2PN Embryos Research Articles

Correlation between the number of cultured human embryos and embryo development in the wow culture dish system

The WOW dish (LinKIDTM culture dish; DNP, Japan) has 25 microwells that allows group culture under a single drop of medium. Through its design, it is possible to manage embryos separately whilst in group culture. Due to paracrine effects associated with group culture, embryo culture results have been reported to be improved in animal species such as the cow and mouse. However, there is scant information on group culture, and any subsequent effects of the WOW dish used for human embryo culture. Therefore, in order to clarify the group effect of WOW culture in human embryos, we investigated whether there is any relationship between the number of cultured embryos and subsequent embryo development. 7039 embryos from 936 IVF cycles (from September 2014 to October 2015) where blastocyst culture was performed in WOW dish or 15μl droplet were analyzed. 814 WOW cycles and 122 droplet cycles were each divided into 5 experimental groups according to number of cultured embryos (4-5, 6-10,11-15,16-20 and 21-25 embryos). The rates of good quality embryos on culture day3 and day5 (defined as ≥3BB, Gardner's score) were analyzed and compared between WOW and droplet groups. 2PN embryos which were obtained from either ICSI or conventional IVF were cultured in WOW (group culture with 60μl single step medium) or a droplet system (single embryo culture with 15μl single step medium) for up to 7 days. The culture medium was exchanged on day3 and day5 in the droplet groups, but not in the WOW groups. Chi-square and Cochran-Armitage tests were used for statistical analysis. The rates of good quality embryos on Day 3 in groups 4-5, 6-10,11-15,16-20 and 21-25 between WOW and droplet groups were 22.8% versus 23.9%, 22.2% versus 17.3%, 25.4% versus 18.3%, 33.7% versus 23.8%, and 33.8% versus 13.1%, respectively. The rates of good quality blastocysts on Day 5 in groups 4-5, 6-10,11-15,16-20 and 21-25 between WOW and droplet groups were 35.5% versus 37.5%, 37.3% versus 35.4%, 39.7% versus 39.4%, 45.5% versus 36.3%, and 48.2% versus 31.1% respectively. The differences between WOW and droplet groups in the 21-25 embryos group were significant (p<0.05) for the two parameters on day3 and day5 respectively. Furthermore, a positive correlation was observed between the number of cultured embryos and embryo development in WOW culture (p<0.01; the Cochran-Armitage Test), but not in droplet culture. A positive correlation was confirmed between the number of cultured embryos and embryo development in WOW culture. In the WOW culture system, increasing the number of cultured embryos, potentially causes elevations in the concentration of paracrine factors. These findings in a human embryo culture system are in line with previous reports from animal species and support the positive role of group culture for human embryo development.

Implantation and live births following transfer of 0pn embryos in natural cycle IVF

Implantation and live births following transfer of 0pn embryos in natural cycle IVF

Gonadotropin-releasing hormone agonist trigger increases the number of oocytes and embryos available for cryopreservation in cancer patients undergoing ovarian stimulation for fertility preservation

To compare the oocyte and embryo yield associated with GnRH-agonist triggers vs. hCG triggers in cancer patients undergoing controlled ovarian stimulation (COS) for fertilization preservation. Retrospective cohort study. Academic center. Cancer patients undergoing COS with letrozole and gonadotropins or gonadotropin-only protocols for oocyte or embryo cryopreservation. Gonadotropin-releasing hormone agonist or hCG trigger. Number of metaphase II (MII) oocytes or two-pronuclei (2PN) embryos available for cryopreservation were primary outcomes. Separate multivariate linear regression models were used to assess the effect of trigger type on the primary outcomes, after controlling for confounders of interest. A total of 341 patients were included, 99 (29.0%) in the GnRH-agonist group and 242 (71%) in the hCG group. There was no difference in the baseline demographics of patients receiving GnRH-agonist or hCG triggers. Within the letrozole and gonadotropins group (n = 269), the number (mean ± SD, 11.8 ± 5.8 vs. 9.9 ± 6.0) and percentage of MII oocytes (89.6% vs. 73.0%) available for cryopreservation was higher with GnRH-agonist triggers compared with hCG triggers. Similar results were noted with GnRH-agonist triggers in the gonadotropin-only group (n = 72) (i.e., a higher number [13.3 ± 7.9 vs. 9.3 ± 6.0] and percentage of MII oocytes [85.7% vs. 72.8%] available for cryopreservation). Multivariate linear regression demonstrated approximately three more MII oocytes and 2PN embryos available for cryopreservation in the GnRH-agonist trigger group, irrespective of cancer and COS protocol type. Utilization of a GnRH-agonist trigger increases the number of MII oocytes and 2PN embryos available for cryopreservation in cancer patients undergoing COS for fertility preservation.

