Background::The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement.Methods::From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model. Results::A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement. Conclusion::Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.

With the rapid changes in lifestyle, natural and social environment, the reproductive health status of couples in childbearing age continues to decline, and long-term outcomes of the rapidly increasing offspring conceived by assisted reproductive technology (ART) needs to be evaluated urgently. Therefore, the focus of research now needs to be extended from death and severe diseases to full life cycle and full disease spectrum. In order to meet the demand for such research, we launched the China National Birth Cohort (CNBC) study, an ongoing prospective and longitudinal study aiming to recruit 30 000 families underwent ART and 30 000 families with spontaneous pregnancies. Long-term follow-up programs will be conducted for both spouses and their offspring. Data of couples and their offspring, such as environmental exposure, reproductive history, psychological and behavioral status, will be collected during follow-up. Peripheral blood, urine, umbilical blood, follicular fluid, semen were also collected at different follow-up nodes. Based on high-quality data and biological samples, CNBC will play an extremely important supporting role and have a far-reaching impact on maternal and children's health care and reproductive health in China. This paper is exactly a brief introduction to the construction and basic design of CNBC.

Objective:To explore the effects of environmental, genetic factors as well as the interactions in early life on the short-term and long-term health of offspring and to systematically evaluate the pregnancy outcomes and health of offspring after birth between families with assisted reproductive technology (ART) conception and families with spontaneous conception.Methods:The China National Birth Cohort (CNBC), a multicenter prospective birth cohort study, includes both families with ART conception and families with spontaneous conception. Since 2016, CNBC has recruited families from 24 hospitals located in 12 provinces, municipalities and autonomous regions throughout China. Information and biospecimens were collected before ART treatment, embryo transfer, at early, second, third trimester and delivery, and at 42 days, 6, 12 and 36 months after birth.Results:By June 2020, CNBC had included 27 044 families with ART conception and 29 589 families with spontaneous conception. The majority of the participants are urban residents. Among the families with ART conception, 65.5% of the men and 63.7% of the women had college degrees or higher. The mean age distribution of men and women was (33.83±5.52) and (32.38±4.67) years. 83.2% of women were primiparas, and the prevalence rates of current regular smokers and current alcohol drinkers were 0.8% and 2.1% in women. Among the families with spontaneous conception, 81.5% of the men and 86.5% of the women had college degrees or higher. The mean age distribution of men and women was (32.06±5.09) and (30.40±4.27) years. 67.2% of women were primiparas, and the prevalence rates of current regular smokers and current alcohol drinkers were 0.1% and 2.2% in women. The baseline characteristics were different between the families with ART conception and spontaneous conception in different regions.Conclusion:CNBC provides a powerful and rich resource in studying the impact of genetic, environmental factors and interactions in early life and ART treatment on the health of offspring after birth.

Objective To compare the etiological constitution of recurrent miscarriage (RM) between patients with consecutive two and three or more miscarriages through combining the routine examination results and embryonic karyotype. Methods Patients with a history of two or more consecutive clinical miscarriages(≤12 weeks of gestation)consulting in the RM clinic of the First Affiliated Hospital of Sun Yat-sen University from March 2011 to January 2016 were collected. Six hundred and ninety-six with detailed history recorded, routine clinical examinations of RM and at least once embryonic karyotype were ultimately enrolled in this study. Their etiological constitution of RM were analyzed in groups of consecutive two and three or more miscarriage. The etiologies of RM in analysis consisted of women age, body mass index (BMI), chromosome abnormalities of couples, uterine abnormalities, endocrinology abnormalities and antiphospholipid syndrome(APS). Results (1)Among 696 patients, the abnormal embryonic karyotypes was 60.6%(422/696)and routine RM etiologies was 32.2%(224/696), leaving the ratio of unexplained RM was only 29.0%(202/696).(2)A total of 717 embryo karyotype were found in 696 patients, included 21 cases with twice embryo karyotype results the percentage of normal embryo was 39.7%(285/717), while abnormal ones was 60.3%(432/717). Among the types of abnormal karyotype, the most common ones (>10%)were trisomy 16(19.2%, 83/432), monosome X(11.3%, 49/432)and trisomy 22(10.9%, 47/432). (3)Among the 696 RM patients, the number of two and three or more miscarriages were respectively 446(64.1%,446/696)and 250(35.9%,250/696). Comparing groups of three or more miscarriages with two miscarriages, there were significant differencein older age as well as uterine adhesion(P<0.05). But no difference was found in body mass index(BMI), the rates of chromosome abnormalities of couples, uterine abnormalities except uterine adhesion, endocrinology abnormalities and APS (all P>0.05) between two groups. Conclusions The abnormal embryonic karyotype is the most common cause of first-trimester RM. The etiological constitution of two and three or more recurrent miscarriages is accordant, suggesting that routine clinical examination and the embryonic karyotype should be started following two consecutive clinical early miscarriages.

