ObjectiveTo investigate the association between endothelin receptor type A gene （EDNRA） and congenital bilateral absence of the vas deferens （CBAVD）. MethodsThis case-control study consisted of 124 subjects with CBAVD and 100 fertile controls.Two single nucleotide polymorphisms （SNPs： rs5335 and rs1801708） in the EDNRA gene were genotyped via PCR， PCR-RFLP analysis， and direct sequencing. ResultsNo significant differences existed between EDNRA polymorphisms and CBAVD phenotype （rs1801708： P=0.220 2， 0.163 2； rs5335： P=0.805 8， 0.818 6）. However， the rs1801708-rs5335 haplotype AG was notably associated with an increased risk of CBAVD （P=0.008 6， OR=2.178， 95% CI： 1.207-3.929）. A significantly protective effect of rs1801708-rs5335 haplotype GG on CBAVD （P=0.038 5， OR= 0.671， 95% CI： 0.460-0.980） was observed. After Bonferroni correction， the result for haplotype A-G remained significant （P =0.008 6<0.012 5）， while haplotype G-G was not significant. ConclusionThe rs1801708-rs5335 AG haplotype of EDNRA is a potential risk factor for CBAVD development in Han Chinese.

Objective To investigate the diagnostic value of serum bromodomain-containing pro-tein 4(BRD4)combined with gastrin-17(G-17)in Helicobacter pylori(Hp)-positive early gastric cancer.Methods A total of 88 patients with Hp-positive early gastric cancer admitted to our hospital from September 2021 to September 2023 were selected as early gastric cancer group.Meanwhile,92 patients with Hp-positive precancerous lesions and 80 patients with Hp-positive gastritis admitted dur-ing the same period were selected as precancerous lesion group and gastritis group,respectively.En-zyme-linked immunosorbent assay(ELISA)was used to detect the serum levels of BRD4 and G-17.Multivariate logistic regression analysis was performed to screen the influencing factors of Hp-positive early gastric cancer.Receiver operating characteristic(ROC)curve analysis was used to evaluate the diagnostic value of serum BRD4 and G-17 in Hp-positive early gastric cancer.Results Compared with the gastritis group,the levels of BRD4 and G-17 in the precancerous lesion group and early gastric cancer group were significantly increased(P＜0.05).Furthermore,compared with the precancerous le-sion group,the levels of BRD4 and G-17 in the early gastric cancer group were also significantly elevated(P＜0.05).The proportion of patients with a family history of gastric cancer,those who preferred hot food,cold food,or high-salt food,as well as the levels of PG Ⅱ,BRD4,and G-17 were signifi-cantly higher in the early gastric cancer group than in the non-gastric cancer group,while the level of pepsinogen(PG)Ⅰ was significantly lower(P＜0.05).Logistic regression analysis revealed that preference for hot food,high-salt food,PG Ⅱ,BRD4,G-17,and PG Ⅰ were all influencing factors for Hp-positive early gastric cancer(P＜0.05).The area under the curve(AUC)values for serum BRD4,G-17,and their combination in diagnosing Hp-positive early gastric cancer were 0.793,0.830,and 0.912,respectively.The diagnostic efficacy of the combined detection was superior to that of single detection(P＜0.05).Conclusion The serum levels of BRD4 and G-17 are elevated in patients with Hp-positive early gastric cancer,and both exhibit certain diagnostic value for Hp-positive early gastric cancer,suggesting their potential as serum biomarkers for the diagnosis of Hp-positive early gastric cancer.

