OBJECTIVE: To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation. METHODS: A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members. Mature MII oocytes were inseminated by intracytoplasmic sperm injection (ICSI), cultured in vitro for 5 to 6 days to the blastocyst stage, and trophoblastocytes were obtained by microbiopsy. Mitochondrial DNA testing (PGT-MT) and chromosomal aneuploidy (PGT-A) analyses were carried out after whole-genome amplification, and the embryos with zero mutation load were selected for transfer. Amniotic fluid and umbilical cord blood samples were collected during middle pregnancy and after birth respectively for mitochondrial DNA testing to verify the reliability of embryo screening. As an attempt, PB1 with good morphology of MII oocytes was selected for transfer into the enucleated oocytoplasm from healthy donors, followed by ICSI fertilization, blastocyst culture and PGT of embryos using the same procedure. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. 2021zhyx-B12). RESULTS: An antagonist protocol was used for ovarian stimulation, and a total of 19 oocytes were obtained, of which 14 MII were fertilized by ICSI, and 2 had developed into blastocysts. PGT-MT was carried out on biopsied trophoblastocytes, in which the mitochondrial DNA 8993T>G mutation load was not detected in one embryo, the other was 100% mutated, and the mutation loads of the remaining unfertilized eggs and developmentally arrested embryos ranged from 0% ~ 100%, presenting a clear biased distribution. With fully informed consent, one PGT-MT zero mutation load blastocyst was transferred and clinical pregnancy was achieved. Mitochondrial DNA and chromosomal testing of amniotic fluid cells during middle pregnancy had revealed no abnormalities. The proband had delivered a healthy boy through Caesarean section at 39⁺⁵ weeks of gestation, and no mutation was detected in the cord blood sample. Five well-formed PBs from 14 eggs were selected for PB1 transfer, followed by ICSI and culture, and two of the reconstituted embryos had formed blastocysts, with none of the above mutations detected in the biopsied samples. CONCLUSION The PGT-MT technology can help families affected with mitochondrial diseases to have healthy offspring. PB1 transfer in combination with ICSI and PGT-MT holds the promise of turning waste into treasure and providing an alternative means of fertility for such families.
Humans ; Preimplantation Diagnosis/methods* ; Female ; DNA, Mitochondrial/genetics* ; Genetic Testing/methods* ; Pregnancy ; Mitochondrial Diseases/genetics* ; Polar Bodies ; Adult ; Feasibility Studies ; Sperm Injections, Intracytoplasmic/methods* ; Embryo Transfer/methods* ; Mutation ; Male ; Blastocyst/metabolism* ; Pedigree

Objective:To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation.Methods:A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members. Mature MII oocytes were inseminated by intracytoplasmic sperm injection (ICSI), cultured in vitro for 5 to 6 days to the blastocyst stage, and trophoblastocytes were obtained by microbiopsy. Mitochondrial DNA testing (PGT-MT) and chromosomal aneuploidy (PGT-A) analyses were carried out after whole-genome amplification, and the embryos with zero mutation load were selected for transfer. Amniotic fluid and umbilical cord blood samples were collected during middle pregnancy and after birth respectively for mitochondrial DNA testing to verify the reliability of embryo screening. As an attempt, PB1 with good morphology of MⅡ oocytes was selected for transfer into the enucleated oocytoplasm from healthy donors, followed by ICSI fertilization, blastocyst culture and PGT of embryos using the same procedure. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. 2021zhyx-B12). Results:An antagonist protocol was used for ovarian stimulation, and a total of 19 oocytes were obtained, of which 14 MⅡ were fertilized by ICSI, and 2 had developed into blastocysts. PGT-MT was carried out on biopsied trophoblastocytes, in which the mitochondrial DNA 8993T>G mutation load was not detected in one embryo, the other was 100% mutated, and the mutation loads of the remaining unfertilized eggs and developmentally arrested embryos ranged from 0% ~ 100%, presenting a clear biased distribution. With fully informed consent, one PGT-MT zero mutation load blastocyst was transferred and clinical pregnancy was achieved. Mitochondrial DNA and chromosomal testing of amniotic fluid cells during middle pregnancy had revealed no abnormalities. The proband had delivered a healthy boy through Caesarean section at 39+ 5 weeks of gestation, and no mutation was detected in the cord blood sample. Five well-formed PBs from 14 eggs were selected for PB1 transfer, followed by ICSI and culture, and two of the reconstituted embryos had formed blastocysts, with none of the above mutations detected in the biopsied samples.Conclusion:The PGT-MT technology can help families affected with mitochondrial diseases to have healthy offspring. PB1 transfer in combination with ICSI and PGT-MT holds the promise of turning waste into treasure and providing an alternative means of fertility for such families.

