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The selection of embryos is a key for the success of in vitro fertilization (IVF). However, automatic quality assessment on human IVF embryos with optical microscope images is still challenging. In this study, we developed a clinical consensus-compliant deep learning approach, named Esava (Embryo Segmentation and Viability Assessment), to quantitatively evaluate the development of IVF embryos using optical microscope images. In total 551 optical microscope images of human IVF embryos of day-2 to day-3 were collected, preprocessed, and annotated. Using the Faster R-CNN model as baseline, our Esava model was constructed, refined, trained, and validated for precise and robust blastomere detection. A novel algorithm Crowd-NMS was proposed and employed in Esava to enhance the object detection and to precisely quantify the embryonic cells and their size uniformity. Additionally, an innovative GrabCut-based unsupervised module was integrated for the segmentation of blastomeres and embryos. Independently tested on 94 embryo images for blastomere detection, Esava obtained the high rates of 0.9940, 0.9121, and 0.9531 for precision, recall, and mAP respectively, and gained significant advances compared with previous computational methods. Intraclass correlation coefficients indicated the consistency between Esava and three experienced embryologists. Another test on 51 extra images demonstrated that Esava surpassed other tools significantly, achieving the highest average precision 0.9025. Moreover, it also accurately identified the borders of blastomeres with mIoU over 0.88 on the independent testing dataset. Esava is compliant with the Istanbul clinical consensus and compatible to senior embryologists. Taken together, Esava improves the accuracy and efficiency of embryonic development assessment with optical microscope images.

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PURPOSE: By exploring the role of miRNAs in human oocyte development, the study was conducted to investigate the epigenetic mechanism contributing to the arrest of oocyte development. METHODS: In total, 140 oocytes from 22 patients were collected in the developmentally arrested oocyte (DAO) group, whereas 420 oocytes from 164 patients were harvested in the control group. The pooled RNA was extracted from all 20 oocytes to establish a RNA library. The total RNA of every ten oocytes was extracted for qPCR validation of miRNA candidates. Bioinformatic software was applied to explore the miRNA candidates and their target genes. RESULTS: Generally, the expression levels of miRNAs altered slightly during normal oocyte development but changed dramatically in the DAOs. Among the top 10 differential miRNAs, let-7a-5p and let-7g-5p, which were abundantly expressed throughout the oocyte development stages, had the broadest biological impact on oogenesis. Validated by qRT-PCR, both miRNAs were profoundly suppressed in the DAOs. During normal oocyte development, the expression levels of let-7a-5p and let-7g-5p at the GV stage were significantly higher than at MI and MII stages. Bioinformatic analysis demonstrated that let-7a-5p and let-7g-5p might regulate oocyte development by targeting PI3K-Akt, P53, cell cycle, and FoxO signaling pathways. CONCLUSIONS: There are dramatic differences in miRNA landscapes between the human oocytes with or without development arrest. In addition, the suppression of let-7a-5p and let-7g-5p might be associated with the occurrence of development arrest. The findings could provide therapeutic targets to correct the arrest of oocyte development in the future.

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The purpose of the present study was to investigate the expression profile of leucine-rich repeat-containing protein 8A (LRRC8A) in osteosarcoma and normal cortical bone, as well as its association with sex, age and tumor malignancy. Immunohistochemical staining of osteosarcoma tissue microarrays (TMAs) was performed to determine the protein expression of LRRC8A and compare them among different subgroups. The expression of LRRC8A in the nuclei and cytoplasm of U2OS tumor cells and MC3T3-E1 osteoblast-like cells was determined using reverse transcription-quantitative PCR. Of all samples of the TMA for patients with osteosarcoma that were tested, 94% featured high cytoplasmic expression of LRRC8A, while in all normal bone tissue control groups, the gene was mainly expressed in the nucleus. In MC3T3-E1 osteoblasts, the expression of LRRC8A at the RNA level was mainly in the cytoplasm. The difference in expression of LRRC8A between microarrays and osteoblasts was statistically significant. In U2OS osteosarcoma cells, LRRC8A mRNA was concentrated in the nuclei and cytoplasm. In osteosarcoma, the expression level of LRRC8A was not significantly associated with sex or age. In conclusion, LRRC8A was highly expressed in the cytoplasm of osteosarcoma cells and the degree of expression may be associated with the degree of tumor malignancy.

