The density of the inner cell mass is a new indicator of the quality of a human blastocyst: a valid supplement to the Gardner scoring system.

STUDY QUESTION: Can the density of the inner cell mass (ICM) be a new indicator of the quality of the human blastocyst? SUMMARY ANSWER: The densification index (DI) developed in this study can quantify ICM density and provide positive guidance for ploidy, pregnancy, and live birth. WHAT IS KNOWN ALREADY: In evaluating the quality of ICM, reproductive care clinics still use size indicators without further evaluation. The main disadvantage of this current method is that the evaluation of blastocyst ICM is relatively rough and cannot meet the needs of clinical embryologists, especially when multiple blastocysts have the same ICM score, which makes them difficult to evaluate further. STUDY DESIGN, SIZE, DURATION: This observational study included data from 2272 blastocysts in 1991 frozen-thawed embryo transfer (FET) cycles between January 2018 to November 2021 and 1105 blastocysts in 430 preimplantation genetic testing cycles between January 2019 and February 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: FET, ICSI, blastocyst culture, trophectoderm biopsy, time-lapse (TL) monitoring, and next-generation sequencing were performed. After preliminary sample size selection, the 11 focal plane images captured by the TL system were normalized and the spatial frequency was used to construct the DI of the ICM. MAIN RESULTS AND THE ROLE OF CHANCE: This study successfully constructed a quantitative indicator DI that can reflect the degree of ICM density in terms of fusion and texture features. The higher the DI value, the better the density of the blastocyst ICM, and the higher the chances that the blastocyst was euploid (P < 0.001) and that pregnancy (P < 0.001) and live birth (P = 0.005) were reached. In blastocysts with ICM graded B and blastocysts graded 4BB, DI was also positively associated with ploidy, pregnancy, and live birth (P < 0.05). ROC analysis showed that combining the Gardner scoring system with DI can more effectively predict pregnancy and live births, when compared to using the Gardner scoring system alone. LIMITATIONS, REASONS FOR CAUTION: Accurate calculation of the DI value places high demands on image quality, requiring manual selection of the clearest focal plane and exposure control. Images with the ICM not completely within the field of view cannot be used. The association between the density of ICM and chromosomal mosaicism was not evaluated. The associations between the density of ICM and different assisted reproductive technologies and different culture conditions in embryo laboratories were also not evaluated. Prospective studies are needed to further investigate the impact of ICM density on clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: ICM density assessment is a new direction in blastocyst assessment. This study explores new ways of assessing blastocyst ICM density and develops quantitative indicators and a corresponding qualitative evaluation scheme for ICM density. The DI of the blastocyst ICM developed in this study is easy to calculate and requires only TL equipment and image processing, providing positive guidance for clinical outcomes. The qualitative evaluation scheme of ICM density can assist embryologists without TL equipment to manually evaluate ICM density. ICM density is a simple indicator that can be used in practice and is a good complement to the blastocyst scoring systems currently used in most centers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research & Development Program of China (2021YFC2700603). The authors report no financial or commercial conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Multiple collapses of blastocysts after full blastocyst formation is an independent risk factor for aneuploidy - a study based on AI and manual validation.

BACKGROUND: The occurrence of blastocyst collapse may become an indicator of preimplantation embryo quality assessment. It has been reported that collapsing blastocysts can lead to higher rates of aneuploidy and poorer clinical outcomes, but more large-scale studies are needed to explore this relationship. This study explored the characteristics of blastocyst collapse identified and quantified by artificial intelligence and explored the associations between blastocyst collapse and embryo ploidy, morphological quality, and clinical outcomes. METHODS: This observational study included data from 3288 biopsied blastocysts in 1071 time-lapse preimplantation genetic testing cycles performed between January 2019 and February 2023 at a single academic fertility center. All transferred blastocysts are euploid blastocysts. The artificial intelligence recognized blastocyst collapse in time-lapse microscopy videos and then registered the collapsing times, and the start time, the recovery duration, the shrinkage percentage of each collapse. The effects of blastocyst collapse and embryo ploidy, pregnancy, live birth, miscarriage, and embryo quality were studied using available data from 1196 euploid embryos and 1300 aneuploid embryos. RESULTS: 5.6% of blastocysts collapsed at least once only before the full blastocyst formation (tB), 19.4% collapsed at least once only after tB, and 3.1% collapsed both before and after tB. Multiple collapses of blastocysts after tB (times ≥ 2) are associated with higher aneuploid rates (54.6%, P > 0.05; 70.5%, P < 0.001; 72.5%, P = 0.004; and 71.4%, P = 0.049 in blastocysts collapsed 1, 2, 3 or ≥ 4 times), which remained significant after adjustment for confounders (OR = 2.597, 95% CI 1.464-4.607, P = 0.001). Analysis of the aneuploid embryos showed a higher ratio of collapses and multiple collapses after tB in monosomies and embryos with subchromosomal deletion of segmental nature (P < 0.001). Blastocyst collapse was associated with delayed embryonic development and declined blastocyst quality. There is no significant difference in pregnancy and live birth rates between collapsing and non-collapsing blastocysts. CONCLUSIONS: Blastocyst collapse is common during blastocyst development. This study underlined that multiple blastocyst collapses after tB may be an independent risk factor for aneuploidy which should be taken into account by clinicians and embryologists when selecting blastocysts for transfer.