Erratum to: Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.

As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos. 3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/μl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos. By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations. This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

Impact of hydrosalpinx fluid on early human embryos

ABSTRACTThis study aimed to investigate the impact of hydrosalpinx fluid (HF) on early human embryonic development. A total of 33 patients who underwent laparoscopic surgery for hydrosalpinx were selected, and the HF specimens obtained from these patients were subjected to bacterial culture, Chlamydia antigen detection, biochemical analysis, and cytokine detection. Meanwhile, human embryos derived from three pronuclei (3PN) were cultured in various HF concentrations. There was no significant difference in the chemical components and physical characteristics between colorless and colored HF specimens, apart from the glucose concentration which was significantly higher in colorless HF. K+ and HCO3− were significantly increased (P < 0.05 and P < 0.01), and Ca2+, Mg2+, and glucose were significantly decreased (P < 0.05, P = 0.006, and P = 0.007) in the two HF specimens, compared to blastocyst culture medium (G-2 medium); no phosphates were detected in the HF specimens. Compared to colorless HF, the concentrations of tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) in the colored HF specimens were significantly increased (P < 0.05). There were no differences in the Chlamydia antigen-positive rate between the HF groups (62.5% vs. 70.6%), and no bacterial growth occurred in the HF specimens. There were no significant differences in the development of the 3PN embryos between the two HF groups (P > 0.05). High-concentration HF (75%) significantly affected the rates of blastulation, blastocyst hatching, and high-quality blastocyst formation (P < 0.05). HF is related to chlamydial infection. Embryonic development may be significantly affected only in high-concentration HF, possibly due to the deficiency of essential elements required for embryonic development. TNF-α and IL-2 concentrations were found to vary between the clear and colored HF specimens; however, TNF-α and IL-2 in HF do not appear to exert adverse effects on embryonic development.

Impact of FMR1 Pre-Mutation Status on Blastocyst Development in Patients Undergoing Pre-Implantation Genetic Diagnosis

Background/Aims: The study aimed to investigate the impact of fragile X mental retardation 1 (FMR1) pre-mutation status on blastocyst development in patients undergoing pre-implantation genetic diagnosis (PGD). Methods: Case-control study of patients <40 years undergoing PGD at blastocyst stage for FMR1 pre-mutation status. Age-matched patients undergoing PGD for other single gene disorders were considered controls. Blastocyst development, calculated per metaphase II (MII) oocyte retrieved and per 2 pronuclear (2PN) embryos, was compared between the 2 groups. Pregnancy outcomes after embryo transfer were also compared. Results: Eighty-one and 791 patients were included in the FMR1 and control groups, respectively. FMR1 pre-mutation carriers had lower indicators of ovarian reserve, required higher gonadotropin doses, and had fewer MII oocytes retrieved. Mean blastocyst development per MII oocyte (12.6 vs. 29.4%; p < 0.001) and per 2PN embryos (21.5 vs. 41.7%; p < 0.001) was lower in the FMR1 group. An inverse correlation between the number of FMR1 CGG repeats and blastocyst development per MII oocyte (ρ = -0.63; p < 0.001) was observed. There was no difference in the rates of clinical pregnancy, spontaneous abortion, or live birth among the groups. Conclusion: Our study suggests lower rates of blastocyst development in patients with FMR1 pre-mutation status and an inverse correlation between the number of FMR1 CGG repeats and blastocyst development.

