OBJECTIVE To establish the ultra-high performance liquid chromatography(UPLC)fingerprint of Jingtian granule,and to evaluate its quality by chemical pattern recognition.METHODS Luna? Omega Polar C18 column(150 mmX2.1 mm,1.6 μm)was used as the chromatographic column,and acetonitrile-0.2％phosphoric acid solution was used as the mobile phase for gradient elution.The flow rate was 0.2 mL/min,the column temperature was 30 ℃,and the detection wavelength was 265 nm.With peak 16 as the reference peak,the UPLC fingerprint of Jingtian granule was established by the Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine(2012 edition).The common peaks were identified,the similarity evaluation was carried out,and the ownership of each common peak was confirmed.Hierarchical cluster analysis(HCA)and principal component analysis(PCA)in chemical pattern recognition methods were used to classify 13 batches of samples(S1-S13),and orthogonal partial least squares-discriminant analysis(OPLS-DA)was used to identify the key components of the differences between different batches of samples.RESULTS RSDs of precision,repeatability and stability of the UPLC method were not more than 4.4％.A total of 25 common peaks were identified in the fingerprints of 13 batches of Jingtian granules.By comparing with the reference substance fingerprint,10 common peaks were identified,namely peak 3(5-hydroxymethyl-2-furaldehyde),peak 5(salidroside),peak 8(chlorogenic acid),peak 15(cinnamic acid),peak 19(aloe-emodin),peak 20(ammonium glycyrrhizinate),peak 21(rhein),peak 23(emodin),peak 24(glycyrrhetinic acid),peak 25(chrysophanol).The similarities of fingerprints of 13 batches of samples were 0.955-0.996.The results of HCA showed that 13 batches of samples could be divided into three categories,among which samples S1,S5,S7,S11-S13 were clustered in one category,S4 and S6 were clustered in one category,S2,S3 and S8-S10 were clustered in one category.PCA results showed that the cumulative variance contribution rate of principal components 1-7 was 92.666％.OPLS-DA further identified 13 differential components,which were mainly derived from Polygonati Rhizoma with wine steaming,Rhodiolae Crenulatae Radix Et Rhizoma,prepared Rhei Radix Et Rhizoma and Glycyrrhizae Radix Et Rhizome Praeparata Cum Melle.CONCLUSIONS The established UPLC fingerprint of Jingtian granule is simple,stable and reproducible.Combined with the chemical pattern recognition method,it can effectively reveal the overall quality difference between different batches of Jingtian granule.The quality of Polygonati Rhizoma with wine steaming,Rhodiolae Crenulatae Radix Et Rhizoma,prepared Rhei Radix Et Rhizoma,Dioscoreae Nipponicae Rhizoma,Polyporus,Cinnamomi Ramulus,Glycyrrhizae Radix Et Rhizome Praeparata Cum Melle is the key to the overall quality of Jingtian granule.

Objective:To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing (PGT) of an ATR-X syndrome pedigree with chromosomal microduplication.Methods:The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022. After chromosome copy number variation sequencing (CNV Seq) detection, it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region, which involved partial sequences of the ATRX gene. The third generation long read sequencing technology was used to detect the female genome sequence, determine the physical location of the insertion of the above repeat into the genome, clarify the pathogenicity of the repeat, and obtain a single nucleotide polymorphism (SNP) haplotype linked to the above micro repeat inheritance. After the couple's full informed consent, PGT was performed to assist pregnancy. One haploid embryo without pathogenic microduplication was selected for transfer. To verify the consistency with PGT test results, amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy, and the fetus was followed up after birth. Results:The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX: 76804463-76804464 (GRCh37/hg19), which is an intra tandem repeat of the ATRX gene, and it is predicted that it may cause damage to the normal function of the ATRX protein. After one cycle of PGT treatment, 27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection (ICSI). Through genetic testing, it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication. After thawing and transferring one of the blastocysts, the pregnancy was achieved, and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results. In November 2023, at 39 +5 weeks of pregnancy, a female live baby was delivered by natural delivery, and she is in good health. Conclusion:The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics. It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity, but also obtain SNP haplotypes that are linked to the target mutation, thus preparing for subsequent embryo detection.