Intermittent short-duration reoxygenation relieves high-altitude pulmonary hypertension via NOX4/H2O2/PPAR-γ axis.

High-altitude pulmonary hypertension (HAPH) is a severe and progressive disease that can lead to right heart failure. Intermittent short-duration reoxygenation at high altitude is effective in alleviating HAPH; however, the underlying mechanisms are unclear. In the present study, a simulated 5,000-m hypoxia rat model and hypoxic cultured pulmonary artery smooth muscle cells (PASMCs) were used to evaluate the effect and mechanisms of intermittent short-duration reoxygenation. The results showed that intermittent 3-h/per day reoxygenation (I3) effectively attenuated chronic hypoxia-induced pulmonary hypertension and reduced the content of H2O2 and the expression of NADPH oxidase 4 (NOX4) in lung tissues. In combination with I3, while the NOX inhibitor apocynin did not further alleviate HAPH, the mitochondrial antioxidant MitoQ did. Furthermore, in PASMCs, I3 attenuated hypoxia-induced PASMCs proliferation and reversed the activated HIF-1α/NOX4/PPAR-γ axis under hypoxia. Targeting this axis offset the protective effect of I3 on hypoxia-induced PASMCs proliferation. The present study is novel in revealing a new mechanism for preventing HAPH and provides insights into the optimization of intermittent short-duration reoxygenation.

Usable blastocysts developed from in-vitro-matured metaphase I oocytes in preimplantation genetic testing cycles.

RESEARCH QUESTION: Are blastocysts derived from in-vitro-matured metaphase I (MI) oocytes less likely to produce usable embryos for transfer compared with those derived from in-vivo-matured oocytes in cycles undergoing preimplantation genetic testing (PGT)? DESIGN: The primary outcome was usable blastocyst rate, which was compared between blastocysts derived from in-vitro-matured MI oocytes after ovarian stimulation and from in-vivo-matured oocytes. Logistic regression analysis using generalized estimating equations was used to control for confounders in the analysis of factors that may influence the chance of a blastocyst being usable and in the comparison of embryological outcomes. Student's t-test, Mann-Whitney U test, chi-squared tests or Fisher's exact tests were used to compare clinical and pregnancy outcomes. RESULTS: A total of 1810 injected metaphase II (MII) oocytes from 154 PGT cycles involving 154 couples were included in this study. A total of 1577 MII oocytes were in-vivo-matured and 233 were in-vitro-matured MI oocytes. The usable blastocyst rate was similar between the in-vitro-matured MI oocyte group and the in-vivo-matured oocyte group (adjusted RR 0.97, 95% CI 0.40 to 2.34). Three live births were achieved using usable blastocysts derived from in-vitro-matured MI oocytes. CONCLUSIONS: If in-vitro-matured MI oocytes can be fertilized and develop into blastocysts, their ability to provide usable embryos for transfer is similar compared with those developed from in-vivo-matured oocytes. These blastocysts could be considered valuable for women with few viable embryos in assisted reproductive technology cycles.

Anti-EGFR ScFv functionalized exosomes delivering LPCAT1 specific siRNAs for inhibition of lung cancer brain metastases.

