A transcriptomic examination of encased rotifer embryos reveals the developmental trajectory leading to long-term dormancy; are they "animal seeds"?

BACKGROUND: Organisms from many distinct evolutionary lineages acquired the capacity to enter a dormant state in response to environmental conditions incompatible with maintaining normal life activities. Most studied organisms exhibit seasonal or annual episodes of dormancy, but numerous less studied organisms enter long-term dormancy, lasting decades or even centuries. Intriguingly, many planktonic animals produce encased embryos known as resting eggs or cysts that, like plant seeds, may remain dormant for decades. Herein, we studied a rotifer Brachionus plicatilis as a model planktonic species that forms encased dormant embryos via sexual reproduction and non-dormant embryos via asexual reproduction and raised the following questions: Which genes are expressed at which time points during embryogenesis? How do temporal transcript abundance profiles differ between the two types of embryos? When does the cell cycle arrest? How do dormant embryos manage energy? RESULTS: As the molecular developmental kinetics of encased embryos remain unknown, we employed single embryo RNA sequencing (CEL-seq) of samples collected during dormant and non-dormant embryogenesis. We identified comprehensive and temporal transcript abundance patterns of genes and their associated enriched functional pathways. Striking differences were uncovered between dormant and non-dormant embryos. In early development, the cell cycle-associated pathways were enriched in both embryo types but terminated with fewer nuclei in dormant embryos. As development progressed, the gene transcript abundance profiles became increasingly divergent between dormant and non-dormant embryos. Organogenesis was suspended in dormant embryos, concomitant with low transcript abundance of homeobox genes, and was replaced with an ATP-poor preparatory phase characterized by very high transcript abundance of genes encoding for hallmark dormancy proteins (e.g., LEA proteins, sHSP, and anti-ROS proteins, also found in plant seeds) and proteins involved in dormancy exit. Surprisingly, this period appeared analogous to the late maturation phase of plant seeds. CONCLUSIONS: The study highlights novel divergent temporal transcript abundance patterns between dormant and non-dormant embryos. Remarkably, several convergent functional solutions appear during the development of resting eggs and plant seeds, suggesting a similar preparatory phase for long-term dormancy. This study accentuated the broad novel molecular features of long-term dormancy in encased animal embryos that behave like "animal seeds".

Evaluation of fragmented embryos implantation potential using time-lapse technology.

AIM: To examine the implantation potential of fragmented embryos that underwent morphokinetic evaluation in a time-lapse incubator. METHODS: A retrospective study analyzing 4210 Day 5 embryos which were incubated in a time-lapse incubator, between 2013 and 2019. Embryos with more than 5% fragmentation (379 embryos) were included in the study. Embryos selected using the general model and re-examined by our in-house model. Embryo fragmentation percentage was documented from the first cell-division (start fragmentation) to its maximal percentage (final fragmentation), and the ratio between them (fragmentation worsening). Data were analyzed with relation to embryo development, embryos transfer or freezing, clinical pregnancy, and live birth rates. RESULTS: Embryo fragmentation and morphokinetics were found to be independent variables for clinical pregnancy achievements. A higher fragmentation worsening was noted among discarded embryos compared to transferred or frozen embryos (p < 0.0001). Advanced maternal age had a significant negative effect on fragmentation (p < 0.001). Missed abortion rates were similar in fragmented embryos that implanted compared with the overall population. Live birth rates were comparable among embryos which were selected for transfer or freezing by their morphokinetics and had different severity of fragmentation. CONCLUSION: Our study shows that fragmented embryos have a potential to implant and therefore should be selected for transfer. Laboratories which do not use time-lapse incubators for embryo selection, should consider transferring fragmented embryos, since they have an acceptable chance for live birth. Calculation of fragmentation worsening may enhance our ability to predict embryo development. Further research with analysis of more fragmented embryo maybe beneficial. This study was approved by the local ethics committee No. 0010-19 CMC on April 18th, 2019.

Should Intra Cytoplasmic Sperm Injection (ICSI) be the primary insemination method in women undergoing IVF cycles with donor sperm?

PURPOSE: To compare efficacy of Intra Cytoplasmic Sperm Injection (ICSI) with conventional in vitro fertilization (IVF) on treatment outcome in women undergoing in vitro fertilization with donor sperm. METHODS: We examined retrospectively the outcome data from 203 patients undergoing fresh cycles of conventional IVF (cIVF) or ICSI and an additional 77 frozen-thawed embryo transfer (FET) cycles during 2003-2014, all using donor sperm. Fertilization, cleavage, pregnancy and live birth rates and number of high-quality embryos were compared between cIVF and ICSI. RESULTS: Altogether 185 women underwent 479 transfer cycles of fresh embryos (237 cIVF vs. 224 ICSI and 18 "rescue ICSI" cycles). In addition, 77 FET cycles were compared (24 cIVF vs. 53 ICSI cycles). No differences were found between cIVF and ICSI in fertilization, cleavage, pregnancy and live birth rates (92.6% vs 92.2%, 73.4% vs 72.4%, 25.3% vs 27.2% and 13.1% vs 14.7%, respectively). Pregnancy and life birth rates remained similar even when FET cycles were included (25.8% vs 26.2% and 13.1% vs 13.7%, respectively). The use of ICSI was associated with lower rates of high-quality embryos (52.7% vs. 63.3%, P < 0.0001). A multivariate logistic regression analysis found that patients' age, number of transferred embryos and smoking were independently associated with the chance to conceive. Patient age correlated inversely with fertilization rate (r = - 0.13, P < 0.006).Non-smokers were more likely to become pregnant (OR = 2.23, P < 0.012). CONCLUSIONS: Our results show that ICSI does not bypass the age-related decrease in oocyte quality in patients using donor sperm for IVF. Use of ICSI was associated with lower rates of high-quality embryos. The findings imply that ICSI should not be the primary method of insemination in patients undergoing IVF with donor sperm.

