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PURPOSE: To investigate whether treatment with commercially available ready-to-use A23187 ionophore (GM508-CultActive) improves embryo development outcome in patients with a history of embryo developmental problems. METHODS: This is a uni-center prospective study in which sibling oocytes of patients with embryos of poor quality on day 5 in the previous cycle were treated or not with CultActive. RESULTS: Two hundred forty-seven metaphase II (MII) oocytes from 19 cycles performed between 2016 and 2019 were included in the study. After ICSI, the sibling oocytes were assigned to the treatment group or to the control group, following an electronically generated randomization list. A number of 122 MII were treated with CultActive and 125 MII had no treatment and were assigned to the control group. No difference in fertilization rate (p = 0.255) or in the capacity of embryos to reach good quality on day 5 (p = 0.197) was observed between the two groups. The utilization rates defined as the number of embryos transferred or cryopreserved per mature oocyte (p = 0.438) or per fertilized oocytes (p = 0.299) were not significantly different between the treated group and the control group. CONCLUSION: The results of the current study do not support the use of CultActive in cases with embryo developmental problems.

PURPOSE: Nucleoporin 37 (NUP37) has been reported to activate the YAP-TEAD signaling, which is crucial for early embryo development. However, whether NUP37 is involved in oocyte meiosis and embryo development remains largely unknown. The study aimed to clarify the function of Nup37 in oocyte maturation and early embryo development, and to explore the mechanism. METHODS: The expression level and subcellular localization of NUP37 were explored. After knocking down of Nup37 by microinjecting interfering RNA (siRNA), the oocyte maturation rate, aberrant PB1 extrusion rate, and blastocyst formation rate were evaluated. In addition, the effect of the downregulation of Nup37 on YAP-TEAD signaling was confirmed by immunofluorescence staining and real-time quantitative PCR. RESULTS: NUP37 was highly expressed in oocytes and early embryos; it mainly localized to the nuclear periphery at mice GV stage oocytes and early embryos. Nup37 depletion led to aberrant PB1 extrusion at the MII stage oocyte and a decreased blastocyst formation rate. The reduction of NUP37 caused YAP1 mislocalization and decreased the expression of Tead1, Tead2, and Tead4 during mice embryo development, thus affecting the YAP-TEAD activity and embryo developmental competence. CONCLUSIONS: In summary, NUP37 played an important role in mice oocyte maturation and preimplantation embryo development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02330-x.

PURPOSE: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. METHOD(S): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um(2)), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. RESULTS: Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009–2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um(2), p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um(2), p = 0.000), with SPN being significantly larger than OPN at each stage (p

PURPOSE: DNA methylation is one of the epigenetic mechanisms that plays critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of methylation, and Dnmt3a, for de novo methylation, are gradually erased from the zygote in succeeding stages and then reestablished in the blastocyst. This study was designed to address the vital role of Dnmt1 and Dnmt3a enzymes by silencing their gene expressions in embryonic development in mice. METHODS: Groups were (i) control, (ii) Dnmt1-siRNA, (iii) Dnmt3a-siRNA, and (iv) non-targeted (NT) siRNA. Knockdown of Dnmt genes using siRNAs was confirmed by measuring the targeted proteins using Western blot and immunofluorescence. Following knockdown of Dnmt1 and Dnmt3a in zygotes, the developmental competence and global DNA methylation levels were analyzed after 96 h in embryo cultures. RESULTS: A significant number of embryos arrested at the 2-cell stage or had undergone degeneration in the Dnmt1 and Dnmt3a knocked-down groups. By 3D observations in super-resolution microscopy, we noted that Dnmt1 was exclusively found in juxtanuclear cytoplasm, while the Dnmt3a signal was preferentially localized in the nucleus, both in trophoblasts (TBs) and embryoblasts (EBs). Interestingly, the global DNA methylation level decreased in the Dnmt1 knockdown group, while it increased in the Dnmt3a knockdown group. CONCLUSION: Precisely aligned expression of Dnmt genes is highly essential for the fate of an embryo in the early developmental period. Our data indicates that further analysis is mandatory to designate the specific targets of these methylation/demethylation processes in mouse and human preimplantation embryos.