Objective: To evaluate the clinical efficacy of preimplantation genetic testing for monogenic disorders(PGT-M) in families with hereditary epilepsy. Methods : Whole-exome sequencing(WES) and familial co-segregation analysis were performed to validate the pathogenicity of variants(PCDH19 c. 1031C > G and LGI1 c. 856T >G) in two monogenic epilepsy families. A clinical PGT-M pathway was implemented,and reproductive outcomes were tracked. Results: In Family 1(PCDH19 likely pathogenic variant),13 blastocysts were biopsied over two ovarian stimulation cycles,yielding 3 unaffected euploid embryos(23. 1%). After the third frozen embryo transfer,a healthy male infant was successfully delivered. Prenatal diagnosis confirmed that the fetus did not carry the pathogenic variant PCDH19. Family 2(LGI1 variant of uncertain significance,VUS) screened 14 blastocysts,identifying 2 unaffected euploid embryos(14. 3%),with the first transfer unsuccessful. A clinical pregnancy was currently ongoing following the second frozen-thawed embryo transfer(FET). Conclusion PGT-M can precisely block the vertical transmission of monogenic epileptic pathogenic variants,offering an effective reproductive intervention strategy for families with hereditary epilepsy.

Objective: To investigate the impact of endometriosis on pregnancy and delivery outcomes in patients undergoing single euploid frozen_thawed blastocyst transfer cycles following preimplantation genetic testing. Methods : A retrospective analysis was performed on clinical data from patients undergoing frozen_thawed blastocyst transfer after preimplantation genetic testing at the reproductive center of The First Affiliated Hospital of Anhui Medical University. The endometriosis group comprised 84 treatment cycles. After 1 : 3 propensity score matching , 252 treatment cycles from non_endometriosis patients were included as the control group. General characteristics and clinical outcomes were compared between the two groups. Results: There were no statistically significant differences between the two groups in terms of general characteristics , human chorionic gonadotropin ( HCG) positive rate , cycle clinical pregnancy rate per cycle , early miscarriage rate , preterm birth rate , live birth rate per cycle , cesarean section rate , delivery weeks , cumulative clinical pregnancy rate , and cumulative live birth rate (all P > 0. 05) . Conclusion Endometriosis may not reduce the pregnancy rate and live birth rate in single frozen euploid blastocyst transfer cycles .

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 investigate the status of allostatic load (AL) in patients with polycystic ovary syndrome (PCOS) and its influence on the clinical outcomes of in vitro fertilization-embryo transfer.Methods:This was a prospective study. By using convenient sampling method, 421 patients with PCOS (PCOS group) and 372 control infertility patients (control group) in the Reproductive Center of the First Affiliated Hospital of Anhui Medical University from April 2022 to January 2024 were investigated for basic information, physical examination, laboratory examination and follow-up of clinical outcomes. The total score of AL was calculated using 16 related indicators of cardiovascular system, metabolic system and immune system, and AL>3 was used as the judgment criteria for the high level AL group and the low level AL group. The differences in general data, embryo development and clinical outcomes between the groups were compared.Results:There were 222 cases (52.7%, 222/421) in PCOS low level AL group and 199 cases (47.3%, 199/421) in PCOS high level AL group. There were 214 patients (57.5%, 214/372) in the control low level AL group and 158 patients (42.5%, 158/372) in the control high level AL group. Embryo development outcomes: number of oocytes retrieved (median: 12, 12, 19, 14, respectively; P<0.001), number of two pronuclei (median: 8, 7, 11, 8, respectively; P<0.001), number of fertilization (median: 9, 9, 13, 10, respectively; P<0.001), number of metaphase of meiosis Ⅱ oocytes (median: 9, 8, 13, 10, respectively; P<0.001), number of transferable embryos (median: 5, 5, 7, 6, respectively; P<0.001), number of high-quality embryos (median: 4, 3, 6, 5, respectively; P<0.001), gonadotropin(Gn) starting dosage (median: 150, 200, 150, 200 U, respectively; P<0.001), total dosage of Gn (median: 1 800, 2 075, 1 575, 2 025 U, respectively; P<0.001), duration of Gn used (median: 10, 10, 10, 10 days, respectively; P=0.027) in the control low level AL group, control high level AL group, PCOS low level AL group and PCOS high level AL group were significantly different. Pairings between groups showed that number of oocytes retrieved, number of two pronuclei, number of fertilization, number of metaphase of meiosis Ⅱ oocytes and number of transferable embryos in PCOS high level AL group were lower than those in PCOS low level AL group (all P<0.05); Gn starting dosage and total dosage of Gn in PCOS low level AL group were lower than those in the other three groups (all P<0.05); duration of Gn used in PCOS high level AL group was higher than that PCOS low level AL group ( P<0.05). Clinical outcomes: the control low level AL group, control high level AL group, PCOS low level AL group and PCOS high level AL group underwent fresh transplantation [27.4% (57/208), 24.4% (38/156), 15.1% (32/212), 17.1% (33/193), respectively; P=0.006] and the proportion of transplanted day 5 embryos [82.7% (172/208), 77.6% (121/156), 91.0% (193/212), 86.5% (167/193), respectively; P=0.018] were statistically significant. There were no significant differences in fertilization rate, biochemical pregnancy rate, clinical pregnancy rate, multiple pregnancy rate and early abortion rate among the four groups (all P>0.05). Conclusion:The high level of AL in PCOS patients may affect the outcomes of embryo development, and more attention should be paid to AL in PCOS patients to reduce stress.

