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PURPOSE: Preimplantation genetic screening (PGS) and assessment of mitochondrial content (MC) are current methods for selection of the best embryos for transfer. Studies suggest that time-lapse morphokinetics (TLM) may also be helpful for selecting embryos more likely to implant. In our study, we sought to examine the relationship between TLM parameters and MC to determine if they could be used adjunctively in embryo selection. We also examined the relationship between MC with ploidy and blastulation. METHODS: Cryopreserved human embryos at the zygote stage were thawed and cultured in a time-lapse system. Blastomere and trophectoderm biopsies were performed on days 3 and 6. Biopsied cells and all whole embryos from day 6 were analyzed for MC (ratio of mitochondrial to nuclear DNA) and ploidy using next-generation sequencing. RESULTS: In embryos, MC per cell declined between day 3 and day 6. While early cleavage parameters did not predict MC, embryos with longer blastulation timing had higher MC on day 6. Day 6 MC was lower in euploid vs. aneuploid embryos and lower in blastocysts vs. arrested embryos. CONCLUSIONS: A lower MC at the blastocyst stage was associated with euploid status and blastocyst formation, indicating better embryo quality compared to those with a higher MC. Higher MC in aneuploid and arrested embryos may be explained by slower cell division or degradation of genomic DNA over time. Blastulation timing may be helpful for selection of higher quality embryos. Combining blastulation timing and MC along with morphologic grading and euploid status may offer a new direction in embryo selection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1113-9) contains supplementary material, which is available to authorized users.

PURPOSE: The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. METHODS: Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 μl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. RESULTS: The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P

PURPOSE: DNA methylation is an epigenetic mechanism that plays critical roles during mammalian oocyte and preimplantation embryo development. It is achieved by adding a methyl group to the fifth carbon atom of cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites using DNA methyltransferase (DNMT) enzymes for de novo and maintenance methylation processes. DNMT1, DNMT3A, and DNMT3B play important roles in establishing methylation of developmentally related genes in oocytes and early embryos. The purpose of this study is to identify the effect of superovulation on the expression and subcellular localizations of these three DNMT enzymes in the mouse oocytes and early embryos. METHODS: Three groups composed of control, normal dose [5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG)], and high dose [7.5 IU PMSG and 7.5 IU hCG] were created from 4–5-week-old female BALB/c mice. The relative expression and subcellular localizations of the DNMT proteins in the control and experiment groups have been characterized by using immunofluorescence staining subsequently analyzed in detailed. RESULTS: DNMT1, DNMT3A, and DNMT3B protein expression in the germinal vesicle and metaphase II oocytes and in one-cell and two-cell embryos differed significantly when some of the normal- and high-dose groups were compared with the control counterparts. CONCLUSION: This study has demonstrated for the first time that superovulation alters expression levels of the DNMT proteins, a finding that indicates that certain developmental defects in superovulated oocytes and early embryos may result from impaired DNA methylation processes.

This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. Culture mouse zygotes to two-cell embryos and test effects after 0.5–1 h AMPK agonists’ (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40–85% in two-cell embryos that was ~ 60–90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30–50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

The purpose of this study is to explore the factors associated with embryo multinucleation, particularly focused on the influence of parental chromosomal polymorphisms in embryo multinucleation. This is a retrospective case-control study involving 1260 infertile couples undergoing their first IVF/ICSI cycles. Couples were screened for abnormalities in their karyotype and were evaluated for blastomere persistence of multinucleation. Demographic characteristics, stimulation protocol, and pregnant outcomes were analyzed using logistic regression analysis. The level of basal FSH was lower in the multinucleated embryos group (5.37 vs 5.72 IU/L). The Multinucleated embryos group received less gonadotropins (1788.5 vs 1891.3 IU), and the level of LH on day of HCG triggering was lower (1.09 vs 1.30 IU/L). More oocytes were recovered in the multinucleated embryos group (11.51 vs 9.23). Chromosomal polymorphisms were seen in at least 1 out of 163 (12.9%) couples. Multivariate logistic regression analysis revealed that chromosomal polymorphisms were independently associated with an increase in the occurrence risk of multinucleated embryos (OR = 1.61, 95% CI, 1.06–2.44) in the first IVF/ICSI cycle. The miscarriage rate in the multinucleated embryos group was 10% higher than that of the control group. Chromosomal polymorphisms were independently associated with multinucleation embryo formation. A higher LH level on the day of HCG triggering was associated with a decreased chance of multinucleation.

This study aimed to test whether there is an association between embryo morphokinetic parameters and ploidy status.Patients with high risk of aneuploidy were analyzed by time-lapse microscopy combined with preimplantation genetic screening (PGS). Accordingly, 256 blastocysts from 75 patients were subjected to trophectoderm biopsy and microarray comparative genomic hybridization (array-CGH). Blastocyst development process was analyzed using time-lapse images.Morphokinetic parameters: tPNf, t2, t3, t4, t5, t8, t9, tcom, tM, tSB, tB, tEB, CC1, CC2, CC3, S2, S3, t5-t2, and tB-tSB showed no significant difference in euploid embryos compared to aneuploid counterparts. In addition, two risk models based on previously published morphokinetic parameters failed to segregate euploid from aneuploid embryos.Morphokinetic parameters subjected to investigation in the present study failed to improve the chance of selecting euploid embryos.

This study aims to describe the role of implementing good laboratory practices to improve in vitro fertilization (IVF) outcomes which are of great interest for practitioners dealing with infertility. Certain modifications were introduced in May 2015 in our IVF laboratory like high-efficiency particulate air CODA system, steel furniture instead of wooden, use of new disinfectants like oosafe, and restriction of personnel entry along with avoidance of cosmetics like perfume to improve pregnancy rates. Volatile organic compound (VOC) meter reading was monitored at two time points and five different places in the laboratory to compare the embryonic development parameters before (group A: July 2014–April 2015) and after (group B: July 2015–April 2016) remodeling. The IVF outcomes from 1036 cycles were associated in this study. Reduction in VOC meter readings, enhanced air quality, improvement in blastocyst formation rate, implantation, and clinical pregnancy rate were observed in the laboratory after implementation of new facilities. Results illustrated that the attention must be focused on potential hazards which expose laboratories to elevated VOC levels. Blastocyst formation rate increased around 18%. Implantation rate, clinical pregnancy rate, and live birth rate increased by around 11, 10, and 8%, respectively. In conclusion, with proper engineering and material selection, we have been able to reduce chemical contamination and adverse effects on culture with optimized IVF results. None.