Objective:To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing (PGT) of an ATR-X syndrome pedigree with chromosomal microduplication.Methods:The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022. After chromosome copy number variation sequencing (CNV Seq) detection, it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region, which involved partial sequences of the ATRX gene. The third generation long read sequencing technology was used to detect the female genome sequence, determine the physical location of the insertion of the above repeat into the genome, clarify the pathogenicity of the repeat, and obtain a single nucleotide polymorphism (SNP) haplotype linked to the above micro repeat inheritance. After the couple's full informed consent, PGT was performed to assist pregnancy. One haploid embryo without pathogenic microduplication was selected for transfer. To verify the consistency with PGT test results, amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy, and the fetus was followed up after birth. Results:The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX: 76804463-76804464 (GRCh37/hg19), which is an intra tandem repeat of the ATRX gene, and it is predicted that it may cause damage to the normal function of the ATRX protein. After one cycle of PGT treatment, 27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection (ICSI). Through genetic testing, it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication. After thawing and transferring one of the blastocysts, the pregnancy was achieved, and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results. In November 2023, at 39 +5 weeks of pregnancy, a female live baby was delivered by natural delivery, and she is in good health. Conclusion:The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics. It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity, but also obtain SNP haplotypes that are linked to the target mutation, thus preparing for subsequent embryo detection.

Objective:To analyze the risk factors of early myocardial injury and the impact of early myocardial injury on prognosis of patients with extensive burns.Methods:A retrospective case series study was conducted. From January 2018 to August 2022, 361 patients with extensive burns who met the inclusion criteria were admitted to Tongren Hospital of Wuhan University & Wuhan Third Hospital, including 231 males and 130 females, aged 50 (36, 58) years, with total burn area of 45% (35%, 60%) total body surface area. According to the highest level of creatine kinase isoenzyme-MB (CK-MB) within 72 h post injury, the patients were divided into early myocardial injury group (CK-MB≥75 U/L, 182 patients) and non-early myocardial injury group (CK-MB<75 U/L, 179 patients). The following data of patients in the 2 groups were collected and analyzed, including gender, age, total burn area, admission time post injury, combination with shock on admission, combination with inhalation injury on admission; the main blood test indexes such as myocardial enzyme spectrum, blood routine, liver and kidney function, and electrolytes within 72 h post injury; and treatment outcomes and fatality rate. Data were statistically analyzed with chi-square test, independent sample t test, or Mann-Whitney U test. The multivariate logistic regression analysis was conducted to screen the independent risk factors for early myocardial injury and for death in patients with extensive burns. Results:There were statistically significant differences in gender, combination with shock on admission, total burn area, and admission time post injury of patients between the two groups (with χ2 values of 6.40 and 6.10, Z values of 5.41 and 3.03, respectively, P<0.05). There were no statistically significant differences in age, combination with inhalation injury on admission of patients between the two groups ( P>0.05). The CK-MB, creatine kinase, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, white blood cell count, neutrophil-to-lymphocyte ratio (NLR), alanine aminotransferase (ALT), aspartate aminotransferase, potassium, and hemoglobin within 72 h post injury were significantly higher than those in non-early myocardial injury group (with Z values of 15.40, 6.26, 7.59, 7.02, 2.64, 4.53, 4.07, 6.32, and 4.12, t=2.34, respectively, P<0.05), while the level of calcium was significantly lower than that in non-early myocardial injury group ( Z=2.72, P<0.05). There were no statistically significant differences in other blood test indexes of patients between the two groups ( P>0.05). The total burn area, admission time post injury, NLR and ALT within 72 h post injury were the independent risk factors for early myocardial injury in patients with extensive burns (with odds ratios of 1.03, 1.07, 1.04, and 1.02, 95% confidence intervals of 1.02-1.05, 1.00-1.11, 1.02-1.07, and 1.00-1.03, respectively, P<0.05). The fatality rate of patients in early myocardial injury group was 8.8% (16/182), which was significantly higher than 2.8% (5/179) in non-early myocardial injury group ( χ2 =5.93, P<0.05). Early myocardial injury, age, combination with shock on admission, and combination with inhalation injury on admission were the independent risk factors for death in patients with extensive burns (with odds ratios of 3.60, 1.04, 6.53, and 3.14, 95% confidence intervals of 1.17-11.05, 1.01-1.07, 1.39-30.68, and 1.15-8.56, respectively, P<0.05). Conclusions:The total burn area, admission time post injury, NLR and ALT within 72 h post injury were the independent risk factors for early myocardial injury in patients with extensive burns. Patients with extensive burns with early myocardial injury have a higher fatality rate, and early myocardial injury is an independent risk factor for the patients' death.