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
APOBEC-1 Deaminase ; genetics ; metabolism ; Base Sequence ; Blastomeres ; cytology ; metabolism ; CRISPR-Cas Systems ; Embryo, Mammalian ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; pathology ; Gene Editing ; methods ; Gene Expression ; HEK293 Cells ; Heterozygote ; Homozygote ; Humans ; Point Mutation ; Primary Cell Culture ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; beta-Globins ; genetics ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; pathology ; therapy

Objective:In this study,a series of experiments were conducted to research the mechanism of anticancer and preliminary molecular effects of PAMs on the HEPG-2 cancer cells.Methods:Morphological observation and MTT assay were used to explore the inhibition and killing effect of PAMs acting on HEPG-2.AO/EB staining and Annexin V-FITC/PI staining were employed to observe the apoptosis of HEPG-2 treated with PAMs.The expression level of Foxm1,bcl-2 and others genes in HEPG-2 cells were detected by using qRT-PCR and western blot.Wound healing and transwell experiments determined if PAMs can inhibit the migration of HEPG-2.Results:PAMs can inhibit and kill HEPG-2 cells in time and dose-dependent manners,and the cytotoxic effects were closely related to the cell apoptosis.The mRNA expression of foxm1,bcl-2 and surviving gene were remarkably decreased in HEPG-2 cells after the treatment of PAMs.PAMs decreased the FoXM1 protein expression in HEPG-2 cells,while up-regulating thep53 protein expression.,and it could also inhibit the migration of cancer cells.Conclusions:The possible molecular mechanism for the killing of HEPG-2 cancer cells by PAMs was proposed.By down-regulating the expression of foxm1 and up-regulating the expression of p53,the transcriptional expression of their downstream target genes survivin and bcl-2 was inhibited or reduced,hence enhancing the cancer cell apoptosis.This study provides an important foundation for the development of anti-cancer Chinese folk medicine based on PAMs.