Comparison of day 2 and overnight day 3 frozen embryo transfers: A prospective randomized controlled trial.

In certain patients cleavage stage embryos may be preferred. The relationship between an additional day in culture and pregnancy outcomes is not well established. We aimed to compare outcomes of day 2 versus overnight day 3 frozen embryo transfer (FET). In this randomized controlled trial, patients with day 2 cryopreserved embryos were allocated to two groups. In group A embryos were transferred on day 2, the same day of thawing. In group B embryos were transferred one day after thawing, on day 3 after overnight incubation. Out of 410 patients eligible, 92 were recruited. Finally, 72 patients participated, 39 in group A and 33 in group B. No significant difference in implantation (11 % in group A and 14 % in group B, p = 0.81), clinical pregnancy (18 % in group A and 21 % in group B, p = 0.73) or live birth rates (13 % in group A and 18 % in group B, p = 0.53) was found. To conclude, no significant difference in reproductive outcomes was found when comparing patients with day 2 or overnight day 3 FET. Considering published data on blastocyst transfer, cleavage stage ET may still be a relevant option and the decision between day 2 or overnight day 3 ET depends on patients' and physicians' preference and recommendation.

Improving embryo selection by the development of a laboratory-adapted time-lapse model.

OBJECTIVE: To study whether a powerful, in-house, embryo-selection model can be developed for a specific in vitro fertilization (IVF) laboratory where embryos were already selected for transfer using general models. DESIGN: In total, 12,944 fertilized oocytes were incubated in an EmbryoScope (Vitrolife, Göteborg, Sweden) at our laboratory. Embryos were selected for transfer or freezing using general models. There were 1,879 embryos with known implantation data (KID), of which 425 had positive KIDs. For the outcome, we set 3 endpoints for KID's definition: gestational sac, clinical pregnancy, and live birth. Results of a comparison between KID-positive and -negative embryos for cell division timings were analyzed separately for intracytoplasmic sperm injection (ICSI) and IVF embryos in patients aged 18-41 years. SETTING: IVF center. PATIENTS: The study included 1,075 women undergoing IVF or ICSI treatment between June 2013 and February 2019. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The KID-positive and -negative embryos were analyzed for statistical differences in cell division timing and cell cycle intervals. We used the EmbryoScope Stats software (Unisense FertiliTech, Aarhus, Denmark) for model development. The statistically different timing parameters were tested for their contribution to scoring in the model. The algorithms were tested for area under the receiver operating characteristic curve (AUC) in the KID embryos for developing day-2, -3, and -5 embryo-selection models. The validation of these algorithms was performed using calibration/validation procedures. RESULTS: Because significant differences in morphokinetics were found between the KID-positive and KID-negative embryos in our laboratory, it was possible to use our specific KID data to develop an in-house model. The algorithms were developed for embryo selection on days 2, 3, and 5 in the ICSI embryos. In most cases, AUC was >0.65, which indicated that these models were valid in our laboratory. In addition, these AUC values were obtained from all gestational sac, clinical pregnancy, and live birth KID embryo databases tested. An increase in the predictability of the models was observed from days 2-3 to day 5 models. The AUC test results ranged between 0.657 and 0.673 for day 2 and day 3, respectively, and 0.803 for the day 5 model. CONCLUSION: A model based on laboratory-specific morphokinetics was found to be complementary to general models and an important additive tool for improving single embryo selection. Developing an in-house laboratory-specific model requires many stages of sorting and characterization. Many insights were drawn about the model developing process. These may facilitate and improve the process in other laboratories.

Dormancy in Embryos: Insight from Hydrated Encysted Embryos of an Aquatic Invertebrate.