Implantation of the mammalian embryo in the uterus is preceded by escape from the zona pellucida. In some species, hatching from the zona occurs preferentially from one or the other poles of the embryo. The situation for the bovine embryo, in which hatching precedes attachment to the uterus by more than a week, is unclear. The purpose was to describe whether hatching of the bovine embryo from the zona pellucida occurs preferentially from the embryonic or abembryonic pole. Bovine blastocysts undergoing hatching were examined by light microscopy (n = 84) and epifluorescence imaging using antibodies for markers of epiblast, hypoblast, and trophectoderm (TE) (n = 26). The location of hatching was classified as being at the embryonic pole, if hatching occurred ipsilateral to the inner cell mass (ICM), or abembryonic, if hatching occurred contralateral to the ICM. A total of 55% of blastocysts exited the zona pellucida through an opening at the embryonic pole. In these cases, 68% of the cells emerging through the zona pellucida were derived from the ICM. The remainder of blastocysts hatched from an opening either contralateral or to the side of the ICM. In these cases, 87% of hatched cells were TE. For the bovine embryo, there is nearly equal probability of hatching from the embryonic or abembryonic poles. Given that the surface area of the zona pellucida in contact with the TE overlying the ICM is less than for the remainder of the blastocyst, there is some preference for hatching through the embryonic pole. Thus, the bovine embryo is distinct from the mouse and human, where hatching occurs preferentially at the abembryonic pole.

The purpose of this study is to explore which part of the trophectoderm best represents the inner cell mass after aCGH analysis. Fifty-one preimplantation genetic diagnosis/preimplantation genetic screening of abnormal blastocysts diagnosed by array comparative genomic hybridization were included in this study. Blastocysts were thawed, incubated for 3 to 4 h, and then biopsied. Four regions were biopsied per blastocyst, including the inner cell mass (ICM), trophectoderm (TE) cells opposite the ICM, TE cells at the upper right of the ICM, and TE cells at the lower right of the ICM. The biopsied pieces were processed through multiple annealing and looping-based amplification cycle sequenced for 24-chromosome aneuploidy screening. The aneuploidy results were compared among the ICM and the different regional trophectoderm cells from the same blastocyst. Fifty of 51 (98.04%) ICM samples were concordant with at least one of the TE biopsies derived from the same embryos. There were 43 blastocysts in which ICM and the other three TE pieces were consistent. Discordance among the four pieces occurred in eight blastocysts. Only one blastocyst was discordant between the ICM and the other three TE pieces, while seven blastocysts were discordant between one of TE and the other three biopsied pieces. There was no special region that the mosaic TE was located. Our findings indicate that TE aneuploidy is an excellent predictor of ICM aneuploidy. The blastocyst mosaic cells are inclined to be located in TE. Moreover, the mosaic TE was not limited to the special region.

This study was conducted to examine the dynamic distribution of polo-like 1 kinase (Plk1) and the possible role it plays in first mitotic division during early porcine embryo development. Indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses were used to study the dynamic expression and subcellular localization of Plk1 protein in pig parthenogenetic embryos. Finally, a selective Plk1 inhibitor, GSK461364, was used to evaluate the potential role of Plk1 during this special stage. The results showed that Plk1 upon expression exhibited specific dynamic intracellular localization, which closely correlated with the α-tubulin distribution during the first mitotic division. GSK461364 treatment resulted in cleavage failure, with majority of the GSK461364-treated embryos being arrested in prometaphase. Further results of the subcellular structure examination showed that GSK461364 treatment led to a significantly higher proportion of the treated embryos having abnormal spindles and misarranged chromosomes at the prometaphase stage. Thus, these results indicated that Plk1 is essential for porcine embryos to complete the first mitotic division. Furthermore, Plk1 regulation was associated with effects on spindle assembly and chromosome arrangement.

The etiology of maternal aging, a common cause of female factor infertility and a rate-limiting step in vitro fertilization (IVF) success, remains still unclear. Proteomic changes responsible for the impaired successful pregnancy outcome after IVF with aged blastocysts have not been yet evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to enlight differences at the protein level in blastocoel fluid of aged and younger woman. Protein composition of human blastocoel fluid isolated by micromanipulation from 46 blastocysts of women aged

OBJECTIVE: To evaluate the feasibility of adjusted mitochondrial DNA quantification in human embryos as a biomarker for implantation potential. DESIGN: Double-blind, observational, prospective analysis of an Asian population in a single university-affiliated in vitro fertilization center. A total of 1617 embryos derived from 380 infertile couples were collected. The DNA from blastomere biopsy (n = 99) or trophectoderm biopsy (n = 1518) were analyzed with next-generation sequencing. RESULTS: The adjusted mtDNA quantification followed a non-normal distribution in both types of the embryos. When stratified by ploidy status, the adjusted mtDNA quantification was significantly higher in aneuploid trophectoderm than in euploid cells, but not in blastomeres. The adjusted mtDNA quantification of embryos showed significant but very weak positive correlation in total trophectoderm cells with maternal age (Spearman’s correlation, r = 0.095, p = 0.0028) but neither in blastomeres nor stratified by ploidy status. The median adjusted mtDNA quantification was also significantly higher in aneuploid blastocysts than in euploid ones while corrected with embryo morphology. Viable embryos did not contain significantly different quantities of adjusted mtDNA compared with nonviable embryos (implanted n = 103, non-implanted n = 164; median 0.00097 vs. 0.00088, p = 0.21) in 267 transferred blastocysts. CONCLUSION: Quantification of adjusted mitochondria DNA in human embryos was significantly lower in euploid blastocysts than in aneuploid blastocysts. However, no statistically significant differences regarding implantation outcome were evident. To our best knowledge, this study provides the largest scale and the first correlation data between mitochondria copy number and human embryo implantation potential in Asians.

Purpose To investigate the global transcriptome and associated embryonic molecular networks impacted with advanced maternal age (AMA). Methods Blastocysts derived from donor oocyte IVF cycles with no male factor infertility (

PURPOSE: The aim of the present study is to investigate role of FoxO transcription factors in preimplantation embryo development by knocking down FoxO1, FoxO3, and FoxO4 genes and also to assess cell cycle arrest related proteins, p53 and p21, and apoptosis-related proteins, fas ligand (FASL), and cleaved caspase 3. METHODS: Knockdown of FoxOs using siRNA was confirmed utilizing RT-PCR and qRT-PCR in gene level and using immunofluorescence in protein level. Following knockdown of FoxO1, FoxO3, and FoxO4 in two-cell mouse embryos with or without resveratrol treatment; developmental competence of embryos and expression patterns of SIRT1, p53, p21, FASL, and CLEAVED CASPASE 3 proteins in embryos by immunofluorescence were assessed after 48 h. ROS levels were measured in knockdown embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine resveratrol dose. RESULTS: Successful knockdown of FoxO genes in mouse embryos utilizing a non-invasive siRNA method was achieved. Significantly, knockdown of FoxO genes impaired preimplantation embryo development which cannot be prevented by resveratrol treatment. Immunofluorescence results showed that resveratrol could protect embryos from cell cycle arrest and apoptosis. FOXO proteins regulate apoptosis and cell cycle related proteins in mouse preimplantation embryos. Moreover, there might be an autofeedback mechanism where FOXO1, FOXO3, and FOXO4 regulate SIRT1 protein expression. CONCLUSIONS: These results suggest that FOXO transcription factors could contribute to mouse preimplantation embryo development, and it remains to investigate whether they have crucial roles in human preimplantation embryo and infertility. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01555-1) contains supplementary material, which is available to authorized users.