Objective:To explore the biological role and clinical significance of ubiquitin-specific protease 7 (USP7) in the carcinogenesis of scar ulcer.Methods:A retrospective observational study combined with bioinformatics analysis was used. The RNA expression profile data of USP7 in tumor and/or its corresponding paracancular normal tissue were obtained from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus database, and the RNA sequencing data were transformed by log 2. The variations of USP7 gene were analyzed by cBioPortal database. The USP7 mRNA expression in tumor and adjacent normal tissue in TCGA database were obtained by using the "Gene_DE" module in TIMER 2.0 database. The survival rates of patients with high and low USP7 expression in cutaneous melanoma (SKCM), cervical squamous cell carcinoma (CESC), lung squamous cell carcinoma (LUSC), and head and neck squamous cell carcinoma (HNSC) were analyzed using the Gene Expression Profile Interactive Analysis 2 (GEPIA2) database, and the Kaplan-Meier survival curves were drawn. Sangerbox database was used to analyze the correlation of USP7 expression in pan-cancer with microsatellite instability (MSI) or tumor mutation burden (TMB) pan-cancer. Through the "correlation analysis" module in the GEPIA2 database, the correlation of USP7 expression in pan-cancer with the expression levels of five DNA mismatch repair genes ( MLH1, MSH2, MSH6, PMS2, and EPCAM) and three essential DNA methyltransferases (DNMT)--DNMT1, DNMT3A, and DNMT3B were evaluated. The USP7 expression in CESC, HNSC, LUSC, and SKCM and its correlation with infiltration of immune cells (B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells) were analyzed by the "Immune-Gene" module in TIMER 2.0 database. The "Similar Genes Detection" module of GEPIA2 database was used to obtain the top 100 protein sets with similar expression patterns to USP7. Intersection analysis was performed between the aforementioned protein sets and the top 50 protein sets that were directly physically bound to USP7 obtained by using the STRING database. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were performed for the two protein sets mentioned above using the DAVID database. The samples of normal skin, hypertrophic scar, scar ulcer, and scar carcinoma with corresponding clinicopathologic features were collected from the Department of Pathology of Tongren Hospital of Wuhan University & Wuhan Third Hospital from October 2018 to October 2022, and the USP7 expression in tissue was detected by immunohistochemical method, with the number of samples of 6. Data were statistically analyzed with Log-rank test, one-way analysis of variance, and Bonferroni test. Results:In pan-cancer, the main gene variations of USP7 were mutation and amplification, and the top 3 tumors with the highest variation frequency (>6%) were bladder urothelial carcinoma, SKCM, and endometrial carcinoma. The main mutation of USP7 gene in pan-cancer was missense mutation. In SKCM with the highest mutation frequency, the main type of mutation was missense mutation in USP7_ICP0_bdg domain. USP7 mRNA expression in breast invasive carcinoma, bile duct carcinoma, colon carcinoma, esophageal carcinoma, HNSC, renal chromophobe cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, LUSC, prostate carcinoma, and gastric carcinoma was significantly higher than that in corresponding paracancer normal tissue ( P<0.05). USP7 mRNA expression in glioblastoma multiforme, renal clear cell carcinoma, renal papillary cell carcinoma, and thyroid carcinoma was significantly lower than that in corresponding