Clinical outcome of cycles with oocyte degeneration after intracytoplasmic sperm injection

ABSTRACTThere are variant rates of oocyte degeneration after intracytoplasmic sperm injection (ICSI) among different patients. Oocyte degeneration after ICSI may reflect the cohort of oocyte quality and subsequent embryo development capacity and clinical outcome. This retrospective study analyzed 255 cycles with at least one degenerated oocyte after ICSI (degeneration group) and 243 cycles with no degenerated oocytes after ICSI (control group). Basic characteristics like female age, body mass index, duration of infertility, hormone (FSH, LH, E2) levels on day 3 of menses, and primary infertility patient rate were similar between the two groups (p > 0.05). Total dose of gonadotropin and length of stimulation were also similar between the two groups (p > 0.05), but the degeneration group exhibited a more exuberant response to ovarian stimulation as reflected by more oocytes retrieved (p < 0.05). The number of 2PN embryos available and high quality embryos were similar between the two groups (p > 0.05), but the high quality embryo rate, early cleavage embryo rate, and available embryo rate were all statistically lower than the control group (p < 0.05). Embryo developmental kinetics seemed to be disturbed and embryo fragmentation rate increased in the degeneration group (p < 0.05). However, there was no statistical difference in the distribution of graded embryos transferred, and there were no statistical differences in the pregnancy rate, implantation rate, and abortion rate between the two groups (p > 0.05). We deduce that the presence of oocyte degeneration after ICSI may be associated with decreased embryo quality with embryo development kinetics disturbed. However, the clinical outcomes may not be affected if the premise that sufficient high quality degeneration group embryos are available for transfer.

Total number of oocytes and 2PN embryos are predictive of livebirth pregnancy in fresh donor oocyte cycles

The association of donor-recipient characteristics and peri-fertilization factors with live birth (LB) remains controversial and warrants further study. The objective of this study is to identify factors predictive of LB following fresh donor oocyte IVF cycles.

Revisiting oolemma characteristics during ICSI in relation to fertilization patterns and embryo development and implantation

The oolemma response during ICSI has been linked to membrane and cytoplasmic maturity. The former is evidenced by the depth of the funnel and can manifest in the unextrusion of the second polar body and oocyte lysis, while the latter in absent fertilization. We investigate the occurrence of normal and abnormal fertilization patterns, as well as oocyte survival in relation to ovarian stimulation and eventual reproductive outcomes. In couples with an adequate cohort of mature oocytes and varying degrees of fertilization, we measured the occurrence of immature membrane and ooplasm (as evident by oocyte lysis), impaired cytoplasmic maturity (as evident by no fertilization), lower membrane maturity (as evident by 3PN embryos) and complete membrane and cytoplasmic maturity (2PN). Only ICSI cycles with >5 oocytes at MII stage were included. Unextrusion of the second polar body, characterized by digynic 3PN, is associated with sudden breakage of the membrane, short funnel, and indicates ooplasmic maturity but membrane dysmaturity. Oocyte lysis is characterized by absence of funnel development and therefore severe membrane dysmaturity. Absent fertilization presented with adequate funnel development up to the center of the oocyte therefore allowing oocyte survival, but unfortunately with ooplasmic immaturity. ICSI cycles were stratified by fertilization rates, in increments of 20%. The rates of 3-PN embryos and oocyte lysis were plotted against increasing fertilization rates. Demographics and ovarian stimulation parameters were compared across fertilization groups. 11911 ICSI cycles were identified. The rate of digynic 3PN embryos decreased with increasing fertilization rate [7.19% (<20%) to 1.10% (81-100%)]. Similar trends in the rate of oocyte lysis were observed as well. There was no difference in the median age of patients (36), number of oocytes (9), or total stimulation days (10) across different fertilization groups. Patients with oocyte lysis had a progressively lower dose of gonadotropins (P=0.007) and peak E2 levels (P<0.001) in comparison to those that had no fertilization, digynic 3PN embryos, and both membrane and cytoplasmic maturity (all 2PN). To assess the impact of oolemma/ooplasmic maturity within the cohort of oocyte injected on the embryo developmental outcome, we compared patients with oocyte lysis to those who had normal fertilization (n=384). Lower implantation (16.7% vs. 32.4%; P<0.001) and clinical pregnancy rates (7.01% vs. 34.6%; P<0.001) were noted in the former group compared to the latter. During ICSI, membrane funnel characteristics may provide insight into oolemma maturity. Oocyte survival, absent/abnormal fertilization provides invaluable information about ooplasmic maturity and embryo developmental competence. Our study highlights the potential link between oolemma behavior, absent/abnormal fertilization and ovarian stimulation.