The aims of the present study are to identify the mechanism(s) whereby obesity impairs fresh embryos and to clarify the effects of vitrification on lipid droplet content within embryos from maternally obese mice. The diet-induced obesity mouse model was established, and the zygotes were captured and cultured to day 3. The eight-cell embryos were selected and divided into fresh and vitrified groups. The blastocysts derived from fresh embryos were used as a control. The expression profiles of endoplasmic reticulum (ER) stress genes (Atf4, Grp78, and Hsp70) and other genes (MnSOD, p53, Gadd45g, caspase-3, IGF-II, ZO-1, and E-cadherin) on day-3 fresh and post-warming eight-cell embryos from obese and control groups were determined. For day-5 fresh blastocysts and blastocysts previously vitrified on day 3, the expression profiles for all of the above genes were also determined. For the fresh group, obesity significantly upregulated Hsp70, p53, IGF-II, and ZO-1 expression in embryos on day 3 and notably upregulated Atf4, MnSOD, Gadd45g, caspase-3, ZO-1, and E-cadherin expression in blastocysts on day 5. For vitrified ones, obesity significantly upregulated Atf4, MnSOD, and Gadd45g expression in embryos on day 3 and notably upregulated Hsp70 expression and downregulated MnSOD in day 5 blastocysts previously vitrified on day 3. Obesity impairs fresh embryos and aggravates embryonic vitrification injury at a molecular level.

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p

The aim was to investigate if improved survival rates could be achieved using a new formulation of solutions for slow freezing of human cleavage stage embryos.The evaluation was divided into two parts. The first part was a retrospective analysis of results obtained after freezing and thawing of day 3 embryos from 400 women using an old formulation of cryopreservation solutions compared to results from 108 women for which cryopreservation had been performed using new compositions of solutions. The second part was prospective, adding cycles until similar numbers of patients had been included in both groups. In total, 2274 embryos from 897 patients were thawed using the old formulation of solutions while 1273 embryos from 542 patients were frozen and thawed using the new solutions. The primary endpoint was survival rate.With the new solutions, the survival rate increased from 82.1 to 94.4 % and the complete embryo survival rate increased from 54.9 to 81.3 %. The implantation rate, clinical pregnancy rate per embryo transfer, and per cycle were 28.2, 45.2, and 43.7 %, respectively, using the old formulations of cryosolutions. With the new solutions, the results reached 33.7, 54.1, and 54.1 %, respectively. All differences in results were statistically significant. The number of cancelled embryo transfers due to no survived embryos was 18 with the old solutions and 0 using the new solutions.With the new composition of solutions for slow freezing and thawing of embryos, significantly improved results were obtained. Additionally, the number of cancelled embryo transfers was reduced.

The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3′-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists’ (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50–85 % Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60–90 % reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60 % reversible with CC, respectively. These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.

The present study evaluated the association between oxidative parameters in embryo cryopreservation medium and laboratory and clinical outcomes. 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. 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

Preimplantation genetic testing (PGT) requires an invasive biopsy to obtain embryonic material for genetic analysis. The availability of a less invasive procedure would increase the overall efficacy of PGT. The aim of the study was to explore the potential of blastocoele fluid (BF) as an alternative source of embryonic DNA for PGT. Collection of BF was performed by aspiration with a fine needle prior to vitrification. BF DNA was subjected to whole-genome amplification (WGA) and analyzed by high-resolution next-generation sequencing (NGS). A high-quality WGA product was obtained from 8 of 11 (72.7 %) samples. Comparison of matching BF and blastomere samples showed that the genomic representation of sequencing reads was consistently similar with respect to density and regional coverage across the 24 chromosomes. A genome-wide survey of the sample sequencing data also indicated that BF was highly representative of known single gene sequences, and this observation was validated by PCR analyses of ten randomly selected genes, with an overall efficiency of 84 %. This study provides further evidence that BF is a promising alternative source of DNA for PGT.

Blastocysts contain a large amount of fluid in the blastocoel, which may pose a risk for ice crystal formation during vitrification. This study aimed to evaluate the effectiveness of laser-induced artificial shrinkage of blastocoel before vitrification on clinical outcome. Patients were divided into two groups: a control group with untreated, expanded blastocysts (n = 115) and a study group with blastocoel artificially eliminated by a laser pulse prior to vitrification (n = 309). Blastocyst survival, clinical pregnancy, and implantation rates were compared. The survival rate was significantly higher in the study group compared with the control group (97.3 and 74.9 %, respectively; p > 0.01). The clinical pregnancy and implantation rates of the study group were significantly higher (p

This study aims to compare implantation, pregnancy, and delivery rates in frozen transfer cycles with blastocysts that were vitrified either with artificial shrinking (AS group) or without (NAS group). Retrospective comparative study of artificial shrinking of blastocysts prior to vitrification and frozen embryo transfer cycles in infertile patients undergoing frozen embryo transfer (FET) was done at the Humanitas Fertility Center between October 2009 and December 2013. Main outcome measure(s) were implantation (IR), pregnancy (PR), and delivery rates (DR) between the two groups. A total of 1028 consecutive warming blastocyst transfer cycles were considered. In 580 cycles (total of 822 blastocysts), artificial shrinking was performed prior to vitrification (AS group), while in the remaining 448 cycles (total of 625 blastocysts), the artificial shrinking was not performed (NAS group). There were no differences in patient age (36.4 ± 3.7 vs. 36.3 ± 3.9) and number of embryos transferred (1.41 ± 0.49 vs. 1.38 ± 0.50) between groups. The IR, PR, and DR in the AS group were significantly higher (p

Along with comparative investigation of the decidualization potential and IL-6 secretion by fresh and frozen ESCs, we also aimed to evaluate the effectiveness of co-culture systems based on fresh or frozen ESCs in terms of clinical pregnancy rates. Outcome analysis of a total of 215 IVF cycles with co-culture with fresh or frozen ESCs was performed. Endometrial tissue was obtained from 17 healthy donors. Concentrations of secreted prolactin, IGFBP-1, and IL-6 in conditioned media from cultured fresh and frozen ESCs (decidualized or not) were measured using ELISA or ECLIA. Embryo co-culture with frozen ESCs resulted in a much lower pregnancy rate compared to the alternative system using fresh ESCs. Furthermore, cultivated frozen ESCs showed considerably decreased release of prolactin, IGFBP-1, and IL-6 compared to fresh ESCs, indicating that cryopreservation negatively affects their decidualization potential and cytokine production. Altogether, this data illustrates the need for optimization and improvement of the existing autologous endometrial co-culture systems.