Brain metastasis (BM) is one of the leading causes of cancer-related deaths in patients with advanced non-small cell lung cancer (NSCLC). However, limited treatments are available due to the presence of the blood-brain barrier (BBB). Upregulation of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in NSCLC has been found to promote BM. Conversely, downregulating LPCAT1 significantly suppresses the proliferation and metastasis of lung cancer cells. In this study, we firstly confirmed significant upregulation of LPCAT1 in BM sites compared to primary lung cancer by analyzing scRNA dataset. We then designed a delivery system based on a single-chain variable fragment (scFv) targeting the epidermal growth factor receptor (EGFR) and exosomes derived from HEK293T cells to enhance cell-targeting capabilities and increase permeability. Next, we loaded LPCAT1 siRNA (siLPCAT1) into these engineered exosomes (exoscFv). This novel scFv-mounted exosome successfully crossed the BBB in an animal model and delivered siLPCAT1 to the BM site. Silencing LPCAT1 efficiently arrested tumor growth and inhibited malignant progression of BM in vivo without detectable toxicity. Overall, we provided a potential platform based on exosomes for RNA interference (RNAi) therapy in lung cancer BM.

Comparison of clinical outcomes for different morphological scores of D5 and D6 blastocysts in the frozen-thawed cycle.

BACKGROUND: Both embryo development speed and embryo morphology score played a significant role in frozen-thawed embryo transfer cycle (FET) outcomes. Most of the literature indicates that D5 embryos performed better than D6 embryos, although a few also indicate that there is no difference in clinical outcomes between D5 and D6 embryos. Clinically, D5 embryos are preferred for equal morphological scores. But how to choose embryos when the morphological score of D6 embryos is better than D5? METHODS: A retrospective study including 8199 frozen-thawed embryo transfers (FETs) was conducted to analyze patients who underwent IVF-FET from January 2018 to December 2020. Patients were divided into 8 groups according to the rate of embryonic development and morphological scores to compare pregnancy outcomes. We further compared clinical pregnancy outcomes and neonatal outcomes between BC embryos on day 5 (D5) and BA/BB embryos on day 6 (D6). RESULTS: Our study found no difference in clinical pregnancy rate (CPR) and live birth rate (LBR) between AA/AB blastocysts in D5 or D6 frozen blastocysts. However, for BA/BB/BC blastocysts, embryonic pregnancy outcome was significantly better in D5 than in D6. In our further analysis and comparison of BC embryos in D5 and BA/BB embryos in D6, we found no difference in clinical pregnancy outcomes and neonatal outcomes, but D6 BA/BB embryos had a higher rate of miscarriage. After adjusting for confounding factors, none of the indicators differed between groups. CONCLUSION: Our study provides suggestions for embryo selection: AA/AB embryos are preferred, regardless of the embryo development day, and the second choice is BA or BB embryos on D5. BA/BB embryos in D6 had a higher miscarriage rate than BC embryos in D5 but were not statistically significant after adjusting for confounding factors.

Transcriptome-wide N6-methyladenine methylation in granulosa cells of women with decreased ovarian reserve.

BACKGROUND: The emerging epitranscriptome plays an essential role in female fertility. As the most prevalent internal mRNA modification, N6-methyladenine (m6A) methylation regulate mRNA fate and translational efficiency. However, whether m6A methylation was involved in the aging-related ovarian reserve decline has not been investigated. Herein, we performed m6A transcriptome-wide profiling in the ovarian granulosa cells of younger women (younger group) and older women (older group). RESULTS: m6A methylation distribution was highly conserved and enriched in the CDS and 3'UTR region. Besides, an increased number of m6A methylated genes were identified in the older group. Bioinformatics analysis indicated that m6A methylated genes were enriched in the FoxO signaling pathway, adherens junction, and regulation of actin cytoskeleton. A total of 435 genes were differently expressed in the older group, moreover, 58 of them were modified by m6A. Several specific genes, including BUB1B, PHC2, TOP2A, DDR2, KLF13, and RYR2 which were differently expressed and modified by m6A, were validated using qRT-PCR and might be involved in the decreased ovarian functions in the aging ovary. CONCLUSIONS: Hence, our finding revealed the transcriptional significance of m6A modifications and provide potential therapeutic targets to promote fertility reservation for aging women.

Rapamycin improves the quality and developmental competence of mice oocytes by promoting DNA damage repair during in vitro maturation.

