Chromatin Accessibility Landscape in Human Early Embryos and Its Association with Evolution.

The dynamics of the chromatin regulatory landscape during human early embryogenesis remains unknown. Using DNase I hypersensitive site (DHS) sequencing, we report that the chromatin accessibility landscape is gradually established during human early embryogenesis. Interestingly, the DHSs with OCT4 binding motifs are enriched at the timing of zygotic genome activation (ZGA) in humans, but not in mice. Consistently, OCT4 contributes to ZGA in humans, but not in mice. We further find that lower CpG promoters usually establish DHSs at later stages. Similarly, younger genes tend to establish promoter DHSs and are expressed at later embryonic stages, while older genes exhibit these features at earlier stages. Moreover, our data show that human active transposons SVA and HERV-K harbor DHSs and are highly expressed in early embryos, but not in differentiated tissues. In summary, our data provide an evolutionary developmental view for understanding the regulation of gene and transposon expression.

Comparison of clinical outcomes of frozen-thawed D5 and D6 blastocysts undergoing preimplantation genetic testing.

BACKGROUND: This study aimed to analyze the clinical outcomes of blastocyst which undergo the preimplantation genetic testing (PGT) transplantation from frozen-thawed D5 and D6. In addition, the effect of blastocyst grade on clinical and neonatal outcomes was also investigated in this study. METHODS: The pregnancy and miscarriage rates of 1130 cycles of frozen embryo transfer, including 784 D5 frozen embryos and 346 D6 frozen embryos in the Reproductive Hospital of Shandong University from January to December 2020 were analyzed. Gardner blastocyst scoring was used for blastocyst evaluation. RESULTS: The pregnancy rate of D5 blastocyst was significantly higher, whereas the miscarriage rate of D5 blastocyst was lower, than that of D6 blastocyst tissue biopsy. No significant difference was observed in birth weight and low birth weight of D5 blastocyst and D6 blastocyst, preterm birth, gestational age, and neonatal sex. Frozen-thawed D5 blastocysts have higher pregnancy success rates and lower miscarriage rates compared to D6 blastocysts. CONCLUSION: Therefore, both blastocyst grade and embryo biopsy date must be considered when transferring frozen embryos.

Conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI): which is preferred for advanced age patients with five or fewer oocytes retrieved?

PURPOSE: To investigate the clinical outcomes of conventional IVF and ICSI in female patients aged 40 years and over with no more than five oocytes retrieved and non-male factor infertility. METHODS: A retrospective study of a cohort of 644 patients undergoing IVF/ICSI treatment. The 534 female patients aged ≧ 40 years with no more than five oocytes retrieved and non-male factor infertility undergoing their first conventional IVF cycles were assigned in IVF group. The rest of 110 patients aged 40 years and over with no more than five oocytes retrieved and non-male factor infertility undergoing first ICSI cycles were recruited in ICSI group. RESULTS: Our results showed the clinical pregnancy, live birth and miscarriage rates were similar between the IVF and ICSI groups (21.59% vs. 13.25%, P > 0.05; 12.16% vs. 6.02%, P > 0.05; 43.68% vs. 54.55%, P > 0.05; respectively), however, the implantation and cumulative live-birth rates were significantly higher in the IVF compared to the ICSI group (15.11% vs. 7.75%, 14.59% vs. 5.56%, P < 0.05), though the IVF group had a lower normal fertilization rate (61.56% vs. 76.00%, P < 0.001). CONCLUSIONS: Our study provides strong evidences that the conventional IVF exhibits advantages over the ICSI method in non-male factor infertility for advanced age patients with five or fewer oocytes retrieved.

Morphokinetic parameters from a time-lapse monitoring system cannot accurately predict the ploidy of embryos.

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

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.

Morphological good-quality embryo has higher nucleus spreading rate/signal resolution rate in fluorescence in situ hybridization.