The aim of this study is to compare the sensitivity of the standard one-cell mouse embryo assay (MEA) to that using in vitro-matured oocytes from hybrid and outbred mice. The study was done by culturing embryos in the presence or absence of two concentrations (0.0005 or 0.001 % v/v) of Triton X-100 (TX100). Embryonic development, blastocyst cell numbers (total and allocation to the trophectoderm [TE] and inner cell mass [ICM]), and blastocyst gene expression were evaluated. Neither concentration of TX100 affected (P > 0.05) cleavage, blastocyst development, or hatching in one-cell embryos from BDF1 mice. However, all cell number endpoints were reduced (P

In in vitro maturation (IVM) cycles primed with human chorionic gonadotropin (hCG), both immature and mature oocytes are retrieved from antral follicles sized 8–12 mm. Using time-lapse microscopy, we compared the morphokinetic behavior of embryos developed from oocytes matured in vivo and in vitro, testing the hypothesis that IVM affects preimplantation development. Furthermore, we extended the morphokinetic analysis of these embryos by a comparison with embryos obtained in stimulated assisted reproduction technology (ART) cycles. In IVM cycles primed with follicle-stimulating hormone (FSH)/hCG, prior to sperm microinjection, oocytes surrounded by an expanded cumulus at retrieval and presumably mature (EC-MII) were incubated for 6 h, while immature oocytes enclosed in a compact cumulus (CC) were matured in vitro for 30 h. The morphokinetics of embryos selected for transfer or cryopreservation, derived from EC-MII and CC oocytes, were comparatively and retrospectively analyzed in terms of cleavage times (t2, t3, t4, t5, and t8) and intervals (cc2, cc3, s2, s3). For further comparison, the morphokinetics of embryos selected for transfer or cryopreservation (ICSI) or giving rise to ongoing pregnancies (model) in stimulated ART cycles was also assessed. The morphokinetic behavior of EC-MII and CC embryos was entirely comparable, as suggested by the absence of statistical differences in the averages of all cleavage times and intervals. Almost all cleavage and interval times were also similar between EC-MII, CC, ICSI, and model groups, with the exception of t4 and s2, which were delayed and longer, respectively, in embryos generated in IVM cycles (EC-MII and CC). These findings do not support the hypothesis that maturation in vitro affects embryo morphokinetics, while they suggest only marginal differences in the morphokinetics of embryos developed from oocytes matured in vivo and in vitro in IVM cycles and embryos developed from mature oocytes recovered in stimulated cycles.

PURPOSE: The aim of the study is to investigate how blastocyst contraction behaviour affects the reproductive competence in high-quality euploid embryos. METHODS: Eight hundred ninety-six high-quality blastocysts derived from 190 patients (mean age 38.05 (SD = 2.9) years) who underwent preimplantation genetic testing for aneuploidies (PGT-A) from January 2016 to October 2017 were included in this study. PGT-A results were reported as euploid or aneuploid. Aneuploid embryos were sub-classified into three categories: monosomy, trisomy and complex aneuploid. Retrospective studies of time-lapse monitoring (TLM) of those embryos were analysed and reproductive outcome of transferred embryos was collected. RESULTS: A total of 234/896 were euploid (26.1%) whilst 662/896 (73.9%) blastocysts were proven to be aneuploid from which 116 (17.6%) presented monosomies, 136 (20.5%) trisomies and 410 (61.9%) were complex aneuploid. The most frequent chromosomal complements were trisomies affecting chromosome 21 and monosomies involving chromosomes 16 and 22. Data analysis showed a statistical difference in the number of contractions being reported greater in aneuploid when compared to euploid embryos (0.6 vs 1.57; p

Purpose Sperm play an essential role in embryonic genome activation and embryonic progression to blastocyst. In the present work, we focus on development of embryos created as a result of ICSI with testicular or epididymal sperm from azoospermic males and compare this to outcomes from normospermic males. The objective of this study was to determine if sperm origin influences clinical outcomes, the kinetics of embryo development, or the incidence of cleavage anomalies and multinucleation. Methods A total of 93 consecutive intracytoplasmic sperm injection cycles (ICSI) performed for 83 couples were included in this study. Observations were made on 594 fertilized oocytes cultured in the EmbryoScope using time-lapse microscopy (TLM). Epididymal sperm (n = 29) cycles or surgically retrieved sperm from the testis (TESE; n = 37 cycles) of men with either obstructive (OA) or non-obstructive azoospermia (NOA) were used to inject oocytes. A further 27 ICSI cycles were performed using ejaculated sperm from normospermic males, designated as our control sperm (CS) group. Kinetic data and cycle outcomes were retrospectively analyzed. Results The clinical pregnancy rate was not different between the three groups (TESE 51.4%, PESA 57.7%, and CS 59.3%). A non-significant decrease was observed in both implantation (30.9%) and live birth rate (43%) with TESE as compared to PESA (35.3%, 58%, respectively) and CS groups (45.1%, 56%, respectively). Failure to compact was significantly higher amongst TESE-NOA embryos (35.2%; P

PURPOSE: To evaluate whether the mitoscore of cleavage stage embryos might correlate with developmental kinetics and the ploidy status. MATERIALS: This retrospective single-center study involved all cycles between April 2016 and April 2018 in which preimplantation genetic testing for aneuploidy (PGT-A) on day 3 was performed. The mitochondrial DNA (mtDNA) content and embryo ploidy were determined on 375 single blastomere biopsies by next generation sequencing (NGS). After intracytoplasmic sperm injection, a time-lapse imaging system (embryoscope) was used to follow the development. The median mtDNA content of cleavage stage embryos (49.4) was used to stratify the embryos into two groups to compare embryo development and ploidy status: low mitoscore group (≤ 49.4) and high mitoscore group (> 49.4). RESULTS: The total number of euploid embryos was equal between both mitoscore groups (32.1% versus 33.5%; p = 0.854). However, embryos in the low mitoscore group had a significantly higher cell number on day 3 (8.13 ± 1.59 versus 7.62 ± 1.5; p = 0.0013) and showed a significantly faster development up until the 8-cell stage. Mitoscore was not different between euploid and aneuploid embryos, with the same blastomere number at the time of biopsy. Furthermore, absence of cavitation within 118 h after insemination was correlated with higher mitoscore values (60.22 ± 42.23 versus 50.97 ± 13.37; p = 0.006) and a lower chance of being euploid (17.1% versus 47.4%; p = 0.001). CONCLUSION: mtDNA content of cleavage stage embryos correlates with time-lapse parameters. Early blastulation is correlated with a lower mtDNA content and a higher chance of euploidy.

Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles.Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes.The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P 0.01). To predict implantation outcomes, we examined the area under the ROC curve (AUCROC), which was significantly higher for the viability than for the morphology score (0.94 vs. 0.55; P 0.01); however, the AUCROCs for the composite and viability scores did not differ significantly (0.92 vs. 0.94; P 0.05).NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

Normally fertilized zygotes generally show two pronuclei (2PN) and the extrusion of the second polar body. Conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI) often result in abnormal monopronuclear (1PN), tripronuclear (3PN), or other polypronuclear zygotes. In this study, we performed combined analyses of the methylation status of pronuclei (PN) and the number of centrosomes, to reveal the abnormal fertilization status in human zygotes. We used differences in DNA methylation status (5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)) to discriminate between male and female PN in human zygotes. These results were also used to analyze the centrosome number to indicate how many sperm entered into the oocyte. Immunofluorescent analysis shows that all of the normal 2PN zygotes had one 5mC/5hmC double-positive PN and one 5mC-positive PN, whereas a parthenogenetically activated oocyte had only 5mC staining of the PN. All of the zygotes derived from ICSI (1PN, 3PN) had two centrosomes as did all of the 2PN zygotes derived from c-IVF. Of the 1PN zygotes derived from c-IVF, more than 50 % had staining for both 5mC and 5hmC in a single PN, and one or two centrosomes, indicating fertilization by a single sperm. Meanwhile, most of 3PN zygotes derived from c-IVF had a 5mC-positive PN and two 5mC/5hmC double-positive PNs, and had four or five centrosomes, suggesting polyspermy. We have established a reliable method to identify the PN origin based on the epigenetic status of the genome and have complemented these results by counting the centrosomes of zygotes.

The purpose of this study was to determine the effect of the protein stabilizer octanoic acid on blastocyst development, implantation, and fetal growth in a murine model. One-cell mouse embryos were collected and individually cultured in medium supplemented with recombinant human serum albumin for 96 h at 5 % oxygen in an EmbryoScope. Embryos were randomly allocated to four octanoic acid groups (0, 400, 800, or 1200 μM). Blastocyst development and cell cycle timings were calculated at 96 h of culture, and experiments were repeated in triplicate. Blastocysts were stained and fixed at 96 h for differential cell counts. Following 96 h of culture, blastocysts were transferred to recipients to determine implantation rates and fetal and placental weights. Blastocyst development, hatching rates, developmental kinetics, and total number of cells were negatively affected by octanoic acid at concentrations commonly used in human IVF. Implantation was not affected by octanoic acid but fetal and placental weights at 800 μM octanoic acid were increased relative to control. Octanoic acid, a standard additive to human protein supplements used in IVF, can have long-term negative effects on embryonic and fetal development. The use of octanoic acid for human embryo culture should be monitored and reduced.

To assess human fertilization and preimplantation embryo development in the presence and in the absence of carbon filtrationThis is a retrospective cohort analysis of fresh, controlled ovarian hyperstimulation cycles as well as previously cryopreserved pronuclear stage embryo transfer cycles in a single IVF center. Embryo development and cycle-based outcomes were compared among three groups: 1) when carbon filtration was present, 2) when carbon filtration was absent, and 3) when carbon filtration had been restored.A total of 524 fresh cycles and 156 cryopreserved embryo cycles were analyzed. Fertilization, cleavage, and blastocyst conversion rates for fresh cycles all declined during the period of absent carbon filtration and recovered after the restoration of carbon filtration. Cryopreserved embryos that were thawed and cultured during the period of absent filtration did not have changes in cleavage or blastocyst conversion rates compared to periods where carbon filtration was present. Clinical pregnancy and live birth rates were unchanged among the three time periods.The absence of carbon filtration in an IVF laboratory air handler is associated with poor fertilization and early embryo development for fresh cycles. Because development of previously frozen pronuclear stage embryos was unaffected, the lack of carbon filtration may preferentially affect embryos in the peri-fertilization period. Carbon filtration is an integral part to a successful human in-vitro fertilization laboratory.

Purpose Oocyte vitrification does not affect embryo quality after oocyte warming, making this method effective in the preservation of female fertility. Morphokinetic parameters can be used to predict the competence of an embryo produced from fresh oocytes. Our aim was to study the effect of oocyte vitrification on zygote-embryo kinetics (pl). Methods The embryo-kinetics of fresh and sibling vitrified/warmed oocytes were compared to determine the consequences of oocyte preservation on the timing of embryo development. A 44-hours time-lapse analysis, from the time of ICSI (t0), of 179 fertilized fresh oocytes was compared to 168 fertilized sibling vitrified/warmed oocytes. Results Oocyte vitrification accelerated pronuclear disappearance, one-cell stage timing and modified nucleoli activity by increasing their number and decreasing their diameter at the zygote stage. In contrast, embryo kinetics during cleavage were similar to those observed for fresh sibling oocytes based on the parameters examined in this study. Conclusions At the zygote stage, oocyte vitrification induces changes in pronuclei stability, probably due to pronuclei envelop instability as well as modifications in nucleoli functionality. Therefore, the predictive morphokinetic parameters on embryo competence found from fresh oocytes must be revised when applied on embryos from vitrified/warmed oocytes.