paracancular normal tissue ( P<0.05). In addition, USP7 mRNA expression in SKCM metastases was much higher than that in primary tumor tissue ( P<0.05). Survival curves showed no significant difference in survival rate between patients with high USP7 expression and patients with low USP7 expression in CESC, HNSC, LUSC, and SKCM (Log-rank P>0.05, with hazard ratios of 1.00, 0.99, 1.00, and 1.30, respectively). USP7 expression in colon cancer, colorectal cancer, thymic cancer, and thyroid cancer was negatively correlated with TMB (with Pearson correlation coefficients of -0.26, -0.19, -0.19, and 0.11, respectively, P<0.05). USP7 expression in glioma, CESC, lung adenocarcinoma, mixed renal carcinoma, and LUSC was positively correlated with MSI expression (with Pearson correlation coefficients of 0.22, 0.14, 0.15, 0.08, and 0.14, respectively, P<0.05), and USP7 expression in colon cancer, colorectal cancer, invasive breast cancer, prostate cancer, HNSC, thyroid cancer, and diffuse large B-cell lymphoma were significantly negatively correlated with MSI expression (with Pearson correlation coefficients of -0.31, -0.27, -0.13, -0.19, -0.16, -0.18, and -0.53, respectively, P<0.05). The expression of USP7 in CESC was positively correlated with that of both MSH2 and MSH6 (with Spearman correlation coefficients of 0.51 and 0.44, respectively, P<0.05), and the expression of USP7 in HNSC was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.39, 0.14, 0.49, 0.54, and 0.41, respectively, P<0.05), and the expression of USP7 in LUSC was positively correlated with the expression of EPCAM, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.20, 0.36, 0.40, and 0.34, respectively, P<0.05), and the expression of USP7 in SKCM was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.11, 0.33, 0.42, 0.55, and 0.34, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was significantly positively correlated with the expression of DNMT1, DNMT3A, and DNMT3B (with Spearman correlation coefficients of 0.42, 0.34, 0.22, 0.45, 0.52, 0.22, 0.36, 0.36, 0.22, 0.38, 0.46, and 0.21, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was positively correlated with CD4 + T cell infiltration (with Partial correlation coefficients of 0.14, 0.22, 0.13, and 0.16, respectively, P<0.05). Being similar to the pattern of USP7 expression and ranked among top 100 protein sets, the top 5 proteins were C16orf72, BCLAF1, UBN, GSPT1, ERI2 (with Spearman correlation coefficients of 0.83, 0.74, 0.73, and 0.72, respectively, all P values<0.05). The top 50 protein sets that directly physically bind to USP7 overlapped with the aforementioned protein set by only one protein, thyroid hormone receptor interaction factor 12. KEGG enrichment analysis showed that USP7 related genes were involved in cell cycle, spliceosome, cell senescence, and p53 signal pathway. GO enrichment analysis showed that USP7 related genes were involved in transcriptional regulation, protein ubiquitination, DNA repair, and cytoplasmic pattern recognition receptor signal pathways. Analysis of clinical samples showed that USP7 expression was significantly higher in hypertrophic scars (0.35±0.05), scar ulcers (0.43±0.04), and scar cancers (0.61±0.03) than in normal skin (0.18±0.04), P<0.05. Conclusions:USP7 may be a clinical biomarker for the progression of cicatricial ulcer cancer.

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). METHODS: A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. RESULTS: Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. CONCLUSION By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.