BACKGROUND: Increasing evidence has shown that the mammalian target of rapamycin (mTOR) pathway plays a critical role in oocyte meiosis and embryonic development, however, previous studies reporting the effects of rapamycin on oocyte IVM showed different or even opposite results, and the specific mechanisms were not clear. METHODS: The immature oocytes from female mice underwent IVM with rapamycin at different concentrations to select an optimal dose. The maturation rate, activation rate, subsequent cleavage and blastocyst formation rates, spindle assembly, chromosome alignment, mitochondrial membrane potential (MMP), ROS levels, and DNA damage levels were evaluated and compared in oocytes matured with or without rapamycin. In addition, the expression levels of genes associated with mTORC1 pathway, spindle assembly, antioxidant function, and DNA damage repair (DDR) were also assessed and compared. RESULTS: Rapamycin at 10 nM was selected as an optimal concentration based on the higher maturation and activation rate of IVM oocytes. Following subsequent culture, cleavage and blastocyst formation rates were elevated in activated embryos from the rapamycin group. Additionally, oocytes cultured with 10 nM rapamycin presented decreased ROS levels, reduced chromosome aberration, and attenuated levels of γ-H2AX. No significant effects on the percentages of abnormal spindle were observed. Correspondingly, the expressions of Nrf2, Atm, Atr, and Prkdc in IVM oocytes were markedly increased, following the inhibition of mTORC1 pathway by 10 nM rapamycin. CONCLUSION: Rapamycin at 10 nM could ameliorate the developmental competence and quality of IVM oocytes of mice, mainly by improving the chromosome alignments. The inhibition of mTORC1 pathway, which involved in activating DDR-associated genes may act as a potential mechanism for oocyte quality improvement by rapamycin.

Semaphorin 4B promotes tumor progression and associates with immune infiltrates in lung adenocarcinoma.

BACKGROUND: Semaphorins have been found to play important roles in multiple malignancy-related processes. However, the role of Semaphorin 4B (SEMA4B) in lung cancer remains unclear. Here, we aimed to explore the biological functions of SEMA4B in through bioinformatic analysis, in vitro and in vivo assays. In the present study, the possible mechanism by which SEMA4B affected the tumor growth and microenvironment of lung adenocarcinoma (LUAD) were investigated. METHODS: The expression of SEMA4B in LUAD was analyzed by bioinformatic analysis and verified by the immunohistochemistry staining. The prognostic value of SEMA4B in LUAD was investigated using the Kaplan-Meier survival and Cox's regression model. After silencing SEMA4B expression, the functions of SEMA4B in LUAD cells were investigated by in vitro experiments, including CCK-8 and plate clone formation. And the effect of SEMA4B on tumor growth and immune infiltration was explored in C57BL/6 mice tumor-bearing models. RESULTS: SEMA4B expression was upregulated in LUAD tissues and correlated with later pathological stages and poor prognosis of LUAD patients. Further study found that SEMA4B silencing suppressed the proliferation of lung cancer cells both in vitro and in vivo. Bioinformatic analysis showed that SEMA4B expression was correlated with the increased infiltration of myeloid-derived suppressor cells (MDSCs), T-regs and the decreased infiltration of CD8+ T cell in LUAD. Importantly, in vivo study verified that the infiltration of T-regs and MDSCs in tumor microenvironment (TME) of Xenograft tissues was decreased after SEMA4B silencing. CONCLUSIONS: These findings demonstrated SEMA4B might play an oncogenic role in LUAD progression, and be a promising therapeutic target for lung cancer.

Intermittent short-duration reoxygenation protects against simulated high altitude-induced pulmonary hypertension in rats.

