Using time-lapse technology to explore vacuolization in embryos on Day 3 and Day 4.

PURPOSE: To investigate the occurrence and development state of embryo vacuoles between the 8-cell and morula stages, and to explore how vacuoles affected the development of embryos. METHODS: A retrospective study of a cohort of 422 patients undergoing conventional in vitro fertilization or intracytoplasmic sperm injection. With the help of time-lapse imaging, the development processes and outcomes of good quality embryos with or without vacuoles were analyzed. RESULTS: Vacuole positive embryos had significantly lower blastulation rate and good quality blastulation rate than vacuole negative embryos, p < 0.05. Compared to vacuole negative embryos, the number of best and good quality blastocysts was significantly reduced, while the number of fair and discarded ones was significantly increased, p < 0.05. The average starting time of vacuolization was 73.7 ± 9.3 h after insemination. The proportion of blastomeres affected by vacuoles was associated with embryonic developmental potential. CONCLUSIONS: Vacuolization on Day 3 and Day 4 was frequently observed and was detrimental to embryo development. The proportion of blastomeres affected by vacuoles may be an indicator of embryo developmental potential.

miR-15a-5p levels correlate with poor ovarian response in human follicular fluid.

Poor ovarian response is a significant problem encountered during in vitro fertilization and embryo transfer procedures. Many infertile women may suffer from poor ovarian response and its incidence tends to be increasing in young patients nowadays. It is a major cause of maternal infertility because it is associated with low pregnancy and live birth rates. However, the cause of poor ovarian response is not clear. In this study, we extracted microRNAs from human follicular fluid and performed miRNA sequencing to investigate a potential posttranscriptional mechanism underlying poor ovarian response. The results showed that many miRNAs were obviously different between the poor ovarian response and non-poor ovarian response groups. We then performed quantitative polymerase chain reaction, Western blot analysis and used an in vitro culture system to verify the sequencing results and to study the mechanism. Notably, we found that miRNA-15a-5p was significantly elevated in the young poor ovarian response group. Furthermore, we demonstrated that high levels of miR-15a-5p in the young poor ovarian response group repressed granulosa cell proliferation by regulating the PI3K-AKT-mTOR signaling pathway and promoted apoptosis through BCL2 and BAD. This could explain the reduced oocyte retrieval number seen in poor ovarian response patients.

Polycystic ovary syndrome susceptibility single nucleotide polymorphisms in women with a single PCOS clinical feature.

STUDY QUESTION: What is the direct genetic contribution of the polycystic ovary syndrome (PCOS) susceptibility single nucleotide polymorphisms (SNPs), identified by previous genome-wide association studies (GWAS) to the definitive clinical features of the syndrome? SUMMARY ANSWER: Each single PCOS clinical feature had a specific genetic association, and rs4385527 in the chromosome 9 open reading frame 3 (C9orf3) conferred a particular risk to the three defined PCOS clinical features in this study, which suggested its fundamental role in the etiology of PCOS. WHAT IS KNOWN ALREADY: PCOS is a heterogeneous disorder characterized by anovulation (OA), hyperandrogenism (HA) and polycystic ovary morphology (PCOM). Two previous GWAS in China have identified 15 independent susceptibility SNPs related to PCOS (PCOS-SNPs). However, little is known about the candidate gene of each clinical feature. STUDY DESIGN, SIZE, DURATION: Case-control study. Three independent groups of women were recruited from 2010 to 2012: 746 subjects with OA only, 278 subjects with HA only and 536 subjects with PCOM only. A total of 1790 healthy women with none of the above pathological characteristics were also enrolled as control subjects during the same time period. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants were women of reproductive age. Genotype and allelic frequencies of 15 PCOS-SNPs were determined in all subjects using direct sequencing and Sequenom Arrays. The allelic frequencies of each case group were compared with the controls. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustment for age and BMI, variants in luteinizing hormone/choriogonadotropin receptor (LHCGR) (rs13405728), C9orf3 (rs4385527) and insulin receptor gene (INSR) (rs2059807) were strongly associated with OA (Padjust < 0.01, <0.001 and <0.05, respectively); rs4385527 in C9orf3 was strongly associated with HA (Padjust< 0.001); variants in the thyroid adenoma associated gene (THADA) (rs13429458 and rs12478601), DENN/MADD domain containing 1A (DENND1A)(rs10818854), and C9orf3 (rs4385527) were significantly associated with PCOM (Padjust < 0.01, <0.001, <0.05 and <0.001, respectively). LIMITATIONS, REASONS FOR CAUTION: The sample size of some case groups was relatively small, which therefore limited the statistical power of the analysis to a certain extent. WIDER IMPLICATIONS OF THE FINDINGS: The present study indicates a potential common genetic basis of three PCOS clinical features. Other specific associated genes may play a synergistic role, leading to heterogeneous pathophysiological changes. Additionally, the increased frequency of PCOS-risk alleles in women with single PCOS clinical features suggests that these subjects have an elevated risk of developing the syndrome, although they cannot be currently diagnosed. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Basic Research Program of China (973 Program) (2012CB944700, 2011CB944502), the National Key Technology Research and Development Program(2011BAI17B00), the National Natural Science Foundation of China (81430029, 81201441, 81490743, 31371453), the Scientific Research Foundation of Shandong Province of Outstanding Young Scientist (2012BSE27089) and the Fundamental Research Funds of Shandong University(2014GN025). There were no competing interests.