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BACKGROUND Recently, ClC-3 chloride channel expression has been noted to be high in some tumors. In chondrosarcoma, which is a malignant tumor with a high incidence in the bone, there has been no previous literature regarding ClC-3 chloride channel expression. Here we evaluated the expression of ClC-3 chloride channel in chondrosarcoma and explored its clinical significance. MATERIAL AND METHODS In this study, 75 chondrosarcoma and 5 normal cartilage tissues were collected. Thereafter, tissue microarray was performed. Immunohistochemistry was also used to observe the level of ClC-3 chloride channel expression between normal and chondrosarcoma tissues. RESULTS Results showed that the expression of ClC-3 chloride channel in the normal chondrocyte was thinner, since it showed distinct differentiation among chondrosarcoma specimens. Interestingly, we noticed that the moderately-differentiated chondrosarcoma (MDC) and the poorly-differentiated chondrosarcoma (PDC) exhibited 94.44% of ClC-3 chloride channel. Besides, the subcellular localization of ClC-3 chloride channel was changed in association with malignant degree changes. The subcellular localization of ClC-3 chloride channel in the MDC and PDC tissue was localized in the cytoplasm and both nucleus and cytoplasm: 83.33% (5 out of 6 cases) and 91.66% (11 out of 12 cases) respectively. On the other hand, we noticed that patient age and gender could have a relation with ClC-3 chloride channel expression; 30- to 60-year-old males showed more expression. CONCLUSIONS These results demonstrated a high frequency of ClC-3 chloride channel overexpression and subcellular localization differences in MDC and PDC tissue, suggesting a specific role of ClC-3 chloride channel in the pathogenesis of chondrosarcoma.

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Silica gel plays an important role in the formation of some biomorphic minerals (e.g. silica-carbonates) with morphologically complex micro/nanostructures in a pure inorganic system. Herein, we demonstrate the potential of strontium zinc silicate (Sr2ZnSi2O7, SZnS) bioceramics as a biomorphic mineral "garden" due to its incongruent dissolution behavior. Briefly, the preferential release of Sr ions from SZnS leaves behind a silica-rich gel on the ceramic surface and leads to an alkaline pH in the localized area close to the silica-rich gel, providing a growth condition similar to that for the conventional synthesis of biomorphic minerals. Based on this unique characteristic of SZnS, a continuous and integrated carbonated calcium-phosphate mineralized layer was formed on 3D porous SZnS scaffolds with the purpose of enhancing scaffold's bioactivity. The mineralized layer not only provides numerous nanotopographic cues for guiding cell behavior, but also avoids burst ion release, thus overcoming side effects caused by the overdose of bioactive ions and the over-high pH. In vitro cell culture experiments and in vivo animal studies demonstrate that the scaffold with nanostructured mineralized layers promotes osteogenic differentiation of osteoblasts and induce more new bone tissues compared to the non-mineralized scaffold.

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PURPOSE: To explore the optimal timing for hCG triggering by investigating the impact of different proportion of dominant follicles on the oocyte developmental competence. METHODS: One hundred ninety-eight infertile women were divided into three groups according to the proportion of dominant follicles on hCG day: (1) low: <15% (n = 66); (2) middle: 15-27% (n = 66); (3) high: >27% (n = 66). The grouping criteria were the bottom and top tertiles of the proportion of dominant follicles. RESULTS: The gonadotropin dosage, duration and maximum follicle diameter in the low proportion group were lower than those in the middle and high proportion groups. Oocyte maturation and the abnormal fertilization rate in the low proportion group were lower than those in the middle and high proportion groups. The normal fertilization rate did not differ among the three groups. The cleavage rate and number of transferable embryos in the low proportion group were significantly higher than those in the high proportion group. The high-quality embryo rate, implantation rate, and pregnancy rate in the low proportion group were significantly higher than those in the middle and high proportion groups. CONCLUSIONS: A high proportion of dominant follicles are closely associated with impaired oocyte developmental competence and low pregnancy rate. These findings suggest that follicular overgrowth induced by delayed hCG triggering may undermine oocyte developmental competence and the proportion of dominant follicles may be a potential parameters for hCG triggering.

