG to recognize various parameters, and areas under the curve were compared using Delong's test for diagnosing pregnancy‐related diseases.ResultsMA had a negative effect on the MA parameter in TEG; EA had a negative effect on the MA and G parameters; HAP had a negative effect on the CI parameter and a positive effect on the R parameter; AA had a negative effect on the CI parameter. Compared with that of the NP group, the G of the EA (p = 0.014) group and the CI of the TA (p = 0.036) MA (p = 0.08) EA (p = 0.026) HAP (p = 0.000004) and AA (p = 0.002) groups were reduced. In the ordinal logistic regression analysis, compared with that of the NP group, the high R value of the HAP group accounted for more than that of the NP group (OR = 48.76, p = 0.001); the high K value of the AA group accounted for more than that of the NP group (OR = 17.00, p = 0.023); the angle value distributions of the TA and AA groups were different from that of the NP group (OR = 3.30, p = 0.039; OR = 0.14, p = 0.029); the low MA value of the MA, EA, and HAP groups accounted for more than that of the NP group (OR = 0.16, p = 0.03; OR = 0.26, p = 0.005; OR = 0.11, p = 0.008); and the low CI value of the HAP group accounted for more than that of the NP group (OR = 0.09, p = 0.005). In the ROC analysis, there were no significant differences in the TEG parameters of pregnant women belonging to the NP and TA, NP and MA, NP and EA, NP and FD, NP and HAP, and NP and AA groups (p > 0.05).

Surface functionalization of poly(dimethylsiloxane) substrates facilitates culture of pre-implantation mouse embryos by blocking non-selective adsorption.

Poly(dimethylsiloxane) (PDMS) is widely used in biomedical settings such as microfluidics for its optical transparency, castability, gas permeability and relative biocompatibility. While PDMS devices with certain modifications or treatments have been used for mammalian pre-implantation embryo culture, it is unclear why native PDMS leads to significant embryo death. In this study, we employ Nile Red as a model hydrophobic small molecule to demonstrate that significant hydrophobic sequestration occurs on native PDMS substrates even with a bovine serum albumin-containing KSOM pre-equilibration. Our results suggest that this small molecule sequestration has detrimental effects on mouse embryo development in PDMS static culture wells, with 0% blastocyst development rates from embryos cultured on native PDMS. We found that prior saturation of the PDMS culture well with water vapour only rescues about 10% of blastocyst development rates, indicating osmolality alone is not responsible for the high rates of embryo arrest. We also present a safe and simple Pluronic F127 pretreatment for PDMS substrates that successfully circumvented the harmful effects of native PDMS, achieving a blastocyst and implantation rate akin to that of our polystyrene controls. Our results call into question how researchers and clinicians can account for the alterations in medium composition and embryo secretions when using hydrophobic substrates, especially in the mammalian embryo culture setting where minimum effective concentrations of peptides and amino acids are commonplace.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

SummaryEmbryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

Functional characterization of a 'plant-like' HYL1 homolog in the cnidarian Nematostella vectensis indicates a conserved involvement in microRNA biogenesis.

While the biogenesis of microRNAs (miRNAs) in both animals and plants depends on the RNase III Dicer, its partner proteins are considered distinct for each kingdom. Nevertheless, recent discovery of homologs of Hyponastic Leaves1 (HYL1), a 'plant-specific' Dicer partner, in the metazoan phylum Cnidaria, challenges the view that miRNAs evolved convergently in animals and plants. Here, we show that the HYL1 homolog Hyl1-like a (Hyl1La) is crucial for development and miRNA biogenesis in the cnidarian model Nematostella vectensis. Inhibition of Hyl1La by morpholinos resulted in metamorphosis arrest in Nematostella embryos and a significant reduction in levels of most miRNAs. Further, meta-analysis of morphants of miRNA biogenesis components, like Dicer1, shows clustering of their miRNA profiles with Hyl1La morphants. Strikingly, immunoprecipitation of Hyl1La followed by quantitative PCR revealed that in contrast to the plant HYL1, Hyl1La interacts only with precursor miRNAs and not with primary miRNAs. This was complemented by an in vitro binding assay of Hyl1La to synthetic precursor miRNA. Altogether, these results suggest that the last common ancestor of animals and plants carried a HYL1 homolog that took essential part in miRNA biogenesis and indicate early emergence of the miRNA system before plants and animals separated.

Adverse pregnancy outcomes among pregnant women living with HIV in Hubei province, China: prevalence and risk factors

ABSTRACT Mother-to-child transmission of Human Immunodeficiency Virus (HIV) has been greatly reduced with the advance of intervention technology. However, adverse pregnancy outcomes (APOs) are still common, and little is known about the driving forces of APOs among pregnant women living with HIV in China. Between January 2004 and December 2020, a total of 638 pregnancies among pregnant women living with HIV were enrolled in this study, 84 (13.2%) pregnancies with 87 APOs were reported. Preterm birth (3.8%), ectopic pregnancy (3.4%), spontaneous abortion (2.0%), and embryo arrest (1.7%) were the most common APOs in pregnant women living with HIV. Exposure to antiretroviral drugs (ARVs) during the first trimester (RR = 4.077, 95% CI: 0.521, 1.484, P<0.001) and the first CD4+ T lymphocyte count (CD4 count)≤ 350/μl (RR = 2.227, 95% CI: 0.063, 0.991, P = 0.026) were risk factors of APOs. The age≤ 30 years (RR = −2.513, 95% CI: −1.067, −0.132, P = 0.012) was associated with the decreasing of APOs. Encouraging people to initiate combination antiretroviral therapy and reach a high CD4 count level before pregnancy would be helpful to prevent APOs. Pregnant women exposed to ARVs in the first trimester needed more attention for APOs.