PURPOSE: The aim of this study was to analyze the metabolic profiles of blastocoel fluid (BF) obtained from bovine embryos produced in vivo and in vitro. METHODS: Expanded blastocysts (20/group) that were in vitro and in vivo derived at day 7 were used. BF was collected and analyzed under direct infusion conditions using a microTOF-Q(®) mass spectrometer with electrospray ionization and a mass range of 50–650 m/z. RESULTS: The spectrometry showed an evident difference in the metabolic profiles of BF from in vivo and in vitro produced embryos. These differences were very consistent between the samples of each group suggesting that embryo fluids can be used to identify the origin of the embryo. Ions 453.15 m/z, 437.18 m/z, and 398.06 m/z were identified as biomarkers for the embryo’s origin with 100% sensitivity and specificity. Although it was not possible to unveil the molecular identity of the differential ions, the resulting spectrometric profiles provide a phenotype capable of differentiating embryos and hence constitute a potential parameter for embryo selection. CONCLUSION: To the best of our knowledge, our results showed, for the first time, an evident difference between the spectrometric profiles of the BF from bovine embryos produced in vivo and in vitro.

PURPOSE: To study embryo morphokinetics in relation to release in spent media of molecules with possible roles in development and implantation (miR-20a, miR-30c, and sHLA-G). METHODS: Data were obtained from embryos generated in standard IVF and ICSI cycles. The Eeva system was used for embryo assessment, based on early morphokinetic parameters and producing a score (1–5, best–worst) corresponding to higher/medium/lower chances of development to blastocyst. miRNAs — mm miR-20a-5p and miR-30c-5p — and sHLA-G were quantified in 25 μl of spent blastocyst media (SBM) collected before vitrification or transfer. Statistical analyses were performed applying Kolmogorov-Smirnov, Shapiro-Wilk, and Spearman’s correlation coefficient tests, where appropriate. RESULTS: SBM were collected from a total of 172 viable blastocysts. Their analysis showed that concentration of miR-20a was progressively lower as Eeva score increased and probability of development to blastocyst decreased (P = 0.016). The opposite trend was observed in the case of miR-30c, i.e., concentration was higher as score increased and chances of development to blastocyst decreased (P = 0.004). Analysis of sHLA-G revealed a negative correlation with Eeva score, i.e., levels were progressively lower as Eeva score increased and probability of development to blastocyst decreased (R = − 0.388, N = 141, P = 0.001). CONCLUSION: Our data suggest that morphokinetic algorithms that predict development to blastocyst stage, in fact, also identify embryos with molecular and cellular profiles more consistent with developmental functions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02162-9.

PURPOSE: Different commercial human embryo culture mediums can alter embryo quality and change birthweight. One component that could be contributing to variations but is not widely investigated is human serum albumin (HSA). HSA plays a multitude of roles during embryo culture and is a carrier for molecules including lipids. It remains unclear if lipid composition of HSA varies among commercial products and its effects on embryo quality, implantation, and fetal outcomes are relatively unknown. METHODS: Utilizing a mouse model of embryo culture, we cultured zygotes until the blastocyst stage (72-h culture) in G1/G2 containing either Vitrolife HSA, Sage HSA, or Recombinant HSA at 10%. Blastocyst quality (development, total cell number, superoxide generation), blastocyst lipid content (neutral lipids, non-esterified fatty acids, phospholipids, and triglycerides), implantation, and fetal lengths and weights were assessed. Fatty acid quantification of HSA source was assessed by standard thin-layer chromatography. RESULTS: Sage HSA had the greatest fatty acid composition, with an eightfold increase in saturated fatty acids. This coincided with reduced blastocyst development, increased superoxide generation, neutral lipids and triglycerides levels of blastocysts, and decreased implantation rates (p < 0.05). Unexpectedly, while Recombinant HSA had the lowest overall lipids it had 70-fold increase in palmitoleic acid and the lowest fetal weights (p < 0.05). CONCLUSION: Indicates the importance of a balance between different types/amount of lipids, and an “optimal ratio” required for embryo and fetal development. Therefore, the lipid content of HSA should be considered when choosing a suitable HSA source for use in clinical IVF. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02255-5.