High-altitude pulmonary hypertension (HAPH) is a severe and progressive disease caused by chronic hypoxia and subsequent pulmonary vascular remodeling. No cure is currently available owing to an incomplete understanding about vascular remodeling. It is believed that hypoxia-induced diseases can be prevented by treating hypoxia. Thus, this study aimed to determine whether daily short-duration reoxygenation at sea level attenuates pulmonary hypertension under high-altitude hypoxia. To this end, a simulated 5000-m hypoxia rat model and hypoxic cultured human pulmonary artery smooth muscle cells were used to evaluate the effect of short-duration reoxygenation. Results show that intermittent, not continuous, short-duration reoxygenation effectively attenuates hypoxia-induced pulmonary hypertension. The mechanisms underlining the protective effects involved that intermittent, short-duration reoxygenation prevented functional and structural remodeling of pulmonary arteries and proliferation, migration, and phenotypic conversion of pulmonary artery smooth muscle cells under hypoxia. The specific genes or potential molecular pathways responsible for mediating the protective effects were also characterised by RNA sequencing. Further, the frequency and the total time of intermittent reoxygenation affected its preventive effect of HAPH, which was likely attributable to augmented oxidative stress. Hence, daily intermittent, not continuous, short-duration reoxygenation partially prevented pulmonary hypertension induced by 5000-m hypoxia in rats. This study is novel in revealing a new potential method in preventing HAPH. It gives insights into the selection and optimisation of oxygen supply schemes in high-altitude areas.

ß-Hydroxyisovalerylshikonin regulates macrophage polarization via the AMPK/Nrf2 pathway and ameliorates sepsis in mice.

CONTEXT: The potential anti-inflammatory bioactivities of ß-hydroxyisovalerylshikonin (ß-HIVS) remain largely unknown. OBJECTIVE: This study investigated the anti-inflammatory effects and underlying mechanisms of ß-HIVS. MATERIALS AND METHODS: RAW 264.7 cells stimulated with LPS (100 ng/mL) for 24 h were treated with the non-cytotoxic doses of ß-HIVS (0.5 or 1 µM, determined by MTT and Trypan blue staining), qRT-PCR and FCM assay were used to examine macrophage polarization transitions. Western blotting was used to evaluate the activation of the AMPK/Nrf2 pathway. In vivo, C57BL/6 mice were randomly divided into vehicle control, LPS (10 mg/kg), and ß-HIVS (2.5 mg/kg) combined with LPS (10 mg/kg) groups, blood samples, BALF, and lung tissues of mice were subjected to ELISA, qRT-PCR, FCM, and H&E staining. RESULTS: ß-HIVS (1 µM) inhibited LPS-induced expression of M1 macrophage markers (TNF-α: 0.29-fold, IL-1ß: 0.32-fold), promoted the expression of M2 macrophage markers (CD206: 3.14-fold, Arginase-1: 3.98-fold) in RAW 264.7 cells; mechanistic studies showed that ß-HIVS increased the expression of nuclear Nrf2 (2.04-fold) and p-AMPK (3.65-fold) compared with LPS group (p < 0.05). In vivo, ß-HIVS decreased the levels of pro-inflammatory cytokines (TNF-α: 1130.41 vs. 334.88 pg/mL, IL-1ß: 601.89 vs. 258.21 pg/mL in serum; TNF-α: 893.07 vs. 418.21 pg/mL, IL-1ß: 475.22 vs. 298.54 pg/mL in BALF), decreased the proportion of M1 macrophages (77.83 vs. 68.53%) and increased the proportion of M2 macrophages (13.55 vs. 19.56%) in BALF, and reduced lung tissue damage and septic mice survival (p < 0.05). CONCLUSIONS: These results indicate that ß-HIVS may be a new potential anti-inflammatory agent.

Repeated cryopreservation process impairs embryo implantation potential but does not affect neonatal outcomes.

RESEARCH QUESTION: Does repeated cryopreservation process affect embryo implantation potential and neonatal outcomes of human embryos? DESIGN: This retrospective cohort study was conducted in the Reproductive Medicine Centre, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All assisted reproductive technology (ART) cycles were carried out between January 2014 and December 2018. Preferentially matched participants were divided into three groups according to the times of embryo cryopreservation: the fresh group (n = 249), the cryopreservation group (n = 244) and the re-cryopreservation group (n = 216). Embryo implantation rate, live birth rate, miscarriage rate and neonatal complication rate were compared among these three groups. RESULTS: The embryo implantation rate, clinical pregnancy rate and live birth rate in the re-cryopreservation group were significantly lower, and the miscarriage rate also slightly increased. Logistic regression analysis indicated that embryos with repeated cryopreservation and lower trophectoderm scores were at higher risk of embryo implantation failure in single embryo transfer cycles (OR 1.79 and 1.56, respectively). No significant differences were observed in gender, gestational age, birthweight, neonatal abnormality and neonatal complications among the groups. CONCLUSIONS: Our findings demonstrate the adverse effect of repeated cryopreservation on embryo implantation potential. The study offers embryologists and reproductive clinicians a warning of detrimental role of repeated cryopreservation. If unnecessary, it is strongly recommended to avoid repeated practice of vitrification and warming on embryos.