PURPOSE: To evaluate the relationship between day 3 embryo quality and nucleus spreading rate/signal resolution rate in Fluorescence in situ hybridization (FISH) during the PGD procedure. METHODS: This study was a retrospective data analysis. 367 day-3 embryos were classified based on morphological scoring: grade 1 to grade 4 were defined from worse to better embryo quality. Day 3 embryos were classified as good quality when the number of blastomeres was between 6 and 10 and grade better than 2'. Nucleus spreading rate, signal rate and the full signal rate were compared between embryos with different morphological scoring. RESULTS: Nucleus spreading rate of blastomeres from morphological high-quality embryos was significantly higher (86.25 %) than from poor-quality embryos (76.53 %) (p < 0.05). The rate of blastomeres with full signals was significantly higher (79.32 %) in the morphological high-quality group than in poor-quality group (64.54 %) (p < 0.05). Similar results were found from day 3 embryos with cell number between 6 cells and 10 cells (nucleus spreading rate 86.01 vs. 76.34 %, p < 0.05; full signal rate 78.72 vs. 62.71 %, p < 0.05). Both have no significant difference in the signal rate (82.67 vs. 89.66 %; 83.10 vs. 89.95 %). CONCLUSIONS: Blastomeres from day 3 embryos with better morphological quality had higher nucleus spreading rate and higher full signal rate during FISH. Through this study, we speculate on whether it should reconsider the necessity of FISH application in embryos with poor quality.

Dynamics of histone acetylation during human early embryogenesis.

It remains poorly understood about the regulation of gene and transposon transcription during human early embryogenesis. Here, we report that broad H3K27ac domains are genome-widely distributed in human 2-cell and 4-cell embryos and transit into typical peaks in the 8-cell embryos. The broad H3K27ac domains in early embryos before zygotic genome activation (ZGA) are also observed in mouse. It suggests that broad H3K27ac domains play conserved functions before ZGA in mammals. Intriguingly, a large portion of broad H3K27ac domains overlap with broad H3K4me3 domains. Further investigation reveals that histone deacetylases are required for the removal or transition of broad H3K27ac domains and ZGA. After ZGA, the number of typical H3K27ac peaks is dynamic, which is associated with the stage-specific gene expression. Furthermore, P300 is important for the establishment of H3K27ac peaks and the expression of associated genes in early embryos after ZGA. Our data also indicate that H3K27ac marks active transposons in early embryos. Interestingly, H3K27ac and H3K18ac signals rather than H3K9ac signals are enriched at ERVK elements in mouse embryos after ZGA. It suggests that different types of histone acetylations exert distinct roles in the activation of transposons. In summary, H3K27ac modification undergoes extensive reprogramming during early embryo development in mammals, which is associated with the expression of genes and transposons.

Optimization of propofol loaded niosomal gel for transdermal delivery.

Propofol is an oily liquid widely used for rapid onset of anaesthesia via intravenous route, which shows major limitations of hypersensitivity, anaphylactic reactions and pain. The aim of the present work was to bypass the above issues by formulating tailored niosomal gel to deliver propofol via non-invasive transdermal route. The niosomes were prepared by film hydration method and sonication using cholesterol and Span 80. The Box Behnken design (BBD) was applied to optimize the size (93.5 nm) and the entrapment efficacy (81.5%) of the niosomes by selecting cholesterol at 139 mg, Span 80 at 0.525% and sonication time at 5.13 min. The scanning electron microscopy image showed spherical shape niosomes with smooth surface without aggregation. The ex vivo release data showed significant improvement in the propofol release (92.2% after 10 h) using niosomes in comparison to the control propofol gel (with 30% methanol) without niosomes (25.3% after 10 h). The in vivo pharmacokinetic parameters in the rat model confirmed the improvement in the relative bioavailability with optimized niosomal gel (relative bioavailability = 12.12) in comparison to the control propofol gel. In conclusion, the niosomal gel offered a potential alternative non-invasive route to deliver propofol for procedural sedation especially in pediatric population.

Application of improved single blastomere fixation technique in preimplantation genetic diagnosis.

To modify a fixation method improving the intensity and clarity of the single blastomeric signal detection by fluorescence in situ hybridization (FISH) in preimplantation genetic diagnosis. 333 cycles of assisted reproduction with preimplantation genetic diagnosis FISH (PGD-FISH) performed in our hospital were analyzed and a total of 3452 single blastomeres were obtained. For the conventional fixation method, the blastomeres were kept in 0.1% sodium citrate with 0.2 mg/ml bovine serum albumin (BSA) for 2-5 min. FISH was performed and the internal relationship between embryo quality and fixed rate, signal detection rate, and signal determination rate was explored. With the modified method, 91.54% of blastomeres were fixed, while 88.30% were fixed with the conventional method. The signal detection rate was significantly increased for the modified group than for the conventional group (compared 98.53% with 94.78%, P < 0.001). Especially, the signal determination rate also showed a significant difference between the two methods (compared 90.51% with 74.17%, P < 0.001). After the development of the fixation method, the fixation efficiency and the signal determination rate were greatly improved, providing more definite diagnosis for the patient. It will hopefully allow more assisted reproduction programs to offer their patients preimplantation genetic diagnosis with FISH.