PURPOSE: This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring. METHODS: The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR. RESULTS: The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta. CONCLUSION: These observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01975-4) contains supplementary material, which is available to authorized users.

PURPOSE: The fertility of women decreases with age because of factors such as an increased incidence of aneuploidies and—possibly—decreased mitochondrial activity in oocytes. However, the relationship between maternal aging and mitochondrial function of their embryos remains unknown. Here, we assessed the relationship between maternal age and mitochondrial functions in their oocytes and embryos METHODS: The relationships between maternal age and oxygen consumption rates (OCRs), mitochondrial DNA (mtDNA) copy numbers, or blastocyst development was investigated using 81 embryos donated from 63 infertility couples. The developmental rates from morulae to blastocysts were retrospectively analyzed using data of 105 patients. RESULTS: The OCRs of morulae decreased with maternal age (r(2) = 0.48, P < 0.05) although there were no relationships between maternal age and mtDNA copy number in any stages. The more oxygen consumed at the morula stage, the shorter time was required for embryo development to the mid-stage blastocyst (r(2) = 0.236, P < 0.05). According to the clinical data analysis, the developmental rate from morulae to blastocysts decreased with maternal age (P < 0.05, < 37 years, 81.1%, vs. ≥ 37 years, 64.1%). CONCLUSIONS: The data of the present study revealed that mitochondrial function at the morula stage of human embryos decreased with their maternal age and a decrease of mitochondrial function led to slow-paced development and impaired developmental rate from morulae to blastocysts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01869-5) contains supplementary material, which is available to authorized users.

PURPOSE: The purpose was to investigate the association between embryonic development or implantation and the content of interleukin-6 and 10 (IL-6, IL-10) and tumor necrosis factor-α (TNF-α) in single-blastocyst conditioned medium (SBCM). METHODS: Thirty-eight SBCM samples (SBCMs) were collected from blastocysts with different morphological scores. IL-6, IL-10, and TNF-α concentration in 38 SBCMs was detected by Single Molecule Array and compared according to the blastocyst quality: top-quality (TQ) and non-top quality (NTQ), or blastulation time: day 5 (D5) and day 6 (D6). In another experiment, 61 SBCMs were collected from TQ blastocyst transplanted on D5, and IL-6 concentration in SBCM was compared based on whether embryos are implanted or not (implanted and non-implanted). RESULTS: In the first experiment, IL-6, IL-10, and TNF-α concentration was not significantly different between the TQ-SBCM and NTQ-SBCM. The D6-SBCM had a higher IL-6 concentration compared with the D5-SBCM, while IL-10 and TNF-α concentration was not significantly different between the D5-SBCM and D6-SBCM. The IL-6 concentration in D5-NTQ or D6-TQ SBCM was higher than that in D5-TQ or D6-NTQ SBCM (P < 0.05), respectively. Furthermore, the spearman analysis demonstrated that IL-6 concentration in SBCM was negatively correlated with the blastocyst quality on D5 and positively correlated with the blastocyst quality on D6. In the second experiment, no significant difference in IL-6 concentration was found between SBCM from implanted and non-implanted blastocyst. CONCLUSION: IL-6 concentration in SBCM was associated with embryo quality depending on the blastulation time, although it might not be associated with the blastocyst implantation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01805-7) contains supplementary material, which is available to authorized users.