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BACKGROUND: Oocyte secreted factors (OSFs), including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play an important role in the process of follicular development and oocyte maturation. Since OSFs are expressed in oocytes and cumulus granulosa cells, the aim of the present study was to explore whether the expression levels of GDF9 and BMP15 mRNAs in cumulus granulosa cells can be used as molecular markers for predicting oocyte developmental potential. METHODS: Cumulus cells of 2426 cumulus-oocyte complexes were collected from 196 female patients who underwent intracytoplasmic sperm injection (ICSI) and were used for mRNA detection on the egg retrieval day. Pearson correlation analysis was used to analyze the correlation between OSF expression and general physiological parameters. Partial correlation analysis was used to analyze the correlation between OSF expression and oocyte developmental potential. Covariance analysis was used to compare OSF expression among different groups. Receiver operating characteristic curves were used to examine the diagnostic value of GDF9 and BMP15 mRNA for predicting pregnancy. RESULTS: The expression levels of GDF9 and BMP15 mRNAs were significantly associated with age, body mass index (BMI), oocyte maturation, normal fertilization, and cleavage rate (P < 0.05). The expression levels of GDF9 and BMP15 mRNAs in the group with high-quality embryos were significantly higher than those in the group without high-quality embryos (P < 0.05). The expression levels of GDF9 and BMP15 mRNAs in the pregnancy group were significantly higher than those in the nonpregnancy group (P < 0.05). The cut-off value of GDF9 mRNA for predicting pregnancy was 4.82, with a sensitivity of 82% and a specificity of 64%. The cut-off value of BMP15 mRNA for predicting pregnancy was 2.60, with a sensitivity of 78% and a specificity of 52%. CONCLUSIONS: The expression levels of GDF9 and BMP15 mRNAs were closely associated with oocyte maturation, fertilization, embryo quality, and pregnancy outcome; therefore, GDF9 and BMP15 mRNAs in cumulus granulosa cells may be considered as new molecular markers for predicting oocyte developmental potential.

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Our previous study has demonstrated that luteinized granulosa cells (GCs) have the potential to proliferate and that the telomerase activity (TA) of luteinized GCs may predict the clinical outcomes of IVF treatment. However, in the field of telomere research, there have always been different opinions regarding the significance of TA and telomere length (TL). Thus, in the present study, we compared the effects of these two parameters on IVF treatment outcomes in the same individuals. TL did not differ significantly between the pregnant group and the non-pregnant group. The TA, number of retrieved oocytes and rate of blastocyst transfer were significantly higher in the pregnant group than in the non-pregnant group (0.8825 OD×mm, 12.75±2.20 and 34.48%, respectively, in the pregnant group vs 0.513 OD×mm, 11.60±0.93 and 14.89%, respectively, in the non-pregnant group (P<0.05)), while basal FSH level was lower in the pregnant group than in the non-pregnant group. The subjects did not differ with regard to ovarian stimulation or other clinical characteristics. A TA increase of 1 OD×mm increased the chance of becoming pregnant 4.769-fold (odds ratio: 5.769, 95% CI: 1.434-23.212, P<0.014). The areas under the receiver operating characteristic curves were 0.576 for TL and 0.674 for TA (P=0.271 and P<0. 012 respectively). The corresponding cut-off points were 4.470 for TL and 0.650 OD×mm for TA. These results demonstrate that TA is a better predictor of pregnancy outcomes following IVF treatment than TL. No other clinical parameters, including age, baseline FSH level or peak oestradiol level, distinguished between the pregnant group and the non-pregnant group as effectively as TA.

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OBJECTIVE: To evaluate male serum anti-Müllerian hormone (AMH) in assessing semen quality and predicting the pregnancy outcomes of assisted reproductive technology (ART). METHODS: A total of 103 male patients under ICSI were allotted to Groups A (normal sperm concentration control, n = 29), B (oligospermia, n = 27), C (obstructive azoospermia, n = 29) and D (non-obstructive azoospermia, n = 18). The contents of serum AMH and other related sexual hormones were determined by ELISA, and their correlations were analyzed with the seminal quality on the day of semen collection and with the pregnancy outcomes after ICSI. RESULTS: The contents of male serum AMH were (5.03 +/- 0.44), (3.70 +/- 0.44), (5.39 +/- 0.71) and (7.31 +/- 1.64) pmol/L, respectively, in Groups A, B, C and D, with no statistically significant differences among the four groups (F = 2.02, P > 0.05). The egg fertilization rate of the 103 couples was (76.13 +/- 23.66) %, not significantly correlated with the male serum AMH level (P > 0.05). The contents of male serum AMH in the pregnancy and non-pregnancy groups were (6.19 +/- 1.05) and (4.72 +/- 1.64) pmol/L, respectively, with no significant difference (t = 1.281, P > 0.05). CONCLUSION: The level of male serum AMH can neither reflect spermatogenesis of men nor predict the egg fertilization rate and pregnancy outcomes after ICSI, and therefore cannot be used alone as a serological predictive marker of ICSI outcomes.

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