Time-lapse observation can help improve workflow and ensure the correct observation of fertilized embryos

In recent years, there have been some reports suggesting that embryos are evaluated as no pronuclei and two polar bodies (0PN2PB) using conventional microscopic observation but the embryos observed by time-lapse shows that these 2PN break down before the time when we normally observe (2PN-BD). However there are very few articles discussing the process and impact from such PN breakdown. Here, we report whether we can reduce the risk for incorrect observation of embryos following insemination by using time-lapse technology. From July 2014 to September 2014, 448 cycles with 2338 oocytes were cultured for 6h following conventional IVF (cIVF) or ICSI and observed at 20h after insemination in Embryo Scope (Vitrolife) then we cultured the embryos into moist incubator until Day5. We used Veeck’s criteria and Gardner’s criteria for embryo evaluation. Treatments used TESE-ICSI and rescue-ICSI and calcium ionophore were excluded. We calculated the ratio for which embryos were evaluated as 0PN2PB by conventional microscopic observation but showed 2PN by time-lapse observation. We also studied the cleavage rate, Day3 good quality embryo rate, Day5 blastocyst rate, and Day5 good quality blastocyst rate of these embryos. Furthermore, we analyzed the time from PN appearance to its breakdown. All statistical analyses were performed using chi squared analysis. Among 281 embryos which were evaluated as 0PN2PB using conventional microscopic observation, 141 embryos were evaluated as 2PN by time-lapse observation. The cIVF group has 2PN-BD embryos of 33.6% (38/113) and the ICSI group has the embryos of 61.3% (103/168). The 2PN-BD embryos show same development compared 2PN embryos in cIVF group. However, in the ICSI group, Day3 good quality rate, Day5 blastocyst rate and Day5 good quality blastocyst rate were significantly higher in the 2PN-BD group (P< 0.01). For the 1825 embryos which were evaluated as 2PN by time-lapse observation, the time from insemination to 2PN appearance was as follows; 8.6h±0.1h (5.5h-19.4h) for the cIVF group, and 6.9h±0.1h (3.6h-22.3h) for the ICSI group. The embryos which developed into 2PN later than 15 hours following insemination did not grow into good quality blastocysts. Finally, the time of 2PN-BD was 15.9h (ICSI) and 17.2h (cIVF) following insemination at earliest. It is very difficult to get the information of correct fertilization without using time-lapse system, because there are some embryos whose pronuclei break down early. Time-lapse technology can also help us improve workflow in the lab since actual observation does not need to be done at a specific time.

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm.

The objective of this study was to evaluate fertility and full-term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co-cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full-term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.

Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.