Purpose The aim of the study was to create a predictive model of blastocyst development based on morphokinetic parameters of time-lapse embryoscope monitoring. Methods Time-lapse recordings of 432 embryos (obtained from 77 patients), monitored in Embryoscope, were involved in the study. Patients underwent in vitro fertilization according to standard procedure between June 2012 and April 2013. A retrospective analysis of morphokinetic features, focused on duration of time from the Intracytoplasmic Sperm Injection (ICSI) procedure to consecutive embryo division for 2, 3, 4 and 5 blastomeres, as well as time intervals between each division, was conducted. All embryos were observed for 5 days. Results Based on the distribution of analyzed morphokinetic parameters and number of embryos developed to blastocyst, a range denoting the possibility of an embryo reaching blastocyst stage was determined. According to the obtained results, univariate and multivariate logistic regression analyses were performed. Based on the times of division for two and five blastomeres and intervals between the second and third division, a multivariate predictive model was created. The predictive equation was constructed based on the parameters of logistic regression analysis (odds ratios). Statistically significant differences (p

To evaluate the effect of the nonsteroidal anti-inflammatory drugs tolfenamic acid and flunixin meglumine in pregnancy rate and embryo survival of recipient mice subjected to embryo transfer.A total of 142 recipient females were transferred with 2,931 embryos and treated with a single injection of tolfenamic acid (1 mg/kg; n = 54 females with 1,129 embryos), flunixin meglumine (2.5 mg/kg; n = 46 females with 942 embryos), or bi-distilled water (10 mL/kg) as control group (n = 42 females with 860 embryos). Pregnancy was checked 2 weeks after embryo transfer, delivery was registered on the due date, and litter size was recorded on Day 7 after birth.Pregnancy rate of tolfenamic acid treated females was significantly higher than flunixin group (P 0.05) and showed a tendency to be higher when compared to the control group (P = 0.06). The number of pups born from transferred embryos in pregnant females was significantly higher for both treatment groups compared to controls (P 0.05). Number of pups from total transferred embryos was higher for both treatment groups (P 0.05) when compared to controls.The use of tolfenamic acid at the time of embryo transfer improves both pregnancy rate and number of live pups in recipient mice, with optimal effects observed with flunixin meglumine. We suggest that the use of tolfenamic acid has beneficial effects on the maintenance of pregnancy and embryo survival in recipient mice, which should be taken into account for further studies in other mammalian females.

The aim of the present randomized, comparative study was to evaluate the effect of reduced culture volumes on sibling human embryo development.Firstly, sibling injected oocytes obtained from 88 out of 165 consenting couples undergoing infertility treatment were cultured either in large (35 μl) or in small drops (15 μl) of culture medium. Secondly, sibling injected oocytes from 77 couples were cultured either in large (35 μl) or in mini drops (7 μl). Embryo quality on day-2 and day-3 and blastocyst formation rate on day-5 were evaluated.No statistically significant difference in terms of embryo quality was detected comparing embryos cultured either in large (35 μl) or small (15 μl) drops until blastocyst stage. Similarly, no difference appeared between large (35 μl) or mini (7 μl) drops until day-3, however a significantly higher blastocyst formation rate was observed in mini (7 μl) drops on day-5.Reduced culture volume seems not to influence early embryo development but a reduction of medium appears to positively affect blastocyst development. This supports the hypothesis that the pre-implantation embryo produces autocrine factors which exert a positive effect on embryo development when culture is performed in a reduced volume.

It was reported that mitochondrial DNA (mtDNA) was significantly increased in aneuploid human embryos compared to euploid embryos and was also associated with maternal age. In this study, we further established the mouse model of mtDNA quantitation in reproductive samples based on whole-genome amplification (WGA) and next-generation sequencing (NGS). WGA followed by NGS-based mtDNA quantitation was first performed on 6 single- and 100-cell samples from a tumor-derived mouse cell line, which was exposed to ethidium bromide to reduce mtDNA content. The relative mtDNA content was normalized to nuclear DNA. This method was then applied to mouse reproductive samples, including 40 pairs of oocytes and polar bodies from 8 CD-1 female mice of advanced reproductive age and 171 blastocysts derived via in vitro maturation (IVM) or in vivo maturation (IVO) from young (6–9 weeks) and reproductively aged (13.5 months) female CF-1 mice. Exposure to ethidium bromide for 3 and 6 days decreased mtDNA levels in both the single- and 100-cell samples as expected. Results demonstrated that the first polar body contained an average of 0.9% of mtDNA relative to oocytes. Compared to the cells in blastocysts, oocytes contained about 180 times as much mtDNA per cell. mtDNA levels were compared among blastocysts from reproductively young and old female mice that had either been produced by IVM or IVO. Cells in blastocysts from younger mice contained significantly lower amounts of mtDNA compared to aged mice (P

PURPOSE: In this current study, our main goal was to establish that EmbryoScope incubation environment is comparable to standard incubation. METHODS: The development of sibling human zygotes was compared after culture in either a benchtop incubator (SI) or an EmbryoScope time-lapse incubator (ES). Between May 2015 to April 2016, a total of 581 normally fertilized 2PN, pronuclear-stage embryos, from 47 patients were allocated to culture in either a benchtop incubator (SI) or an EmbryoScope incubator (ES). RESULTS: The development of embryos to cleavage (up to day 3) and blastocyst stages (day 5/6) was compared between the two different incubators. The proportion of good quality embryos was higher in the ES group compared to the SI on day 2 (66.8 vs. 50.5%, P = 0.014) and on day 3 (75.1 vs. 56.0%, P = 0.006). Those differences were statistically significant. A higher proportion of embryos developed to good quality blastocysts when cultured in the EmbryoScope compared to the benchtop (49.4 vs. 42.0%, P = 0.24), but this was not significant. Finally, no significant differences were noted with the proportion of blastocysts chosen for cryopreservation on day 5/6 in the two incubators. CONCLUSIONS: The findings support the view that the EmbryoScope incubator supports at least equivalent in vitro development of human embryos compared to other standard incubation methods and may promote improved development during early cleavage stages.

The aim of this study was to compare timings of key events of embryo development from those originating from either fresh or cryopreserved ejaculate sperm using time-lapse technology. In this retrospective observational cohort study, time-lapse technology was used to monitor 1927 embryos from 234 women undergoing intracytoplasmic sperm injection (ICSI) and utilizing either fresh (n = 172 cycles) or cryopreserved ejaculate sperm (n = 62 cycles) for insemination were included in the study. Key developmental events as described in time-lapse were compared with the use of generalized estimating equations (GEE) to adjust for any auto-correlation between the observations. In addition, multivariable logit regression models were used to account for any known baseline differences between the two groups. There were no differences in conventional embryo development such as number of 8-cell embryos by 72 h (p = 0.359), the number of blastocysts by 120 h (p = 0.417), and the number of top quality blastocysts (p = 0.956) between the two groups compared. There were no statistical differences in the timings of any of the key embryo developmental events (PN_t1, NEBD, cytokinesis, t2, t3, t4, t5, t6, t7, t8, tM, tSB, tEB, tHB, s1, s2, s3, cc2, and cc3) when either fresh or cryopreserved ejaculate sperm was used for ICSI. This was also confirmed with conventional morphological assessment. This observational cohort study has shown that there are no differences in the morphokinetic parameters of early embryo development when either fresh or frozen ejaculate sperm are used for ICSI insemination.