PURPOSE: To describe an interesting not previously described morphokinetic finding. METHODS: Retrospective case report of a couple undergoing controlled ovarian stimulation (COS) followed by in vitro fertilization and blastocyst transfer. RESULTS: We identified a unique finding of blastulation of a fertilized human zygote after conventional in vitro fertilization. The fertilized zygote did not show any clear cytokinesis until approximately 107 h post insemination, when it started dividing into a blastocyst. By 113 h post insemination, inner cell mass and trophectoderm cells could be clearly distinguished and the blastocyst was completely hatched by 136 h post insemination. CONCLUSION: Time-lapse systems offer more detailed observations of embryonic development. Here, we report an atypical development of an embryo that was not described previously. We hope to become an insightful discussion among peers and incentive the publication of such findings in the future. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01704-x) contains supplementary material, which is available to authorized users.

PURPOSE: A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. METHODS: Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. RESULTS: WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. CONCLUSIONS: In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1252-z) contains supplementary material, which is available to authorized users.

Purpose To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. Methods Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0–1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. Results More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. Conclusions Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency. Electronic supplementary material The online version of this article (10.1007/s10815-018-1156-y) contains supplementary material, which is available to authorized users.

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 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

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

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.

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.

In an attempt to improve in vitro embryo production, we investigated the effect of fibroblast growth factor 10 (FGF10) during in vitro maturation on the developmental capacity of bovine oocytes. Cumulus–oocyte complexes (COCs) were aspirated from follicles of 3–8 mm diameter. After selection, the COCs were matured in medium with or without 0.5 ng/mL of FGF10. The effect of FGF10 during in vitro maturation (IVM) on nuclear maturation kinetics and expansion of the cumulus cells was investigated. Oocyte competence was assessed by the production and development speed of embryos and the relative expression of genes associated with embryo quality. FGF10 delayed the resumption of meiosis from 8 h onwards, but did not affect the percentage of oocytes reaching metaphase II, nor did it increase cumulus expansion at 22 h of maturation. We found no difference between treatments regarding embryo production, developmental speed, and gene expression. In conclusion, the presence of FGF10 during IVM had no effect on embryo production, developmental speed, and gene expression.

The goal of this study was to evaluate the ultrastructure of cytoplasmic fragments along with the effect of cytoplasmic fragment and perivitelline space coarse granulation removal (cosmetic microsurgery) from embryos before embryo transfer on ART outcomes. One hundred and fifty intracytoplasmic sperm injection cycles with male factor infertility were included in this prospective study. Patients were divided into three groups of case (n = 50), sham (n = 50), and control (n = 50). Embryos with 10–50 % fragmentation were included in this study. Cosmetic microsurgery and zona assisted hatching were only performed in case and sham groups respectively. Extracted fragments were evaluated ultrastructurally by transmission electron microscopy (TEM). Rates of clinical pregnancy, live birth, miscarriage, multiple pregnancies, and congenital anomaly in the three groups were also compared. Micrographs from TEM showed that mitochondria were the most abundant structures found in the fragments along with mitochondria-vesicle complexes, Golgi apparatus, primary lysosomes, and vacuoles. There were no significant differences in demographic characteristics, laboratory and clinical data, or embryo morphological features between the groups. The rate of clinical pregnancy in control, sham, and case groups had no significant differences (24, 18, and 18 %, respectively). The rates of live birth, miscarriage, multiple pregnancy, and congenital anomaly were also similar between the different groups. Our data demonstrated that cosmetic microsurgery on preimplantation embryos had no beneficial effect on ART outcomes in unselected groups of patients. As mitochondria are the most abundant organelles found in cytoplasmic fragments, fragment removal should be performed with more caution in embryos with moderate fragmentation.

Author, PURPOSE: Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy. METHODS: Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28h after fertilization. PB2s from embryos at 4-6 (1-cell), 24 (2-cell), 48 (4-cell), and 72h (morula) were fused with MII oocytes to induce premature chromosome condensation. Caspase and TUNEL assays were used to detect apoptotic PB2s at 24, 48, and 72h. PB2s were fused with one of the blastomeres of the 2-cell embryos to produce mixoploid embryos. RESULTS: DNA synthesis in the PB2s continued until 22h after fertilization. At 4-6h, nearly all of the PB2s showed G1-type chromosomes and there was no significant increase in chromosome damage. At 24, 48, and 72h, S-type chromatin predominated. Few PB2s showed apoptotic response until 72h. Regardless of the fusion with the PB2, more than 90% of the embryos developed to 4-cell stage, and over 80% of the resultant 4-cell embryos had daughter blastomeres with a morphologically normal nucleus. Some of the daughter blastomeres displayed triploidy. CONCLUSIONS: The PB2 is viable for at least 72h after fertilization, with slow progression through the cell cycle. Once the PB2 has been incorporated into a blastomere, the cell cycle of the PB2 might be synchronized with that of the host resulting in diploid/triploid mixoploidy.