As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos. 3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100ng/μl) and guide RNA (gRNA) (50ng/μl). For oligo-injections, donor oligo-1 (99bp) or oligo-2 (99bp) (100ng/μl) or dsDonor (1kb) was mixed with Cas9 mRNA (100ng/μl) and gRNA (50ng/μl) and injected into the embryos. By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations. This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

Sequential versus Monophasic Media Impact Trial (SuMMIT): a paired randomized controlled trial comparing a sequential media system to a monophasic medium

To determine whether sequential or monophasic media is the more optimal formulation for blastocyst development and sustained implantation rates (SIR) in IVF. Paired randomized controlled trials. Academic. Infertile couples (N = 192) with female partner ≤42years old and normal ovarian reserve. Fertilized zygotes from each patient were randomly divided into two groups: [1] cultured in sequential media and [2] cultured in monophasic medium. Sequential media consisted of Quinn's Advantage Cleavage Medium (SAGE) followed by Blast Assist (Origio). The monophasic medium used was Continuous Single Culture (Irvine Scientific). Paired ETs were accomplished by transferring the best euploid blastocyst from each media group. DNA fingerprinting was used to link outcomes. The primary outcome measure was the proportion of blastocysts suitable for clinical use. Secondary outcome measures included timing of blastulation, aneuploidy rates, and SIR. Sustained implantation rate is defined as the number fetal heart beats at 8-9weeks of gestation, divided by the number of embryos transferred. A total of 192 patients had their 2PN embryos (N = 2,257) randomized to each culture system. Sequential media had higher blastulation rate than monophasic medium (55.2% vs. 46.9%). No differences were found in the day of blastulation or aneuploidy rate. Of the 168 patients who had euploid blastocysts suitable for transfer, 126 completed a paired ET. Among the double ETs, there was no difference in implantation between groups. This is the first randomized controlled trial to examine paired euploid transfers of sibling zygotes cultured in sequential versus monophasic media. This study demonstrates that the usable blastocyst rate is greatest after culture in the sequential media tested in comparison with the monophasic formulation selected for study. However, no difference exists in timing of blastulation, aneuploidy, or SIR. Whether these observations are generalizable to other media systems remains to be determined. NCT01917240.

Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography.

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.

IVF-ICSI Split Insemination Reveals those Cases of Unexplained Infertility Benefitting from ICSI Even when the DNA Fragmentation Index is Reduced to 15% or Even 5%

IVF-ICSI Split Insemination Reveals those Cases of Unexplained Infertility Benefitting from ICSI Even when the DNA Fragmentation Index is Reduced to 15% or Even 5% Purpose The aim of the study was to assess the fertilization rate (FR) of randomized sibling oocytes inseminated by conventional IVF or ICSI in couples with unexplained infertility. Methods The 16-month study was conducted at an established private IVF facility. Oocytes recovered from couples with normal semen parameters and normal DNA fragmentation index (DFI; <30%), were randomly allocated to IVF or ICSI and the FR (2PN/MII) was assessed. Pregnancy outcome following embryo transfers were analyzed with regards to either IVF-embryo vs. ICSI-embryo, and in relation to DFI levels. Results Of 585 oocytes retrieved from 38 patients, 463 were mature (MII). The ICSI group generated a significantly higher number of 2PN embryos with a mean FR of 83.4% vs. 67.6% (p<0.05). There were no cases of complete fertilization failure (CFF) in the ICSI group, but there were 7.9% in the IVF group. The significant difference of FR was observed only when the DFI level was ≥ 15% and if such cutoff was applied, the CFF cases would be reduced to 2.6%. Of the 30 patients who had fresh embryo transfers performed, the ICSI group showed a higher pregnancy rate (69.2% vs. 58.8%; N.S.) with a significantly higher mean DFI value in the non-pregnant group (p<0.05). Conclusions IVF-ICSI split insemination can reveal those cases which will benefit from ICSI even where semen parameters and DFI are normal; however if the DFI is reduced to a 15% cut-off level, the rate of CFF will be minimized, but not completely excluded, even at 5%..