Recently, we established a noninvasive system for selecting human blastocysts with a high pre-transfer implantation potential based on first and second division patterns. The present study was carried out to improve the selection system. Embryos that completed first and second divisions within 25.90 and 37.88 h after culture, respectively, were selected using a time-lapse incubator. We examined the effects of compaction and blastocyst formation times on pregnancy rates after transferring these embryos at the blastocyst stage. The completion of compaction and blastocyst formation times (79.93 and 97.47 h after culture, respectively) of embryos resulting in pregnancies after transfer were significantly (P

For translocation carriers, preimplantation genetic diagnosis (PGD) provides the opportunity to distinguish between normal/balanced and unbalanced embryos prior to implantation and, as such, increases the likelihood of a successful ongoing pregnancy. The data presented here compares autosomal reciprocal and Robertsonian translocation segregation patterns in day 3 versus day 5/6 IVF-PGD embryos to determine if there is a difference in the chromosome segregation patterns observed at these developmental time points.A retrospective analysis on PGD translocation carriers at Monash IVF was performed. Segregation patterns were compared between day 3 and day 5/6 embryos to ascertain whether selection against malsegregants exists.For reciprocal translocations, 1649 day 3 embryos (139 translocations) from 144 couples and 128 day 5/6 embryos (59 translocations) from 60 couples were analysed. Day 3 segregation analysis showed that 22.3% of embryos were normal/balanced (consistent with 2:2 alternate segregation) and 77.7% were unbalanced (malsegregation). Day 5/6 segregation analysis showed that 53.1% of embryos were normal/balanced and 46.9% were unbalanced. For Robertsonian translocations, 847 day 3 embryos (8 translocations) from 54 couples and 193 day 5/6 embryos (6 translocations) from 31 couples were analysed. Day 3 segregation analysis showed that 38.7% of embryos were normal/balanced (consistent with 2:1 alternate segregation) and 61.3% were unbalanced. Day 5/6 segregation analysis showed that 74.1% of embryos were normal/balanced and 25.9% were unbalanced.This data demonstrates an increase in the proportion of genetically normal/balanced embryos at day 5/6 of development. This suggests a strong natural selection process between day 3 and day 5/6 in favour of normal/balanced embryos. These findings support performing PGD testing on day 5/6 of embryo development.

The oxygen consumption rates (OCRs) in mice and cattle have been reported to change during preimplantation embryogenesis. On the other hand, mitochondrial DNA (mtDNA) copy number has been shown to be unchanged in mice and changed in cattle and pigs. The interactions between mitochondrial functions and mtDNA copy numbers in human embryos during preimplantation development remain obscure.Sixteen oocytes and 100 embryos were used to assess mtDNA copy numbers and OCR. Three oocytes and 12 embryos were used to determine cytochrome c oxidase activity. All specimens were obtained between July 2004 and November 2014, and donated from couples after they had given informed consent. Mature oocytes and embryos at 2-14-cell, morula, and blastocyst stages were used to assess their OCR in the presence or absence of mitotoxins. The mtDNA copy number was determined using the samples after analysis of OCR. The relationships between developmental stages and OCR, and developmental stages and mtDNA copy number were analyzed. Furthermore, cytochrome c oxidase activity was determined in oocytes and 4-cell to blastocyst stage embryos.The structure of inner mitochondrial membranes and their respiratory function developed with embryonic growth and the mtDNA copy numbers decreased transiently compared with those of oocytes. The undifferentiated state of inner cell mass cells appears to be associated with a low OCR. On the other hand, the mtDNA copy numbers increased and aerobic metabolism of mitochondria increased in trophectoderm cells.The mitochondrial respiratory function of human embryos developed along with embryonic growth although the copy numbers of mtDNA decreased transiently before blastulation. OCRs increased toward the morula stage ahead of an increase of mtDNA at the time of blastulation. Data regarding changes in mitochondrial function and mtDNA copy number during preimplantation development of human embryos will be useful for the development of ideal culture media.

The objective of this work was to determine which embryonic morphokinetic parameters up to D3 of in vitro development have predictive value for implantation for the selection of embryos for transfer in clinical practice based upon information generated from embryo transfers with known implantation data (KID). A total of 800 KID embryos (100% implantation rate (IR) per transfer and 0% IR per transfer) cultured in an incubator with Time-Lapse system were retrospectively analysed. Of them, 140 embryos implanted, whereas 660 did not. The analysis of morphokinetic parameters, together with the embryo morphology assessment on D3, enabled us to develop a hierarchical model that places the classical morphological score, the t4 and t8 morphokinetic values, as the variables with the best prognosis of implantation. In our decision tree, the classical morphological score is the most predictive parameter. Among embryos with better morphological scores, morphokinetics permits deselection of embryos with the lowest implantation potential.

This study evaluated and compared survival, re-expansion, and percentage of live cells of individual Days 5 and 6 human blastocysts that were vitrified and warmed with the Vit Kit Freeze/Thaw (Irvine Scientific, CA), or with two protocols using the Global Fast Freeze/Thaw Kits (LifeGlobal, Canada). Frozen/thawed Day 2–3 or discarded embryos were cultured to blastocyst (culture day 5–6). Group 1 blastocysts were vitrified with the Vit Kit (n = 29) and High Security Vitrification (HSV) devices. Group 2 (n = 47) and Group 3 (n = 48) blastocysts were cryopreserved with the Global Fast Freeze Kit and 0.25 ml straws, using a direct plunge or a −100 °C holding step, respectively. Group 4 (Controls, n = 30) were not vitrified. Blastocysts were subsequently cultured for 24 h, assessed for survival and expansion, and then stained individually with propidium iodide and Hoechst. Live and total cell number was assessed with ImageJ (NIH), and the percentage of live cells calculated for each blastocyst. The percentage of live cells was not different between vitrified and control (non-vitrified) blastocysts, thus vitrification did not affect cell survival. Survival (following thawing and after 24 h culture), re-expansion, and percentage of live cells were not different for blastocysts vitrified and warmed between the two vitrification/warming kits, or between the two protocols for the Global Fast Freeze/Thaw Kits. Blastocyst vitrification can be achieved with equal success using simplified protocols and cheaper and easy to load freezing straws, providing simultaneously increased safety, and efficiency with lower cost, when compared with vitrification using specialized embryo vitrification devices.