Humans ; Pregnancy ; Female ; Child ; Male ; Preimplantation Diagnosis ; East Asian People ; Semen ; Genetic Testing ; Muscular Atrophy, Spinal/genetics* ; Aneuploidy ; Blastocyst/pathology* ; High-Throughput Nucleotide Sequencing ; Spermatozoa

Objective:To explore the value of polar body sequencing in preimplantation genetic testing (PGT) for monogenic disease of a female patient with Van der Woude syndrome.Methods:PGT based on polar body sequencing was performed for a female patient with Van der Woude syndrome caused by a de novoIRF6 pathogenic variant. Totally six oocytes were fertilized by intracytoplasmic sperm injection (ICSI). The first, second polar bodies and the trophoblast ectoderm cells of blastocysts were biopsied respectively. Sanger sequencing was used to detect the pathogenic variant in the biopsied cells after genome-wide amplification. The genotypes and pathogenic possibilities of the embryos were inferred according to the genotypes of corresponding tested polar bodies. In order to prevent the absence of transplantable embryos due to the failure of blastocyst culture, vitrification was performed on an embryo with good morphology and low pathogenic possibility before blastocyst formation. The 175 single nucleotide polymorphisms (SNPs) within the 1M region upstream and downstream from the pathogenic variant location were tested by targeted capture sequencing in the couple and selected polar bodies and embryos to construct the haplotypes. An embryo with low pathogenic possibility was transferred. Prenatal diagnosis was strongly recommended after successful pregnancy. Prenatal and postnatal follow-up were performed. Results:Totally six first polar bodies and six second polar bodies were obtained. The pathogenic variant was successfully sequenced in 11 polar bodies. Among the six embryos, one embryo with low pathogenic possibility was vitrified on day 4 (D4) after fully informed consent of the couple; one embryo developed to blastocyst was detected with high pathogenic possibility; the other four embryos were degenerated during blastocyst culture. The SNP haplotypes closely linked to the pathogenic variant location were successfully constructed by linkage analysis. The haplotype analysis of the embryos was in consistent with Sanger sequencing. The D4 embryo with low pathogenic possibility was transferred. The couple refused to conduct invasive prenatal diagnosis after pregnancy. None orofacial clefts were detected after the baby was born, and the pathogenic variant was not detected in the neonatal cord blood either.Conclusion:This study successfully blocked a female patient with Van der Woude syndrome caused by a de novoIRF6 pathogenic variant give birth to an affected baby by polar body sequencing based preimplantation genetic testing for monogenic disease.

Objective:To explore the value of polar body sequencing in preimplantation genetic testing (PGT) for monogenic disease of a female patient with Van der Woude syndrome.Methods:PGT based on polar body sequencing was performed for a female patient with Van der Woude syndrome caused by a de novoIRF6 pathogenic variant. Totally six oocytes were fertilized by intracytoplasmic sperm injection (ICSI). The first, second polar bodies and the trophoblast ectoderm cells of blastocysts were biopsied respectively. Sanger sequencing was used to detect the pathogenic variant in the biopsied cells after genome-wide amplification. The genotypes and pathogenic possibilities of the embryos were inferred according to the genotypes of corresponding tested polar bodies. In order to prevent the absence of transplantable embryos due to the failure of blastocyst culture, vitrification was performed on an embryo with good morphology and low pathogenic possibility before blastocyst formation. The 175 single nucleotide polymorphisms (SNPs) within the 1M region upstream and downstream from the pathogenic variant location were tested by targeted capture sequencing in the couple and selected polar bodies and embryos to construct the haplotypes. An embryo with low pathogenic possibility was transferred. Prenatal diagnosis was strongly recommended after successful pregnancy. Prenatal and postnatal follow-up were performed. Results:Totally six first polar bodies and six second polar bodies were obtained. The pathogenic variant was successfully sequenced in 11 polar bodies. Among the six embryos, one embryo with low pathogenic possibility was vitrified on day 4 (D4) after fully informed consent of the couple; one embryo developed to blastocyst was detected with high pathogenic possibility; the other four embryos were degenerated during blastocyst culture. The SNP haplotypes closely linked to the pathogenic variant location were successfully constructed by linkage analysis. The haplotype analysis of the embryos was in consistent with Sanger sequencing. The D4 embryo with low pathogenic possibility was transferred. The couple refused to conduct invasive prenatal diagnosis after pregnancy. None orofacial clefts were detected after the baby was born, and the pathogenic variant was not detected in the neonatal cord blood either.Conclusion:This study successfully blocked a female patient with Van der Woude syndrome caused by a de novoIRF6 pathogenic variant give birth to an affected baby by polar body sequencing based preimplantation genetic testing for monogenic disease.