Supplementing single step culture media with insulin for continuous uninterrupted in vitro culture of human embryos and monitoring the outcome: prospective randomized clinical trial

ulation and are less expensive. Both formulations have attained good outcomes, but an adequately powered assessment including usable blastulation rates (USBR), ploidy risk, and SIR is lacking. DESIGN: Paired RCT. MATERIALS AND METHODS: Patients with normal ovarian reserve were recruited. A paired design allowed each patient to serve as their own control, eliminating many confounding variables seen in prior studies. After confirming fertilization, patients’ zygotes were randomized (1:1 ratio) to either Seq media (Quinn’s Advantage Cleavage Medium, Sage then Blast Assist, Origio) or Mono media (Continuous Single Culture; Irvine Scientific). Each culture system used separate incubators. Assessed endpoints for all embryos included USBR, blastulation timing (arrest vs day 5 vs 6) and ploidy status. Paired euploid blastocyst transfers, one from each group, were performed. DNA fingerprinting of concepti was used as needed to link each embryo to a definitive outcome. SIR was defined as the presence of a fetal heart beat at 8-9 weeks gestation. Statistical analysis performed via McNemar’s Chi Square and Wilcoxon sum rank tests. RESULTS: 186 patients had their 2PN embryos (N1⁄42257) randomized to each culture system. Seq media had a higher blastulation rate then Mono media (p1⁄40.001, 55.5% vs. 46.0%). No differences were found in the day of blastulation (p1⁄40.4063) or in the aneuploidy rate (p1⁄40.5518). Of the 168 patients who had euploid blastocysts suitable for transfer, 126 completed a paired embryo transfer and 42 had a SET. Amongst the SETs, the SIR was equivalent (p1⁄41.0). Of the 126 double embryo transfers, 36 patients had one fetal heart beat at discharge, however there was no statistical difference in the likelihood of implantation between groups (p1⁄40.8642). CONCLUSIONS: This is the first randomized controlled trial to systematically examine paired euploid transfers of sibling zygotes cultured in in Seq vs. Mono media. This study demonstrates that the usable blastocyst rate is greatest after culture in Seq media in comparison to a Mono formulation; however no difference exists in SIR. Supported by: Irvine scientific provided Mono media.

Genome-wide uniparental disomy screen in human discarded morphologically abnormal embryos.

Uniparental disomy (UPD) has been shown to be rare in human normal blastocysts, but its frequency in discarded morphologically abnormal embryos and its relevance to embryonic self-correction of aneuploid remains unknown. The aim of this study was to detect UPD in discarded morphologically abnormal embryos. Both discarded morphologically abnormal embryos, including zero-pronuclear zygotes (0PN), one-pronuclear zygotes (1PN), three-pronuclear zygotes (3PN) and 2PN embryos scored as low development potential were cultured into blastocysts then underwent trophectoderm biopsy. Genome-wide UPD screening of the trophectoderm of 241 discarded morphologically abnormal embryo sourced blastocysts showed that UPD occurred in nine embryos. Five embryos exhibited UPDs with euploid chromosomes, and four displayed UPDs with chromosomal aneuploid. The percentage of UPDs among the morphologically abnormal sourced blastocysts was 3.73%, which is significant higher than the percentage observed in normal blastocysts. The frequency of UPD in 3PN-sourced blastocysts was 7.69%, which is significantly higher than that in normal blastocysts. This study provides the first systematic genome-wide profile of UPD in discarded morphologically abnormal embryos. Our results indicated that UPD may be a common phenomenon in discarded morphologically abnormal embryos and may be relevant to human embryonic self-correction.

Birth of nine normal healthy babies following transfer of blastocysts derived from human single-pronucleate zygotes.