Numerous aquatic invertebrates remain dormant for decades in a hydrated state as encysted embryos. In search for functional pathways associated with this form of dormancy, we used label-free quantitative proteomics to compare the proteomes of hydrated encysted dormant embryos (resting eggs; RE) with nondormant embryos (amictic eggs; AM) of the rotifer Brachionus plicatilisA total of 2631 proteins were identified in rotifer eggs. About 62% proteins showed higher abundance in AM relative to RE (Fold Change>3; p = 0.05). Proteins belonging to numerous putative functional pathways showed dramatic changes during dormancy. Most striking were changes in the mitochondria indicating an impeded metabolism. A comparison between the abundance of proteins and their corresponding transcript levels, revealed higher concordance for RE than for AM. Surprisingly, numerous highly abundant dormancy related proteins show corresponding high mRNA levels in metabolically inactive RE. As these mRNAs and proteins degrade at the time of exit from dormancy they may serve as a source of nucleotides and amino acids during the exit from dormancy. Because proteome analyses point to a similarity in functional pathways of hydrated RE and desiccated life forms, REs were dried. Similar hatching and reproductive rates were found for wet and dried REs, suggesting analogous pathways for long-term survival in wet or dry forms. Analysis by KEGG pathways revealed a few general strategies for dormancy, proposing an explanation for the low transcriptional similarity among dormancies across species, despite the resemblance in physiological phenotypes.

Oxidative markers in cryopreservation medium from frozen-thawed embryos: a possible tool for improved embryo selection in in vitro fertilization?

PURPOSE: The present study evaluated the association between oxidative parameters in embryo cryopreservation medium and laboratory and clinical outcomes. METHODS: This prospective laboratory study was conducted in an IVF unit in a university-affiliated hospital with 91 IVF patients undergoing a frozen-thawed embryo transfer cycle. Following thawing, 50 µL of embryo cryopreservation medium was retrieved from each cryotube and tested by the thermochemiluminescence (TCL) assay. TCL amplitudes after 50 (H1), 150 (H2), and 280 s (H3) were recorded in counts per second (CPS) and the TCL ratio determined for comparison with implantation and pregnancy rates. RESULTS: A total of 194 embryos were transferred in 85 frozen-thaw cycles. Twenty-one pregnancies (24.7 %) occurred. Implantation and overall and clinical pregnancy rates were higher when the median TCL H1 amplitude was <32 CPS compared to ≥32 CPS (14.6 vs. 5.3 %, 37.5 vs. 17 %, 28.1 vs. 9.4 %, respectively). No pregnancies occurred when the H1 amplitude was ≥40 CPS. Logistic regression multivariate analysis found that only the median TCL H1 amplitude was associated with the occurrence of pregnancy (OR = 2.93, 95 % CI 1.065-8.08). The TCL ratio inversely correlated with the duration of embryo cryopreservation (r = -0.37). CONCLUSIONS: The results indicate that thawed embryos may express oxidative processes in the cryopreservation medium, and higher oxidative levels are associated with lower implantation rates. These findings may aid in the improved selection of frozen-thawed embryos for IVF.

[CHALLENGING THE OPTIMAL NUMBER OF RETRIEVED OOCYTES AND ITS IMPACT ON PREGNANCY AND LIVE BIRTH RATES IN IVF/ICSI CYCLES].

INTRODUCTION: Large numbers of retrieved oocytes are associated with higher chances of having cryopreservation of embryos. However, the process entailed exposes women to increased risk for ovarian hyperstimulation syndrome. Furthermore, mild ovary stimulation protocols are more patient-friendly and with less adverse effects. Only limited reports exist on the significance of the number of retrieved oocytes achieved in a single stimulation cycle. AIM: To investigate the optimal number of retrieved oocytes to achieve pregnancy and live birth. METHODS: This retrospective analysis included 1590 IVF cycles. Oocytes maturation, fertilization, cleavage, as well as pregnancy and live birth rates were analyzed according to the number of retrieved oocytes. RESULTS: Oocyte maturation, fertilization and cleavage rates were lower in cycles with more than 10 retrieved oocytes compared with other groups. Live birth rates were highest when the number of retrieved oocytes was 11-15. CONCLUSIONS: Retrieval of more than 15 oocytes was not associated with a significant increase in chances of conception and birth. DISCUSSION: The better oocyte quality with 10 or less oocytes retrieved could be the result of a possible interference with the natural selection, or the minimized exposure of growing follicles to the potentially negative effects of ovarian stimulation. Although the average number of available embryos was higher when more than 10 oocytes were retrieved, achievement of more than 15 oocytes did not improve IVF outcome in terms of pregnancy and delivery rates. SUMMARY: Analysis of 1590 IVF cycles including the frozen-thawed transfers shows that the best outcomes were achieved with an optimal number of 11-15 oocytes.

Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells.

Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.

Long-term impact of early olfactory experience on later olfactory conditioning.

This study examined the duration of the effect of early olfactory experience in rats by determining the ease of conditioning and then reconditioning to an early-experienced odor. Rat pups (experimental group) were exposed to aniseed odor sprayed on the mother's belly from day 1 to 20 after birth. A control group was exposed only to water. At the ages of 21 and 40 days all the rats (experimental and control) were tested for preference for the odor of aniseed. Starting from day 41 after birth they were conditioned in a Y-maze to approach the odor of aniseed for a reward. We then divided both groups into five subgroups each. Each subgroup was retrained to approach aniseed after 5, 6, 7, 8 or 9 months, and their speeds of reconditioning to the odor were compared. The results showed that all rats in the early exposed group had remembered the odor and did not require reconditioning, unlike those in the group that had not had the early olfactory conditioning. The effect of the early experience was still detectable at least 5 months after last exposure to the odor.