This is a retrospective analysis of the morphokinetics, prevalence, and implantation potential of embryos with irregular first and second cleavages as identified by time-lapse microscopy.The study included 253 women who underwent 387 assisted reproduction treatments with intracytoplasmic sperm injection (ICSI). Each patient was assigned to one of three groups based on embryo cleavage results. In group I, one to two embryos per cycle showed irregular cleavage; group II, at least three embryos with abnormal cleavage; and in group III (the control group), all embryos cleaved normally. The number of embryos that cleaved from 1 to ≥3 cells or from 2 to ≥5 cells for each patient was recorded. Their prevalence and association with women's characteristics and pregnancy outcome were evaluated.The prevalence of irregular cleavage was 15.6 % among 1772 ICSI embryos. In 101 cycles, 1-2 embryos per cycle showed irregular cleavage (group I). In 32 cycles, at least 3 embryos showed abnormal cleavage (group II). In 254 cycles, all embryos cleaved normally (group III). The average age of the women in group II was significantly lower in comparison with groups I and III (32.5 ± 4.2 vs. 35.1 ± 4.9 and 35.5 ± 5.1, respectively, p 0.02). In comparison of groups I and II, the odds ratio for ≥3 embryos with irregular cleavage in women younger than 35 was 3.48 (95 % CI, 1.28 to 9.46). Embryos with irregular cleavage were transferred in 16 women. Three live births were achieved following the transfer of single blastocysts derived from embryos with irregular cleavage from two to five cells.Early embryos with irregular cleavage are significantly more prevalent in younger women. When these embryos develop to the blastocyst stage, they may have normal implantation potential, leading to the birth of healthy babies.

PURPOSE: The aim of this prospective sibling oocyte study was to evaluate whether reduced culture volume improves blastocyst formation. METHODS: Twenty-three patients with extended embryo culture until day 5 were selected for the study. After injection, 345 sibling oocytes were individually cultured in either 25 or 7 μl droplets of Origio cleavage medium under oil. On day 3 of development, embryos were transferred to droplets with the corresponding volume of Origio blastocyst culture medium. Fertilization and embryo quality on day 3 and day 5/6 were evaluated. RESULTS: No statistically significant difference (p = 0.326) in fertilization rate was observed (81.3 versus 83.0 %). There was no significant difference in terms of the number of excellent and good-quality embryos obtained on day 3 between both groups (p = 0.655). Embryo culture in 25 μl droplets led to more embryos with a higher cell number when compared to 7 μl culture (p = 0.024). On day 3, 132 and 131 embryos were considered for further culture until day 5/6. Blastulation rates were significantly higher in the 25 μl group (75.0 versus 61.6 %; p = 0.017) and significantly more day 5 embryos with excellent and good quality were found in this group (54.5 versus 40.5 %; p = 0.026). Finally, the utilization rates expressed per mature oocyte (41.4 versus 29.8 %; p = 0.043), per fertilized oocyte (50.7 versus 36.6 %; p = 0.023), and per day 3 embryo undergoing extended culture to day 5/6 (54.5 versus 39.7 %; p = 0.019) were all significantly higher in the 25 μl group. CONCLUSION: Reduced culture volume (7 μl) negatively impacts early development by reducing the cell number on day 3 and both blastocyst formation and quality.