Objective To construt and validate a risk prediction model for hepatocellular carcinoma(HCC)in patients with chronic liver disease based on age-male-ALBI-platelets(aMAP)score combined with RAR and PIV.Methods A total of 143 patients with chronic liver disease were divided into the HCC group(32 cases)and the non-HCC group(111 cases)according to whether HCC occurred.General clinical data,aMAP score and peripheral blood indicator level were compared between two groups.Multivariate Logistic regression was used to analyze influencing factors of HCC in inpatients with chronic liver disease.A nomogram risk prediction model was constructed and validated.Results Compared with the non-HCC group,there were higher age,higher proportion of males,higher levels of total bilirubin(TBIL),red blood cell distribution width(RDW),neutrophil count(NEU)and monocyte count(MON),lower levels of albumin(ALB)and lymphocyte count(LYM),higher levels of aMAP score,RDW to ALB(RAR)and pan-immune inflammation value(PIV)in the HCC group(P＜0.05).Multivariate Logistic regression showed that higher levels of aMAP score,RAR and PIV were independent risk factors for HCC in inpatients with chronic liver disease(P＜0.05).The area under receiver operator characteristic(ROC)curve(AUC)of the nomogram risk prediction model constructed based on above factors was 0.823(95%CI:0.747-0.899).The calibration curve showed that the predicted value was basically consistent with the actual observed value,and the Brier score was 0.125.The decision curve showed that the model had a clear positive net benefit.The AUC of internal validation of the prediction model by Bootstrap method was 0.823(95%CI:0.820-0.825),indicating that the model had a good degree of differentiation.Conclusion The nomogram risk prediction model based on aMAP score,RAR and PIV showed a good predictive performance of HCC in patients with chronic liver disease,which could benefits the individualized treatment and follow-up.

Objective:To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation.Methods:A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members. Mature MII oocytes were inseminated by intracytoplasmic sperm injection (ICSI), cultured in vitro for 5 to 6 days to the blastocyst stage, and trophoblastocytes were obtained by microbiopsy. Mitochondrial DNA testing (PGT-MT) and chromosomal aneuploidy (PGT-A) analyses were carried out after whole-genome amplification, and the embryos with zero mutation load were selected for transfer. Amniotic fluid and umbilical cord blood samples were collected during middle pregnancy and after birth respectively for mitochondrial DNA testing to verify the reliability of embryo screening. As an attempt, PB1 with good morphology of MⅡ oocytes was selected for transfer into the enucleated oocytoplasm from healthy donors, followed by ICSI fertilization, blastocyst culture and PGT of embryos using the same procedure. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. 2021zhyx-B12). Results:An antagonist protocol was used for ovarian stimulation, and a total of 19 oocytes were obtained, of which 14 MⅡ were fertilized by ICSI, and 2 had developed into blastocysts. PGT-MT was carried out on biopsied trophoblastocytes, in which the mitochondrial DNA 8993T>G mutation load was not detected in one embryo, the other was 100% mutated, and the mutation loads of the remaining unfertilized eggs and developmentally arrested embryos ranged from 0% ~ 100%, presenting a clear biased distribution. With fully informed consent, one PGT-MT zero mutation load blastocyst was transferred and clinical pregnancy was achieved. Mitochondrial DNA and chromosomal testing of amniotic fluid cells during middle pregnancy had revealed no abnormalities. The proband had delivered a healthy boy through Caesarean section at 39+ 5 weeks of gestation, and no mutation was detected in the cord blood sample. Five well-formed PBs from 14 eggs were selected for PB1 transfer, followed by ICSI and culture, and two of the reconstituted embryos had formed blastocysts, with none of the above mutations detected in the biopsied samples.Conclusion:The PGT-MT technology can help families affected with mitochondrial diseases to have healthy offspring. PB1 transfer in combination with ICSI and PGT-MT holds the promise of turning waste into treasure and providing an alternative means of fertility for such families.