The purpose of this study is to examine the clinical outcomes of blastocysts derived from human single-pronucleate (1PN) embryos after conventional in vitro fertilization (cIVF) and intracytoplasmic sperm injection (ICSI) cycles. This was a retrospective study at a reproductive center of a hospital. To evaluate embryo quality and clinical outcomes, cIVF or ICSI cycles with one or more 1PN embryos were compared with same cycles with 2PN embryos (control cycles). A total of 623cycles (426 cIVF cycles and 197 ICSI cycles) were treated with cIVF or ICSI. The single pronuclear status rate was similar between cIVF (22.1%) and ICSI (25.1%) cycles. Although the development rates of 1PN embryos on day 3 and day 5/6 in cIVF were significantly higher than those in ICSI, those of 1PN embryos in cIVF were significantly lower compared to 2PN embryos (p < 0.01). Nonetheless, the ongoing pregnancy rates achieved with 1PN blastocysts in 1PN embryos did not significantly differ from the control group. Thirty-three transfer cycles with 33 blastocysts derived from 1PN embryos in cIVF resulted in nine deliveries with no newborn malformations; however, no implantation was observed in three ICSI cycles. Although the blastocyst formation rate of 1PN embryos was significantly lower than 2PN embryos in cIVF and ICSI cycles, 1PN blastocysts in cIVF, and not from ICSI, demonstrated an adequate ongoing pregnancy rate. These results suggested that 1PN blastocysts in cIVF are available for clinical use and may lead to an increase in the chance of pregnancy in patients receiving assisted reproductive technology with 1PN embryos.

Does early cleavage correlate with chromosome constitution in human preimplantation embryos?

IntroductionThe purpose of this study is to analyze the correlation between the occurrence of early cleavage and chromosome constitution of embryos from preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Material and methodsA total of 595 embryos from 96 cycles were included in this retrospective study. ResultsWhen clinical pregnancy rates per transfer were compared, statistically significant differences were observed between patients that had at least one early cleavage (ECpos) embryo and patients no EC (ECneg) (35.7% vs. 6.7%; p<.001). Statistically significant differences were found between EC, No PN and 2PN embryos at 26h, good embryo quality at day 2 (71.4%, 55.6% and 40.7%, respectively; p<.05) and in blastocyst rate (43.3% EC vs. no early cleavage embryos (NEC) 27.1%; p<.001) in PGS cycles. These differences were not found in the PGD group. Early-cleaved embryos exhibited less chromosome abnormalities than No PN and 2PN group in PGS (61%, 69.4% and 77.2%; p<.05) and differences in PGD group (69.8%, 76.2% and 66.7%; p<.05). DiscussionEarly cleavage has shown to correlate with embryo quality, with the capacity to develop up to blastocyst stage, as well as with euploid chromosome constitution.

Value of transferring embryos that show no evidence of fertilization at the time of fertilization assessment

To determine the value of transferring embryos formed from nonpronuclear (0PN) zygotes. A case-control study. Not applicable. The current study was a retrospective analysis of embryo transfers of just 0PN embryos using fresh cleavage-stage embryos (0PN cleavage fresh), frozen-thawed cleavage-stage 0PN embryos (0PN cleavage frozen), and frozen 0PN blastocyst-stage embryos (0PN blast frozen). To study the effect of 0PN transfer, comparison groups were used: fresh cycles of 2PN (2PN cleavage fresh-C) and frozen-thawed cycles cleavage-stage (2PN cleavage frozen-C) and blastocyst-stage (2PN blast frozen-C). Comparison groups were matched for cycle and patient characteristics to the 0PN group. Implantation rate (IR), pregnancy rate, and transferable embryo rate. For fresh cycles, the IR in the 0PN cleavage fresh was lower than that in the 2PN cleavage fresh-C (8.04% vs. 19.50%, respectively). For frozen-thawed cycles, the IR in the 0PN cleavage frozen was lower than that in the 2PN cleavage frozen-C (15.38% vs. 28.24%, respectively), but the IR in 0PN blast frozen was comparable to that of 2PN blast frozen-C (39.56% vs. 48.18%, respectively). Transfer of 0PN embryos from fresh or frozen-thawed cycles results in pregnancies and live births. Nonpronuclear embryos have a lower IR than 2PN embryos, but if the embryos are cultured to the blastocyst stage and then are frozen, their IRs approach that of 2PN embryos in subsequent frozen-thawed cycles. The culture of 0PN embryos to the blastocyst stage may select for embryos with a near-normal IR.