We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis).ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining.In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM.GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

To analyze differences in morphokinetic parameters of chromosomally normal and aneuploid embryos utilizing time-lapse imaging and CGH microarray analysis.This retrospective cohort study included patients undergoing IVF treatment and preimplantation genetic diagnosis for sex selection. A total of 460 embryos cultured in incubators with time-lapse imaging system (EmbryoScope) were selected for biopsy on day 3 of development. Subsequently, CGH microarray analysis was performed for aneuploidy screening of 24 chromosomes. Kinetic parameters including time for appearance of second polar body (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPBf), time to division to 2(t2), 3(t3), 4(t4), 5(t5) cells, length of second and third cell cycle (CC2= t3 t2, CC3=t5-t3), synchrony of cell division from 2 to 4 cells (S2=t4-t3) and interval t5-t2 were analyzed to compare chromosomally normal and abnormal embryos.The mean time durations for tPNf, t2, t5, CC2, CC3, t5-t2 differed significantly between normal and abnormal embryos.Time-lapse imaging morphokinetics may play a role in early prediction of aneuploid embryos due to differences in kinetic behavior that may aid in improving clinical outcome.

To determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities.We retrospectively applied key time lapse imaging events of embryos (Campbell et al.[5, 6]) to stratify embryos into 3 groups: low, medium and high risk of aneuploidy. The actual ploidy results (from array comparative genomic hybridization) were compared with expectations [5, 6]. Sources of variability in morphokinetic parameters were determined using Analysis of Variance (ANOVA).The model failed to segregate euploid embryos from aneuploid embryos cultured at our facility. Further analysis indicated that the variability of embryos among patients was too great to allow selection of euploid embryos based on simple morphokinetic thresholds. Clinical selection of embryos based on morphokinetics alone is unlikely to identify euploid embryos accurately for transfer or yield higher rates of live delivery.The use of non-invasive morphokinetics is unlikely to discriminate aneuploid from euploid embryos. Further, it does not approach the accuracy of preimplantation genetic screening with array comparative genomic hybridization.

Antioxidant and anti-apoptotic effects of melatonin on development of in vitro fertilization (IVF)/vitrified two-cell mouse embryos were evaluated in this study.The IVF two-cell embryos were vitrified by cryotop, and were cultured in KSOM medium in different concentrations of melatonin (10(-6), 10(-9), 10(-12) M) and without melatonin. The blastocyst cell number, apoptotic cells and glutathione (GSH) level were evaluated by differential, TUNEL and cell tracker blue staining, respectively. The expression of Bax and Bcl-xl genes was evaluated by qPCR. The expression of melatonin receptors (Mtnr1a and Mtnr1b) in mouse 2-cell embryos and blastocysts was evaluated by RT-PCR.Melatonin increased the rate of cleavage and blastulation at 10(-12) M concentration (p 0.05). The number of trophectoderm and inner cell mass showed a significant increase (p 0.05) in 10(-9) M melatonin. The 10(-9) M and 10(-12) M melatonin treatments significantly reduced (p 0.05) the apoptotic index. The significant increase in the expression of Bcl-xl observed at 10(-9) M concentration however, reduced expression of Bax was not statistically significant. The levels of GSH in 10(-9) and 10(-12) M groups were significantly improved relative to the control group (p 0.05). The Mtnr1a was expressed in 2-cell embryos and blastocysts in all groups, but the expression of Mntr1b was not detected.Melatonin may have a special role against oxidative stress in protection of IVF/vitrified embryos.

Myo-inositol (myoIns) has a positive role in mammalian development and human reproduction. Since experiments on farming species suggest a similar role in preimplantation development, we evaluated the hypothesis that the inclusion of myoIns in human embryo culture media would produce an increase in embryo quality in IVF cycles, using the mouse embryo assay.To determine the effect of myoIns on completion of preimplantation development in vitro, one-cell embryos of the inbred C57BL/6N mouse strain were produced by ICSI, cultured in human fertilization media in the presence of myoIns (myoIns+) or in its absence (myoIns-) and evaluated morphologically. Daily progression through cleavage stages, blastocyst production and expansion and blastomere number at 96 hours post fertilization were assessed.Compared to myoIns- embryos, myoIns+ embryos displayed a faster cleavage rate and by the end of preimplantation development, the majority of myoIns+ blastocysts was expanded and formed by a higher number of blastomeres.The presence of myoIns resulted in both an increase in proliferation activity and developmental rate of in vitro cultured early mouse embryos, representing a substantial improvement of culture conditions. These data may identify myoIns as an important supplement for human embryo preimplantation culture.