Objective To construt and validate a risk prediction model for hepatocellular carcinoma(HCC)in patients with chronic liver disease based on age-male-ALBI-platelets(aMAP)score combined with RAR and PIV.Methods A total of 143 patients with chronic liver disease were divided into the HCC group(32 cases)and the non-HCC group(111 cases)according to whether HCC occurred.General clinical data,aMAP score and peripheral blood indicator level were compared between two groups.Multivariate Logistic regression was used to analyze influencing factors of HCC in inpatients with chronic liver disease.A nomogram risk prediction model was constructed and validated.Results Compared with the non-HCC group,there were higher age,higher proportion of males,higher levels of total bilirubin(TBIL),red blood cell distribution width(RDW),neutrophil count(NEU)and monocyte count(MON),lower levels of albumin(ALB)and lymphocyte count(LYM),higher levels of aMAP score,RDW to ALB(RAR)and pan-immune inflammation value(PIV)in the HCC group(P＜0.05).Multivariate Logistic regression showed that higher levels of aMAP score,RAR and PIV were independent risk factors for HCC in inpatients with chronic liver disease(P＜0.05).The area under receiver operator characteristic(ROC)curve(AUC)of the nomogram risk prediction model constructed based on above factors was 0.823(95%CI:0.747-0.899).The calibration curve showed that the predicted value was basically consistent with the actual observed value,and the Brier score was 0.125.The decision curve showed that the model had a clear positive net benefit.The AUC of internal validation of the prediction model by Bootstrap method was 0.823(95%CI:0.820-0.825),indicating that the model had a good degree of differentiation.Conclusion The nomogram risk prediction model based on aMAP score,RAR and PIV showed a good predictive performance of HCC in patients with chronic liver disease,which could benefits the individualized treatment and follow-up.

Objective To investigate the application value of single nucleotide polymorphism(SNP)linkage analysis based on next-generation sequencing(NGS)technology in preimplantation genetic testing(PGT)of families with autosomal recessive polycystic kidney disease(ARPKD).Methods A family with ARPKD was selected,where the female member had a pregnancy ultrasound revealing polycystic kidney in the fetus.Genetic testing showed compound heterozygous mutations of the polycystic kidney/polycystic liver disease 1 gene(PKHD1),c.10444C>T(paternal)and c.4303del(maternal),with the c.4303del mutation being reported for the first time.Targeting the coding region of the PKHD1 gene,335 high-density tightly linked SNP sites were selected in the upstream and downstream 2M regions using multiplex polymerase chain reaction(PCR)and NGS.The couple′s SNP risk haplotypes carrying gene mutations were constructed.After in vitro fertilization,blastocyst culture was performed.Trophoblastic cells obtained from the biopsy were subjected to whole-genome amplification,and NGS was used for linkage analysis and low-depth chromosomal aneuploidy screening of the embryos.Sanger sequencing was used to verify the results of embryo linkage analysis.Results Among the 6 biopsied embryos,4 were mutation-free and euploid,1 exhibited heterozygous for the mutation and mosaic while another unstable sequencing data,making it impossible to judge.One of the mutation-free and developmentally healthy euploid embryos was implanted into the maternal uterus,resulting in the full-term delivery of a healthy baby.Conclusion Application of NGS-based SNP linkage analysis in PGT can effectively blocking the vertical transmission of ARPKD within families,while avoiding abortion issues caused by aneuploid embryos.This study is also the first PGT report target-ing the PKHD1 gene c.4303del mutation.

Objective Infertility affects approximately one-sixth of the people of childbearing age worldwide,causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development.Premature ovarian insufficiency(POI)refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles,constituting a significant cause of female infertility.In recent years,with the help of the rapid development in genetic sequencing technology,it has been demonstrated that genetic factors play a crucial role in the onset of POI.Among the population suffering from POI,genetic studies have revealed that genes involved in processes such as meiosis,DNA damage repair,and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified.FA complementation group M(FANCM)is a group of genes involved in the damage repair of DNA interstrand crosslinks(ICLs),including FANCA-FANCW.Abnormalities in the FANCM genes are associated with female infertility and FANCM gene knockout mice also exhibit phenotypes similar to those of POI.During the genetic screening of POI patients,this study identified a suspicious variant in FANCM.This study aims to explore the pathogenic mechanisms of the FANCM genes of the FA pathway and their variants in the development of POI.We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.Methods One POI patient was included in the study.The inclusion criteria for POI patients were as follows:women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L(with a minimum interval of 4 weeks inbetween tests),alongside clinical symptoms of menstrual disorders,normal chromosomal karyotype analysis results,and exclusion of other known diseases that can lead to ovarian dysfunction.We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics(ACMG).Subsequently,the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis.Plasmids containing wild-type and mutant FANCM genes were constructed and introduced into 293T cells.The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group,the EGFP FANCM-MUT group,and the EGFP group,respectively.To validate the production of truncated proteins,cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody.In order to investigate the impact on DNA damage repair,immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM's ability to localize on chromatin.Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT group,which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.Results In a POI patient from a consanguineous family,we identified a homozygous variant in the FANCM gene,c.1152-1155del:p.Leu386Valfs*10.The patient presented with primary infertility,experiencing irregular menstruation since menarche at the age of 16.Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone(AMH)level of 0.07 ng/mL.Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia.The patient's parents were a consanguineous couple,with the mother having regular menstrual cycles.The patient had two sisters,one of whom passed away due to osteosarcoma,while the other exhibited irregular menstruation,had been diagnosed with ovarian insufficiency,and remained childless.Bioinformatics analysis revealed a deletion of four nucleotides(c.1152-1155del)in the exon 6 of the patient's FANCM gene.This variant resulted in a frameshift at codon 386,introducing a premature stop codon at codon 396,which ultimately led to the production of a truncated protein consisting of 395 amino acids.In vitro experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization,with the protein being present only in the cytoplasm.Following treatment with mitomycin C,there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid(P<0.01),indicating a statistically significant impairment of DNA damage repair capability caused by this variant.Conclusions The homozygous variant in the FANCM gene,c.1152-1155del:p.Leu386Valfs*10,results in the production of a truncated FANCM protein.This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex,preventing its entry into the nucleus and the subsequent recognition of DNA damage.Consequently,the localization of the FA core complex on chromatin is disrupted,impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs.By disrupting the rapid proliferation and meiotic division processes of primordial germ cells,the reserve of oocytes is depleted,thereby triggering premature ovarian insufficiency in females.