EGFR-specific single-chain variable fragment antibody-conjugated Fe3O4/Au nanoparticles as an active MRI contrast agent for NSCLC.

Overexpression of epidermal growth factor receptor (EGFR) is closely associated with a poor prognosis in non-small cell lung cancer (NSCLC), thus making it a promising biomarker for NSCLC diagnosis. Here, we conjugated a single-chain antibody (scFv) targeting EGFR with Fe3O4/Au nanoparticles to form an EGFR-specific molecular MRI bioprobe (scFv@Fe3O4/Au) to better detect EGFR-positive NSCLC tumors in vivo. In vitro, we demonstrated that the EGFR-specific scFv could specifically deliver Fe3O4/Au to EGFR-positive NSCLC cells. In vivo experiments showed that the accumulation of scFv@Fe3O4/Au in tumor tissue was detectable by magnetic resonance imaging (MRI) at the indicated time points after systemic injection. The T2W signal-to-noise ratio (SNR) of EGFR-positive SPC-A1 tumors was significantly decreased after scFv@Fe3O4/Au injection, which was not observed in the tumors of mice injected with BSA@Fe3O4/Au. Furthermore, transmission electron microscopy (TEM) analysis showed the specific localization of scFv@Fe3O4/Au in the SPC-A1 tumor cell cytoplasm. Collectively, the results of our study demonstrated that scFv@Fe3O4/Au might be a useful probe for the noninvasive diagnosis of EGFP-positive NSCLC.

A novel mutation in ZP3 causes empty follicle syndrome and abnormal zona pellucida formation.

PURPOSE: To identify disease-causing genes involved in female infertility. METHODS: Whole-exome sequencing and Sanger DNA sequencing were used to identify the mutations in disease-causing genes. We performed subcellular protein localization, western immunoblotting analysis, and co-immunoprecipitation analysis to evaluate the effects of the mutation. RESULTS: We investigated 17 families with female infertility. Whole-exome and Sanger DNA sequencing were used to characterize the disease gene in the patients, and we identified a novel heterozygous mutation (p.Ser173Cys, c.518C > G) in the ZP3 gene in a patient with empty follicle syndrome. When we performed co-immunoprecipitation analysis, we found that the S173C mutation affected interactions between ZP3 and ZP2. CONCLUSIONS: We identified a novel mutation in the ZP3 gene in a Chinese family with female infertility. Our findings thus expand the mutational and phenotypical spectrum of the ZP3 gene, and they will be helpful in precisely diagnosing this aspect of female infertility.

Computed Tomographic Imaging of 3 Patients With Coronavirus Disease 2019 Pneumonia With Negative Virus Real-time Reverse-Transcription Polymerase Chain Reaction Test.

We reported computed tomographic (CT) imaging findings of 3 patients with coronavirus disease 2019 (COVID-19) pneumonia with initially negative results before CT examination and finally confirmed positive for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse-transcription polymerase chain reaction assay.

Prediction model for clinical pregnancy for ICSI after surgical sperm retrieval in different types of azoospermia.