Pregnancy with oocytes characterized by narrow perivitelline space and heterogeneous zona pellucida: is intracytoplasmic sperm injection necessary?

PURPOSE: This retrospective study analyzed fertilization protocols and pregnancy outcomes for oocytes with with narrow perivitelline space and heterogeneous zona pellucid (NPVS/HZP). METHODS: In 63 in-vitro fertilization cycles filled with NPVS/HZP oocytes (abnormal oocytes group) and 521 cycles with normal oocytes (normal oocytes group), major clinical and laboratory parameters were recorded and compared in different fertilization cycles (conventional IVF cycles, rescue ICSI cycles, and traditional ICSI cycles). RESULTS: NPVS/HZP oocytes meant lower MIIoocytes rates in both IVF and ICSI cycles compared with normal oocytes (p < 0.05). The 2PN rates for abnormal oocytes were significantly lower than those for normal oocytes in both conventional IVF cycles (58.8% VS 71.3%, P < 0.05) and rescue ICSI cycles (58.0% VS 78.0%, P = 0.0000). The high-quality embryo rates in normal oocytes groups were significantly higher than those in abnormal oocytes groups in different fertilization cycles (52.2% VS 35.0%, P < 0.01; 42.9% VS 23.9%, P < 0.001; 50.6% VS 31.0%, P = 0.0000, respectively). No clinical pregnancy was obtained from abnormal oocytes in 11 conventional IVF cycles. The clinical pregnancy rates in rescue ICSI and traditional ICSI cycles were comparatively lower in abnormal oocytes groups, but there was no significant difference as compared with normal oocytes groups (35.0% VS 48.1% and 26.7% VS 50.7%, P > 0.05, respectively). CONCLUSIONS: Retrieval of oocytes characterized by NPVS/PZP from cycle to cycle was one of the reasons for obscure infertility. ICSI may be the right way to avoid fertilization failure and get pregnancy in women with NPVS/HZP oocytes.

Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles.

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3-5 h (Group B) or 24-28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P<0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P<0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.

Human cleaving embryos enable robust homozygotic nucleotide substitutions by base editors.

Base editing installs a precise nucleotide change in specific gene loci without causing a double-strand break. Its efficiency in human embryos is generally low, limiting its utility in functional genetic studies. Here, we report that injecting base editors into human cleaving two-cell and four-cell embryos results in much higher (up to 13-fold) homozygotic nucleotide substitution efficiency as opposed to MII oocytes or zygotes. Furthermore, as a proof-of-principle study, a point mutation can be efficiently corrected by our method. Our study indicates that human cleaving embryos provide an efficient base editing window for robust gene disruption and correction.

Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells.

The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies.

[Comparison of vitrification and slow-freezing of human day 3 cleavage stage embryos: post-vitrification development and pregnancy outcomes].

OBJECTIVE: To compare the effects of vitrification with slow-freezing on the developmental ability of day 3 cleavage stage embryos. METHODS: Patients who had no less than 4 high quality embryos were included in this study. These embryos were cryopreserved using the methods of vitrification or slow-freezing. In the cryopreserved embryo transfer cycles, the embryos which were cryopreserved using one of the methods were chosen randomly. The developmental ability of embryos was compared between these two groups. RESULTS: A total of 80 patients were included in this study with 160 embryos. In the group of slow-freezing, 73 (91%) embryos were survived and achieved 15 (38%) clinical pregnancies. Among these, 3 were twins and the implantation rate was 25% (18/73). In the group of vitrification, 71 (89%) embryos were survived and achieved 19 (48%) clinical pregnancies. Among these, 9 were twins and the implantation rate was 39% (28/71), which was significantly higher than the slow-freezing group (P < 0.05). Otherwise, the clinical pregnant rate and multiple pregnant rate was higher in the group of vitrification than the slow-freezing group, but had no significance. CONCLUSION: Vitrification is more benefit for the developmental ability of the thawed embryos and more suitable for the cryopreservation of day 3 cleavage stage embryos.

Synthesis, biological evaluation and modeling studies of terphenyl topoisomerase IIα inhibitors as anticancer agents.

We report the synthesis and evaluation of a series of novel terphenyls. Compound 17 had the most potent anticancer activity, indicating that the phenolic hydroxyl was a key group. A DNA relaxation test showed that compound 17 had a strong inhibitory effect on TOP2α, but not on TOP1, which was consistent with the docking analysis results. We performed a 3D-QSAR study using CoMFA and CoMSIA to determine, for the first time, the chemical-biological relationship in the inhibition of TOP by terphenyls. The CoMFA and CoMSIA model had good modeling statistics: leave-one-out q(2) of 0.605 and 0.622, r(2) of 0.998 and 0.994, and r(2)pred (test set) of 0.742 and 0.660. These results suggest that the ortho-phenolic hydroxyl on ring A is important for producing terphenyls with more efficacious activity.

Elective single blastocyst transfer is more suitable for normal responders than for high responders.

BACKGROUND: Embryo quality and receptivity of the endometrium are two factors that determine the results of in vitro fertilization/intra-cytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). There is no consensus of the optimal transfer strategy for normal responders or high responders. The current study aimed to find the optimal transfer strategy for different subgroups of patients. METHODS: From April 2010 to December 2010, patients who meet the following criteria were included in this study; primary infertility, female age ≤ 35 years, FSH level on female cycle day 2 - 3 ≤ 12 mIU/ml, at least six good quality embryos available on day three. The clinical outcomes using different transfer strategies between normal responders and high responders were reviewed and compared. RESULTS: For the normal responders, the clinical pregnancy rate of day three double-embryo transfer (DET) was comparable to that of day five elective single blastocyst transfer (eSBT), 64.04% vs. 60.33% (P > 0.05). For the high responders, the clinical pregnancy rate of day five eSBT was significantly lower than that of day three DET, 43.35% vs. 57.21% (P < 0.05). For the high responders, the rates of clinical pregnancy and implantation in frozen-thawed embryo transfer (FET) cycles were notably higher than in eSBT cycles (64.56% vs. 43.35% and 62.11% vs. 43.35% respectively) (P < 0.05). CONCLUSIONS: For normal responders, eSBT might be an applicable strategy to reduce multiple pregnancy rates while maintaining acceptable overall pregnancy rates. And in order to reduce multiple pregnancies and increase the chance of pregnancy of high responders, FET may be a preferable strategy.