Zinc oxide nanoparticle causes toxicity to the development of mouse oocyte and early embryo

Zinc oxide nanoparticle (ZnO NP) is one of the most widely used nanomaterial in industrial and commercial products. Here, we reported hazardous effects of ZnO NP on the development of mouse oocyte and pre-implantation embryo. ZnO NP compromises meiosis partially by induction of oxidative stress as antioxidant rescues the development of ZnO NP-exposed oocytes, albeit with limited efficiency. It causes mitochondrial- and endoplasmic reticulum stresss which thereby activates autophagy and apoptosis to trigger oocyte demise. Examining ZnO NP-exposed oocytes that complete M-phase entry witnesses a disruption in meiotic cytoskeleton architecture. Intriguingly, loss of Grp78, a chaperone in the ER, phenocopies ZnO NP-induced meiotic defects and cytoskeleton disorganization. Importantly however, ZnO NP commences cytotoxicity by more than releasing of Zn2+ in that ZnCl2 to a much less extent recapitulates ZnO NP-induced phenomena. The prevailing DNA damage is another causative to developmental arrest and degeneration of oocytes and early embryos, but compared with oocytes, embryos are more sensitive to ZnO NP and succumb to death. ZnO NP is demonstrated in this study to be toxic for oocytes and enbryos in mammals, which warrants careful evaluation of human exposure with regard to its influence on reproductive health.

Sperm-specific protein ACTL7A as a biomarker for fertilization outcomes of assisted reproductive technology.

Obtaining high-quality embryos is one of the key factors to improve the clinical pregnancy rate of assisted reproductive technologies (ART). So far, the clinical evaluation of embryo quality depends on embryo morphology. However, the clinical pregnancy rate is still low. Therefore, new indicators are needed to further improve the evaluation of embryo quality. Several studies have shown that the decrease of sperm-specific protein actin-like 7A (ACTL7A) leaded to low fertilization rate, poor embryo development, and even infertility. The aim of this study was to study whether the different expression levels of ACTL7A on sperm can be used as a biomarker for predicting embryo quality. In this study, excluding the factors of severe female infertility, a total of 281 sperm samples were collected to compare the ACTL7A expression levels of sperms with high and low effective embryo rates and analyze the correlation between protein levels and in-vitro fertilization (IVF) laboratory outcomes. Our results indicated that the ACTL7A levels were significantly reduced in sperm samples presenting poor embryo quality. Furthermore, the protein levels showed a significant correlation with fertilization outcomes of ART. ACTL7A has the potential to be a biomarker for predicting success rate of fertilization and effective embryo and the possibility of embryo arrest. In conclusion, sperm-specific protein ACTL7A has a strong correlation with IVF laboratory outcomes and plays important roles in fertilization and embryo development.

Current and future therapies for abnormal early embryogenesis with assisted reproductive technology

Each stage of embryonic development, including normal gamete maturation, fertilization, zygotic genome activation, and cleavage, is crucial for human reproduction. Early embryo arrest is a common phenomenon. It is estimated that about 40%–70% of human embryos are arrested at early developmental stages. However, the exact mechanism remains largely uncertain. Embryos can be investigated in vitro by way of the development of in vitro fertilization/intracytoplasmic sperm injection. In addition to iatrogenic factors related to abnormal oocyte/embryo development, multiple gene mutations have been found to be involved in such phenotypes. Based on the knowledge of known etiological factors, several therapies are proposed to improve clinical outcomes. Here, we shed light on current and potential therapies for treating these conditions through reviewing articles and combining with our clinical and research experience.

High Temperature After Anthesis Releases Embryo Arrest in the Mukaku Kishu–type Seedless Cultivar ‘Southern Yellow’

High Temperature After Anthesis Releases Embryo Arrest in the Mukaku Kishu–type Seedless Cultivar ‘Southern Yellow’

Inhibition of DRP1 Impedes Zygotic Genome Activation and Preimplantation Development in Mice.

Mitochondrion plays an indispensable role during preimplantation embryo development. Dynamic-related protein 1 (DRP1) is critical for mitochondrial fission and controls oocyte maturation. However, its role in preimplantation embryo development is still lacking. In this study, we demonstrate that inhibition of DRP1 activity by mitochondrial division inhibitor-1, a small molecule reported to specifically inhibit DRP1 activity, can cause severe developmental arrest of preimplantation embryos in a dose-dependent manner in mice. Meanwhile, DRP1 inhibition resulted in mitochondrial dysfunction including decreased mitochondrial activity, loss of mitochondrial membrane potential, reduced mitochondrial copy number and inadequate ATP by disrupting both expression and activity of DRP1 and mitochondrial complex assembly, leading to excessive ROS production, severe DNA damage and cell cycle arrest at 2-cell embryo stage. Furthermore, reduced transcriptional and translational activity and altered histone modifications in DRP1-inhibited embryos contributed to impeded zygotic genome activation, which prevented early embryos from efficient development beyond 2-cell embryo stage. These results show that DRP1 inhibition has potential cytotoxic effects on mammalian reproduction, and DRP1 inhibitor should be used with caution when it is applied to treat diseases. Additionally, this study improves our understanding of the crosstalk between mitochondrial metabolism and zygotic genome activation.