Antinociceptive and anti-inflammatory effects of cryptotanshinone through PI3K/Akt signaling pathway in a rat model of neuropathic pain.

Peripheral nerve injuries often induce neuropathic pain through inflammation. Cryptotanshinone isolated from Salvia miltiorrhiza Bunge has been found to exert anti-inflammatory and analgesic activities. Thus, this study aimed to determine whether cryptotanshinone inhibits chronic constriction injury (CCI)-induced neuropathic pain in rats and its mechanism of action. CCI was performed by applying four loose ligatures to rat sciatic nerve. Cryptotanshinone was orally administered using pre-surgery, acute or repeated post-surgery treatment. The pain behaviors were determined by recording paw withdrawal mechanical threshold (PWMT) and thermal withdrawal latency (TWL). ELISA kits were used to measure interleukin (IL)-6, IL-1ß and tumor necrosis factor (TNF)-α levels. qRT-PCR were performed to detect IL-6, IL-1ß, TNF-α, PI3K and Akt expression. The phosphorylation of PI3K/Akt signaling was assessed using western blotting. PWMT and TWL in CCI group were higher than those in the control and sham groups. The acute post-CCI cryptotanshinone treatment but not pre-surgery treatment reduced PWMT and TWL. The effect of cryptotanshinone is more prominent when it was repeatedly administered after CCI. The CCI-induced increase in IL-6, IL-1ß, TNF-α, PI3K/Akt signaling and their phosphorylation was also suppressed by repeated post-CCI cryptotanshinone treatment. This study suggested that post-CCI cryptotanshinone treatment reduced the surgery-induced neuropathic pain by suppressing PI3K/Akt signaling therefore inhibited inflammation.

Application of testicular spermatozoa cryopreservation in assisted reproduction.

OBJECTIVE: To evaluate the efficiency of spermatozoa cryopreservation and to compare the clinical pregnancy outcomes in intracytoplasmic sperm injection (ICSI) using fresh versus cryopreserved spermatozoa collected by testicular sperm aspiration (TESA). METHODS: A retrospective study was performed to compare the outcomes of men who accepted frozen-spermatozoa-based TESA-ICSI with those of men who underwent TESA-ICSI using fresh spermatozoa between January 1, 2015, and December 30, 2016. The groups were matched for age. The rates of fertilization, good-quality embryos, blastocyst formation, and clinical pregnancy outcomes were obtained from clinical records and were compared between the groups. RESULTS: There were no significant differences between the frozen TESA group (n=79) and the fresh TESA group (n=194) in the rates of fertilization (71.4% vs 73.4%), good-quality embryos (55.3% vs 54.5%), blastocyst formation (60.9% vs 60.1%), clinical pregnancy (61.7% vs 55.1%), and live delivery (51.1% vs 45.7%) (P>0.05 for all comparisons). CONCLUSION: Freezing low-count sperm collected by TESA with a cryoprotectant was an efficient method in the treatment of male factor infertility.

ARA 290 relieves pathophysiological pain by targeting TRPV1 channel: Integration between immune system and nociception.

ARA 290 is an erythropoietin-derived polypeptide that possesses analgesic and tissue protective effect in many diseases such as diabetes and cancer. The analgesic effect of ARA 290 is mediated by its anti-inflammatory and immunomodulatory functions, or more specifically, by targeting the innate repair receptor (IRR) to down-regulate inflammation to alleviate neuropathic pain. However, whether other mechanisms or pathways are involved in ARA 290-mediated analgesic effect remains elusive. In this study, we are particularly interested in whether ARA 290 could directly target peripheral nociceptors by blocking or influencing receptors in pain sensation. Using calcium imaging, cell culture and behavioral tests, we demonstrated that ARA 290 was able to specifically inhibit TRPV1 channel activity, and relieve the mechanical hypersensitivity induced by capsaicin. Our study suggested that ARA 290 could potentially function as a novel antagonist for TRPV1 channel. This finding would not only contribute to the development of new pain treatment using ARA 290, but also help to improve our understanding of the integration between the immune system and the peripheral nervous system.

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.