Polar bodies are the byproducts of oocyte meiosis, which contain the corresponding genetic material with the egg cell. Genetic testing of the polar bodies can infer the chromosomal and genetic state of the corresponding egg cell. Polar body biopsy is a safe and effective method for preimplantation genetic testing (PGT), which has the characteristics of less damage to the embryo and easy to be accepted ethically. Polar body analysis is mainly used for PGT of maternal monogenic diseases, chromosomal aneuploidy and structure rearrangement, with significant advantages especially for the females with de novo mutations, absent of family members, and gonadal mosaic mutation, preventing the transmission of mitochondrial diseases, and screening chromosomal aneuploidy with advanced age. In addition, compared with blastocyst detection, preimplantation detection in the polar body stage avoids embryo waste caused by failure of blastocyst culturing, improves the utilization of embryos. This characteristic may take more advantages for females with ovarian insufficiency. Moreover, polar body biopsy moves the detection time forward and makes fresh embryo transfer available. This paper summarized the application of polar body biopsy in PGT aiming to reveal its clinical value.

Objective:To evaluate the association between polycystic ovary syndrome (PCOS)-related symptom combinations and ovarian stimulation high response in infertile patients with PCOS symptoms and controlled ovarian stimulation treatment by recombinant Human Follitropin Alfa (r-hFSHα) solution for injection, and to evaluate the efficacy and safety outcomes of using the r-hFSHα prefilled injection pen in high-risk patients with ovarian hyperstimulation syndrome (OHSS).Methods:This prospective, observational, phase Ⅳ study enrolled 1055 patients with at least one symptom/sign of PCOS using the r-hFSHα prefilled pen for over 4 months follow-up observation from December 2015 to September 2017 in the Second Hospital of Hebei Medical University, Center for Reproductive Medicine, Shandong University, Tangdu Hospital of the Air Force Military Medical University, Dalian Maternity and Child Health Care Hospital, Nanjing Maternity and Child Health Care Hospital, Jiangxi Maternity and Child Health Care Hospital, the First Affiliated Hospital of Xinjiang Medical University, the First Affiliated Hospital of Anhui Medical University, the Affiliated Hospital of Inner Mongolia Medical University. The primary endpoints assessed included the development of polycystic ovaries, elevated serum testosterone levels, menstrual cycle disturbances, development of hirsutism, and completion of egg retrieval. The efficacy endpoints of the study included the number of ocoytes retrieved, the number of M Ⅱ oocyte, the biochemical pregnancy rate, the clinical pregnancy rate, and the implantation rate. Results:In the full analysis set ( n=997), polycystic ovary rate was 54.5% (543/997), serum testosterone level was (0.4±0.2) μg/L, menstrual cycle disorder rate was 45.0% (449/997), hirsutism rate was 10.5% (105/997). The average number of oocytes retrieved after ovarian stimulation was 14.4. The clinical pregnancy rate per transfer cycle was 53.6% (251/468), the live birth rate was 45.3% (212/468), the biochemical pregnancy rate was 60.9% (285/468), the implantation rate was 39.1% (349/893), and the fresh embryo transfer cancellation rate was 24.0% (239/997). OHSS incidence was diagnosed in 1.8% (19/1054) of patients (safety set, n=1054), including 8 (0.8%) mild cases, 10 (0.9%) moderate cases and 1 (0.1%) severe case. According to the results of exploratory analysis, a decrease in body mass index (BMI) was associated with an increased risk of high response. For every 1 kg/m 2 decrease in BMI, the risk of high response (number of retrieved oocytes >15) increased by approximately 9%, the risk of high response (number of retrieved oocytes >20) increased by approximately 9%. For every 1 increase in antral follicle count (AFC), the risk of high response(number of retrieved oocytes >15) increased by approximately 6% and the risk of high response (number of retrieved oocytes >20) by approximately 4%. Conclusion:Patients with at least one symptom/sign of ovarian hyperstimulation achieved good clinical outcomes with the use of the r-hFSHα prefilled pen, and high response was associated with lower BMI and AFC.