Objective To investigate the efficacy and feasibility of preimplantation genetic diagnosis (PGD) with human leukocyte antigen (HLA) matching for beta-thalassemia. Methods A total of 43 referred beta-thalassemia couples, with at least on child in need of hematopoietic stem cell transplantation (HSCT), underwent PGD for HLA matching at the First Affiliated Hospital of Sun Yat-sen University from 2010 to 2015. PGD cycles of these couples were retrospectively analyzed, and 15 infants born from PGD-HLA were followed up. Results A total of 84 oocyte retrieval cycles were performed, providing 14±7 oocytes per cycle. Fifty nine embryos biopsied cycles were done, including 24 cleavage stage and 35 blastocyst stage biopsy cycles. In cleavage stage, 259 embryos were biopsied, 93.4% (242/259) of them with conclusive molecular diagnosis, and the percentage of unaffected embryos (normo-homozygote and heterozygote) was 71.4%(185/259). The percentage of HLA matched unaffected embryos was 9.3%(24/259). In blastocyst stage, 306 embryos were biopsied, while 93.8% (287/306) of them were conclusive, and the percentage of unaffected embryos was 70.6% (216/306). The percentage of HLA matched unaffected embryos in blastocyst stage biopsy was 14.4%(44/306), which was higher than in cleavage stage biopsy (P<0.05). Forty three female carriers underwent 48 embryo transfer cycles including 3 fresh and 45 frozen-thawed embryo transfer cycles. Three fresh embryo transfer cycles were done after cleavage stage biopsy, resulted in a birth of healthy twins born at 36 weeks′gestation. All the embryos were frozen after blastocyst biopsied. Totally, 54 frozen-thawed embryos that were transferred in 45 frozen-thawed embryo transfer cycles included 25 embryo from cleavage stage biopsy and 29 embryo from blastocyst stage biopsy, and 42 of them were HLA matched. Clinical pregnancy rate and implantation rate per cycle in frozen-thawed embryo transfer were 38%(17/45) and 37%(20/54) respectively. A total of 15 infants were born, 2 were from a fresh embryo transfer cycle, and 13 were from frozen-thawed embryo transfer cycles. Results of prenatal diagnosis from delivered cases were matched to that of PGD. Four sick children have been cured by HSCT from these HLA matched born siblings. Conclusion PGD with HLA matching is an established method for conceiving a child who may donate hematopoietic stem cells to save an ill sibling.

Objective To investigate the premature spontaneous ovulation rates in in vitro fertilization-embryo transfer (IVF-ET) cycles using gonadotropin-releasing hormone antagonist (GnRH-ant) and gonadotropin-releasing hormone agonist (GnRH-a), as well as the risk factors for premature spontaneous ovulation. Methods The rates of premature spontaneous ovulation in a total of 10 612 cycles using GnRH-ant or GnRH-a were compared. Matched case-controlled study and binary logistic regression model were conducted to analyze the risk factors for premature spontaneous ovulation. Results The spontaneous ovulation rate in the whole for GnRH-a cycles was 0.15%(13/8 514), compared with a 1.62%(34/2 098) in GnRH-ant cycles (P<0.01). Further matched controlled study and regression analyze found out that higher basal FSH level was a predominant risk and prediction factor for spontaneous ovulation (OR=1.20, P=0.009). Conclusions In GnRH-ant cycles, spontaneous ovulation rate is about 10 times than which in GnRH-a cycles. Diminished ovarian function is a predominate risk factor for premature spontaneous ovulation.

OBJECTIVETo provide preimplantation genetic diagnosis(PGD) for two couples carrying thalassemia mutations and chromosomal abnormalities.

METHODSCouple 1 were both carriers of β 41/42 thalassemia mutations, while the husband has carried a reciprocal translocation with a karyotype of 46,XY,inv(9)(p11;q13),t(11;22)(q25;q13). Couple 2 were both carriers of α (-SEA) thalassemia mutation. Their chromosome karyotypes were both normal, but had two spontaneous abortions. The couples had received 1 and 3 blastocysts respectively through in vitro fertilization(IVF) cycles. Following the biopsy, the cells underwent whole genome amplification, and the amplified DNA from each embryo was subjected to genetic testing and a 23-chromosome single nucleotide polymorphism(SNP) microarray assay.

RESULTSThe embryo of couple 1 was diagnosed as carrier of β 41/42 thalassemia with euploid chromosomes. The embryo was transferred and resulted in intrauterine pregnancy. Similarly, an embryo of couple 2 was verified as carrier of α (-SEA) thalassemia with euploid chromosomes.

CONCLUSIONPGD for aneuploidy coupled with testing for single gene disorders via trophectoderm biopsy and whole genome amplification is feasible. The approach can attain diagnosis with minimal damage with sound clinical outcome.

Adult ; Aneuploidy ; Blastocyst ; cytology ; Chromosome Aberrations ; Embryo Transfer ; Female ; Fertilization in Vitro ; Genetic Testing ; Heterozygote ; Humans ; Male ; Mutation ; Pregnancy ; Preimplantation Diagnosis ; beta-Thalassemia ; diagnosis ; embryology ; genetics