Purpose Morphology alone is not enough for the selection of the embryo (s) with the highest implantation potential and time-lapse imaging has added embryo development kinetics as another selection criterion. Therefore, a combination of morphology with kinetics has inspired a new field termed “morphokinetics”, providing a new way of evaluating and selecting embryos. The aim of the study was to identify a criterion solely based on morphokinetic data and available up to the 8-cell stage (t8) to predict blastocyst formation and quality. Methods The study included 3,354 embryos, with annotations up to t8, and cultured until day 5 from 626 infertile patients. A total of 17 kinetic expressions, either absolute cleavage timings and time intervals or time ratios were tested retrospectively for the prediction of blastocyst formation and quality. Results Relative timings (t8-t5, the cleavage synchronicity from 4 to 8 cells and from 2 to 8 cells) were found to be better indicators of blastocyst formation and quality when compared to absolute time-points. Especially, the cleavage synchronicity from 2 to 8 cells (CS2-8) = ((t3-t2) + (t5-t4))/(t8-t2)) was found to be the best predictor available on day 3 for blastocyst formation and quality (AUC:0.786; sensitivity: 83.43; specificity: 62.46). Conclusions Time intervals and relative ratios based on selected cleavage cycles defining synchronicity allowed a specific analysis providing high predictivity of blastocyst formation and quality. Electronic supplementary material The online version of this article (doi:10.1007/s10815-014-0341-x) contains supplementary material, which is available to authorized users.

To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model.Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed.Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS.The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

To compare the expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocyte extracts and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized (IVF) embryos.Hand-made cloned embryos were produced using oocyte extracts treated donor cells and brilliant cresyl blue (BCB) stained recipient cytoplasts. IVF embryos were produced by culturing 15-20 COCs in BO capacitated sperms from frozen thawed buffalo semen and the mRNA expression patterns of genes implicated in metabolism (GLUT1), pluripotency (OCT4), DNA methylation (DNMT1), pro- apoptosis (BAX) and anti-apoptosis (BCL2) were evaluated at 8- to16- cell stage embryos.A significantly (P 0.05) higher number of 8- to16- cell and blastocyst stages (73.9 %, 32.8 %, respectively) were reported in hand-made cloning (HMC) as compared to in vitro fertilization (49.2 %, 24.2 %, respectively). The amount of RNA recovered from 8- to 16- cell embryos of HMC and in vitro fertilization did not appear to be influenced by the method of embryo generation (3.76 ± 0.61 and 3.82 ± 0.62 ng/μl for HMC and in vitro fertilization embryos, respectively). There were no differences in the expression of the mRNA transcripts of genes (GLUT1, OCT4, DNMT1, BAX and BCL2) were analysed by real-time PCR between hand-made cloned and IVF embryos.Pre-treatment of donor cells with oocyte extracts and selection of developmentally competent oocytes through BCB staining for recipient cytoplast preparations may enhance expression of developmentally important genes GLUT1, OCT4, DNMT1, BAX, and BCL2 in hand-made cloned embryos at levels similar to IVF counterparts. These results also support the notion that if developmental differences observed in HMC and in vitro fertilization produced foetuses and neonates are the results of aberrant gene expression during the pre-implantation stage, those differences in expression are subtle or appear after the maternal to zygotic transition stage of development.

To evaluate: (i) the influence of morphology at cleavage stage on blastocyst formation and implantation, and (ii) whether the transfer of low-quality embryos on day-three would be a better approach than the transfer at blastocyst stage.This study included 8,444 embryos obtained from 1,125 patients undergoing ICSI cycles between January/2011 and September/2013. The influence of the quality of the embryo on days-two and -three on blastocyst formation and implantation success was evaluated. Moreover, the implantation potential of low-quality embryos, at cleavage stage, transferred on day-three was compared with the implantation potential of low-quality embryos, at cleavage stage, transferred on day-five.Low-quality embryos on day-two had an approximate 20 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-two had a nearly 40 % decrease in the implantation chance. Low-quality embryos on day-three had a 30 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-three had an almost 40 % decreased implantation chance. The implantation rate didn't differ when low-quality embryos on the cleavage stage were transferred on day-three or left in culture and transferred on day-five.The transfer of low-quality embryos on day-three is a better approach than transfer at the blastocyst stage. In addition, the embryo morphology evaluation at the cleavage stage is still needed for the selection of the embryo with the best implantation potential in extended embryo culture programmes.

Morphological assessment of human blastocysts has been effective for selecting embryos with high potential. However, they often show repeated shrinkage and expansion toward their hatching. Here we assessed whether capturing morphological changes over time of vitrified-warmed blastocysts could lead to a better selection of viable embryos from shrunken blastocysts.The implantation rates of vitrified-warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729 cycles that were subjected to single blastocyst transfer. Vitrified (107) and fresh blastocysts (17) were donated for the experimental study. To assess the relationship between morphology (expanded vs. shrunken) and the mitochondrial respiration of blastocysts, the oxygen consumption rate (OCR) was analyzed for 55 specimens using an uncoupler of oxidative phosphorylation. The remaining 69 blastocysts were used for recording morphological changes every 15 min for 48 h after warming.Because there were no surplus embryos, 7 % of the vitrified-warmed blastocysts were shrunken and transferred. The shrunken embryos had sufficient implantation ability (40 %). The OCR of the shrunken embryos was significantly lower than that of their expanded counterparts. Upon exposure to the uncoupler, the OCR of some shrunken embryos increased to levels similar to the expanded specimens. Time-lapse images revealed some shrunken embryos which formed blastocoel by 5 h following warming exhibited developmental competence to the hatched stage.Data of the present study suggest a group of shrunken blastocysts contains many viable and clinically available embryos and time-lapse observation of vitrified-warmed blastocysts is a potential method to distinguish viable embryos from shrunken blastocysts.

The levels and timing of expression of genes like BCLXL, HDAC1 and pluripotency marker genes namely, OCT4, SOX2, NANOG and KLF4 are known to influence preimplantation embryo development. Despite this information, precise understanding of their influence during preimplantation embryo development is lacking. The present study attempts to compare the expression of these genes in the in vivo and in vitro developed preimplantation embryos.The in vivo and in vitro developed rabbit embryos collected at distinct developmental stages namely, pronuclear, 2 cell, 4 cell, 8 cell, 16 cell, Morula and blastocyst were compared at the transcriptional and translational levels using Real Time PCR and immunocytochemical studies respectively.The study establishes the altered levels of candidate genes at the transcriptional level and translational level with reference to the zygotic genome activation (ZGA) phase of embryo development in the in vivo and in vitro developed embryos. The expression of OCT4, KLF4, NANOG and SOX2 genes were higher in the in vitro developed embryos whereas and HDAC1 was lower. BCLXL expression had its peak at ZGA in in vivo developed embryos. Protein expression of all the candidate genes was observed in the embryos. BCLXL, KLF4 and NANOG exhibited diffused localisation whereas HDAC1, OCT4, and SOX2 exhibited nuclear localisation.This study leads to conclude that BCLXL peak expression at the ZGA phase may be a requirement for embryo development. Further expression of all the candidate genes was influenced by ZGA phase of development at the transcript level, but not at the protein level.