STUDY QUESTION: Can a counselling tool be developed for couples with different types of azoospermia to predict the probability of clinical pregnancy in ICSI after surgical sperm retrieval? SUMMARY ANSWER: A prediction model for clinical pregnancy in ICSI after surgical sperm retrieval in different types of azoospermia was created and clinical type of azoospermia, testicular size, male FSH, male LH, male testosterone, female age, female antral follicle count (AFC) and female anti-Müllerian hormone (AMH) were used as predictors. WHAT IS KNOWN ALREADY: Prediction models are used frequently to predict treatment success in reproductive medicine; however, there are few prediction models only for azoospermia couples who intend to conceive through surgical sperm retrieval and ICSI. Furthermore, no specific clinical types of azoospermia have been reported as predictors. STUDY DESIGN, SIZE, DURATION: A cohort study of 453 couples undergoing ICSI was conducted between 2016 and 2019 in an academic teaching hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples undergoing ICSI with surgically retrieved sperm were included, with 302 couples included in the development set and 151 couples included in the validation set. We constructed a prediction model using multivariable logistic regression analysis. The internal validation was based on discrimination and calibration. MAIN RESULTS AND THE ROLE OF CHANCE: We found that for male patients involved in our model, different clinical types of azoospermia are associated with different clinical pregnancy outcomes after ICSI. Considering the clinical type of azoospermia, larger testicular volume and higher levels of FSH, LH and testosterone in the body are associated with higher clinical pregnancy success rates. For women involved in our model, younger age and higher AFC and AMH levels are associated with higher clinical pregnancy success rates. In the development set, the AUC was 0.891 (95% CI 0.849-0.934), indicating that the model had good discrimination. The slope of the calibration plot was 1.020 (95% CI 0.899-1.142) and the intercept of the calibration plot was -0.015 (95% CI -0.112 to 0.082), indicating that the model was well-calibrated. From the validation set, the model had good discriminative capacity (AUC 0.866, 95% CI 0.808-0.924) and calibrated well, with a slope of 1.015 (95% CI 0.790-1.239) and an intercept of -0.014 (95% CI -0.180 to 0.152) in the calibration plot. LIMITATIONS, REASONS FOR CAUTION: We found that BMI was not an effective indicator for predicting clinical pregnancy, which was inconsistent with some other studies. We lacked data about the predictors that reflected sperm characteristics, therefore, we included the clinical type of azoospermia instead as a predictor because it is related to sperm quality. We found that almost all patients did not have regular alcohol consumption, so we did not use alcohol consumption as a possible predictor, because of insufficient data on drinking habits. We acknowledge that our development set might not be a perfect representation of the population, although this is a common limitation that researchers often encounter when developing prediction models. The number of non-obstructive azoospermia patients that we could include in the analysis was limited due to the success rate of surgical sperm retrieval, although this did not affect the establishment and validation of our model. Finally, this prediction model was developed in a single centre. Although our model was validated in an independent dataset from our centre, validation for different clinical populations belonging to other centres is required before it can be exported. WIDER IMPLICATIONS OF THE FINDINGS: This model enables the differentiation between couples with a low or high chance of reaching a clinical pregnancy through ICSI after surgical sperm retrieval. As such it can provide couples dealing with azoospermia a new approach to help them choose between surgical sperm retrieval with ICSI and the use of donor sperm. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the National Natural Science Foundations of China (81501246 and 81501020 and 81671443). The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Association between blastocyst morphology and live birth rate following frozen-thawed single blastocyst transfer: Results from a 5-year retrospective analysis of 2593 cryopreserved blastocysts.

AIM: Our aim was to investigate associations between blastocyst morphology parameters and live birth outcome and to make possible additional recommendations for existing embryo selection strategies. METHODS: This retrospective cohort study included 2593 frozen-thawed single blastocyst transfers (SBT) cycles from 2012 to 2016. Multivariable logistic regression model was used to analyze the independent predictive effectiveness of blastocyst parameters for live birth rate (LBR). RESULTS: The participants enrolled in the present study were 32 (28-35) years old with a median body mass index of 21.20 (19.60-23.40) kg/m2 , among whom 1058 (40.8%) women had live births. Among the three blastocyst morphology parameters, we found only inner cell mass grade and trophectoderm cell grade had significant effects on LBR (P < 0.001). When adjusting for potential confounders in a multivariable logistic regression model, the expansion and hatching (EH) stage of blastocoel also showed obvious correlation with LBR. Blastocysts at EH stage 4-5 had a significantly higher LBR than that at stage 3 (P < 0.05). Additionally, the timing of blastulation was also an important predictor of LBR. Blastocysts vitrified on day 6 and day 7 yielded a lower LBR than that vitrified on day 5 (P < 0.001). CONCLUSION: The timing of blastulation and all blastocyst morphology parameters were associated with LBR independently. Although the most important parameter for predicting clinical outcomes remains undetermined, the timing of blastulation was a stable predictor of live birth for frozen-thawed SBT.

Clinical and genetic analysis of cytochrome P450 oxidoreductase (POR) deficiency in a female and the analysis of a novel POR intron mutation causing alternative mRNA splicing : Overall analysis of a female with POR deficiency.