The decline of ovarian function due to aging is an irreversible natural physiological process, manifested by the progressive depletion of follicles and the decrease of oocyte quality in the ovary. Existing studies have shown that the Sirtuins family is involved in the development of ovarian aging through various pathways, such as regulation of nicotinamide adenine dinucleotide levels, oxidative stress, chromosome segregation and genomic stability. In this paper, we review the current roles of Sirtuins family proteins in ovarian aging and their molecular mechanisms.

Objective:To investigate the embryo development and clinical pregnancy outcomes in patients with ovarian hyperstimulation syndrome (OHSS) undergoing assisted reproductive technology (ART).Methods:A retrospective cohort study was conducted on data from 4 080 cycles of in vitro fertilization/intracytoplasmic sperm injection-embryo transfer treatments performed at the Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University from August 2019 to August 2021. Patients were divided into OHSS group ( n=524) and non-OHSS group (control group, n=3 556) based on whether OHSS occurred, and the OHSS group was further divided into OHSS combined with polycystic ovary syndrome (PCOS) subgroup ( n=231) and OHSS combined with non-PCOS subgroup ( n=293) based on the presence of PCOS. General information, embryo developmental data and clinical outcomes were compared between the two groups. Results:1) Patients in the OHSS group [(30.7±3.6) years] were younger than those in control group [(31.5±4.8) years, P<0.001], and the number of retrieved oocytes (28.2±5.7), rates of high-quality embryos [52.7% (4 982/9 463)], blastocyst formation [54.0% (5 059/9 371)], biochemical pregnancy [75.0% (393/524)], clinical pregnancy [69.5% (364/524)], and live birth [58.0% (304/524)] were significantly higher in the OHSS group than in control group [12.5±6.7, 49.8% (14 042/28 204), 51.4% (14 279/27 797), 59.5% (2 115/3 556), 54.1% (1 924/3 556), 43.6% (1 550/3 556), respectively; all P<0.001]. 2) Patients in the OHSS combined with PCOS subgroup [(30.2±3.1) years] were younger than those in the OHSS combined with non-PCOS subgroup [(31.1±4.0) years, P=0.009], and the estradiol level [165.0 (101.0, 222.5) pmol/L] was higher than that in the OHSS combined with non-PCOS subgroup [141.0 (81.0,202.0) pmol/L, P=0.005]; rates of high-quality embryos [56.3% (2 413/4 284)], blastocyst formation [67.1% (2 846/4 239)], and high-quality blastocysts [57.7% (2 445/4 239)] were also significantly higher in the OHSS combined with PCOS subgroup than in the OHSS combined with non-PCOS subgroup [49.6% (2 569/5 179), 60.3% (3 092/5 132), 50.9% (2 614/5 132), respectively; all P<0.001]. Conclusion:There is a certain correlation between OHSS and female age. The occurrence of OHSS does not affect embryo development and does not increase adverse pregnancy outcomes in infertile patients. The presence of PCOS does not affect the pregnancy outcomes of OHSS patients. However, in ART, we still strive to avoid the occurrence of adverse events such as OHSS as much as possible.

The decline of ovarian function due to aging is an irreversible natural physiological process, manifested by the progressive depletion of follicles and the decrease of oocyte quality in the ovary. Existing studies have shown that the Sirtuins family is involved in the development of ovarian aging through various pathways, such as regulation of nicotinamide adenine dinucleotide levels, oxidative stress, chromosome segregation and genomic stability. In this paper, we review the current roles of Sirtuins family proteins in ovarian aging and their molecular mechanisms.