To investigate the correlation between the ooplasmic volume and the number of mitochondrial DNA (mtDNA) copies in embryos and how they may affect fecundity. Using real-time PCR, mtDNA quantification was analyzed in unfertilized oocytes and uncleaved embryos. The size of the ovum was also assessed by calculating the ooplasmic volume at the time of granulosa cell removal for IVF or ICSI. Quantification analysis of the mtDNA in blastomeres was performed by real-time PCR at the 7–8 cell stage of the cleaved embryos at 72 h after oocyte retrieval. We calculated the cytoplasmic volume of the blastomeres. Our studies showed a significantly lower mtDNA copy number in unfertilized oocytes and uncleaved embryos in women who were older than 40 years of age (p

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly.(1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining.The blastocyst development was significantly higher (P 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P 0.05) in single embryo culture with the WOW system than without.Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.

To evaluate human sperm nuclear chromatin decondensation in a heterologous ICSI system using hamster ova injected with human sperm.Frozen hamster oocytes were injected with Triton X-100 treated sperm and fixed at different time points post ICSI. Oocytes injected with non-treated sperm served as controls. Male pronuclear decondensation was evaluated after staining with DAPI.Sperm cells with partially destroyed membranes and depletion of the acrosome decondense more rapidly and to a greater extent than membrane/acrosome intact cells. Marked variability in pronuclear size was observed for any time point post ICSI, which most probably reflects the heterogeneity in the mature human sperm population.Remodeling of male gamete nuclei in this heterologous ICSI mimics events that occur during natural fertilization in humans and therefore this approach may be used for studies of human sperm chromosomes transformations.

We examined whether there is a correlation among early embryo cleavage, speed of cleavage, and implantation potential for in-vitro fertilization (IVF) treatment and intracytoplasmic sperm injection (ICSI). This retrospective study examined 112 cycles of IVF and 82 cycles of ICSI in patients less than 40 years of age. Early cleavage was defined as embryonic mitosis occurring 25-27 h after insemination. These day-3 embryos were then grouped according to cleavage speed (rapid, normal, and slow) and morphological quality (good or poor). A larger proportion of early-cleavage embryos developed normally compared to non-early-cleavage embryos (IVF: 69.1 % vs. 47.1 %, respectively; ICSI: 63.0 % vs. 45.6 %, respectively). The early-cleavage embryos also produced more good quality embryos than the non-early-cleavage embryos (IVF: 80.2 % vs. 56.4 %, respectively; ICSI: 73.4 % vs. 59.4 %). The implantation rate was significantly higher with early-cleavage embryos in both IVF (42.9 % vs. 19.7 %) and ICSI (48.1 % vs. 24 %). These results indicate that early-cleavage embryos have a higher rate of normal development and develop into better quality embryos on day 3, resulting in more and higher quality embryos to choose from for day-3 embryo transfer. Thus, early cleavage may be a useful criterion when selecting embryos for IVF or ICSI.

Purpose The aim of this study was to determine which genes and gene pathways are differentially expressed when comparing human blastocysts with cleavage-stage embryos. Methods We individually assessed gene expression in preimplantation human embryos at cleavage (n = 3) and blastocyst (n = 3) stages. Gene expression patterns were then validated in publically available datasets and then independently validated in vitro with additional human embryos using TaqMan gene expression assays. Immunolocalization studies were conducted to identify protein expression in intact blastocyst-stage embryos. Results Compared to cleavage-stage embryos, blastocyst-stage embryos differentially expressed 51 genes (p

Purpose To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. Methods One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Results Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. Conclusions The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).