OBJECTIVE: To characterize the clinical features of a female with P450 oxidoreductase (POR) deficiency and to investigate the underlying mechanisms of POR inactivation. METHODS: The proband was a 35-year-old woman with primary infertility and menstrual irregularity. The reproductive endocrine profile was evaluated. DNA sequencing was conducted for the identification of POR gene mutation. RT-PCR was performed to confirm the impact of the mutation on POR mRNA. A molecular model was built for the structural analysis of mutant POR protein. RESULTS: The evaluation of reproductive endocrine profile revealed elevation of serum follicle-stimulating hormone (11.48 mIU/ml), progesterone (11.00 ng/ml), 17α-hydroxyprogesterone (24.24 nmol/l), dehydroepiandrosterone (6300 nmol/l), and androstenedione (3.89 nmol/l) and decreased estradiol (36.02 pg/ml). Sequencing of the POR gene showed the female was a compound heterozygote of the paternal P399_E401 deletion and a novel maternal IVS14-1G>C mutation. Functional analysis revealed IVS14-1G>C mutation caused alternative splicing of POR mRNA, with the loss of 12 nucleotides in exon 15 (c.1898_1909delGTCTACGTCCAG). Also, the resulting mutant POR protein had a V603_Q606 deletion, which inactivated the nucleotide-binding domain of NADPH in POR protein (K602_Q606). CONCLUSION: The mutation IVS14-1G>C of the POR gene could cause alternative splicing of POR mRNA and dysfunction of the resulting POR protein. Under proper IVF strategy with glucocorticoid therapy and endometrial preparation, females with mild POR deficiency still have the opportunity to have a live birth.

[Application of mapping allele with resolved carrier status technology in preimplantation genetic testing].

OBJECTIVE: To assess the value of mapping allele with resolved carrier status (MaReCs) technology for the determination of balanced translocation carrier status for embryos. METHODS: Blastocysts produced by 25 reciprocal translocation carriers and 15 Robertsonian translocation carriers were detected by MaReCs. After genetic counseling, transplantable blastocysts were selected. Amniocentesis was performed to check fetal chromosomes at 16 to 20 gestational weeks, and the consistency of amniocentesis and MaReCs was determined. RESULTS: No significant difference was found in the normal rate for chromosome copy number variations (CNVs) in blastocysts between reciprocal translocation carriers and Robertsonian translocation carriers (28.6% vs. 32.0%, P> 0.05). For 12 (48%) reciprocal translocation carriers and 8 (32%) Robertsonian translocation carriers, the status of translocation carrier of embryos was successfully determined. The results of amniocentesis were consistent with that of MaReCs in all 11 pregnancies. CONCLUSION: MaReCs is a reliable method to distinguish the translocation carrier status of embryos of balanced translocation carriers. It can help a certain proportion of balanced translocation carriers to select completely normal embryos while reduce transfer of embryo carrying a balanced translocation.

Is differences in embryo morphokinetic development significantly associated with human embryo sex?†.

To date, very few studies have reported on the relationship between live birth gender and embryo development kinetics. This study included 1735 women undergoing in vitro fertilization or intracytoplasmic sperm injection by using a time-lapse system. Finally, a total of 228 qualified patients with 100% implantation and known live birth information were included in the analysis. There were 174 male live births and 134 female live births. The time to 3 (t3), 4 (t4), and 5 (t5) cell development of male embryos was significantly shorter/earlier than female embryos (P < 0.05). The duration of the second cell cycle (cc2) in male embryos was significantly shorter than female embryos (P = 0.002). Multivariate logistic regression showed that only t3 had a significant correlation with live birth gender; the odds ratio (OR) was 0.786, 95% confidence interval (CI) was 0.625-0.988 (P < 0.05). When morphokinetic parameters were divided into groups based on quartiles, embryos within the sex ranges were observed to have significantly different proportions of male and female live births (P < 0.05). The results showed that t3 (<14 h) was the most relevant parameter related to live birth gender (OR 2.452, 95% CI 1.071-5.612, P = 0.03). These findings support the idea that embryo morphokinetic parameters were affected by the sex of the embryo. Currently, embryologists use embryo morphokinetics to establish models of development, in order to improve accurate selection of viable embryos. Thus, this factor needs to be considered when embryologists use embryo morphokinetics to select embryos.