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 investigate the status of allostatic load (AL) in patients with polycystic ovary syndrome (PCOS) and its influence on the clinical outcomes of in vitro fertilization-embryo transfer.Methods:This was a prospective study. By using convenient sampling method, 421 patients with PCOS (PCOS group) and 372 control infertility patients (control group) in the Reproductive Center of the First Affiliated Hospital of Anhui Medical University from April 2022 to January 2024 were investigated for basic information, physical examination, laboratory examination and follow-up of clinical outcomes. The total score of AL was calculated using 16 related indicators of cardiovascular system, metabolic system and immune system, and AL>3 was used as the judgment criteria for the high level AL group and the low level AL group. The differences in general data, embryo development and clinical outcomes between the groups were compared.Results:There were 222 cases (52.7%, 222/421) in PCOS low level AL group and 199 cases (47.3%, 199/421) in PCOS high level AL group. There were 214 patients (57.5%, 214/372) in the control low level AL group and 158 patients (42.5%, 158/372) in the control high level AL group. Embryo development outcomes: number of oocytes retrieved (median: 12, 12, 19, 14, respectively; P<0.001), number of two pronuclei (median: 8, 7, 11, 8, respectively; P<0.001), number of fertilization (median: 9, 9, 13, 10, respectively; P<0.001), number of metaphase of meiosis Ⅱ oocytes (median: 9, 8, 13, 10, respectively; P<0.001), number of transferable embryos (median: 5, 5, 7, 6, respectively; P<0.001), number of high-quality embryos (median: 4, 3, 6, 5, respectively; P<0.001), gonadotropin(Gn) starting dosage (median: 150, 200, 150, 200 U, respectively; P<0.001), total dosage of Gn (median: 1 800, 2 075, 1 575, 2 025 U, respectively; P<0.001), duration of Gn used (median: 10, 10, 10, 10 days, respectively; P=0.027) in the control low level AL group, control high level AL group, PCOS low level AL group and PCOS high level AL group were significantly different. Pairings between groups showed that number of oocytes retrieved, number of two pronuclei, number of fertilization, number of metaphase of meiosis Ⅱ oocytes and number of transferable embryos in PCOS high level AL group were lower than those in PCOS low level AL group (all P<0.05); Gn starting dosage and total dosage of Gn in PCOS low level AL group were lower than those in the other three groups (all P<0.05); duration of Gn used in PCOS high level AL group was higher than that PCOS low level AL group ( P<0.05). Clinical outcomes: the control low level AL group, control high level AL group, PCOS low level AL group and PCOS high level AL group underwent fresh transplantation [27.4% (57/208), 24.4% (38/156), 15.1% (32/212), 17.1% (33/193), respectively; P=0.006] and the proportion of transplanted day 5 embryos [82.7% (172/208), 77.6% (121/156), 91.0% (193/212), 86.5% (167/193), respectively; P=0.018] were statistically significant. There were no significant differences in fertilization rate, biochemical pregnancy rate, clinical pregnancy rate, multiple pregnancy rate and early abortion rate among the four groups (all P>0.05). Conclusion:The high level of AL in PCOS patients may affect the outcomes of embryo development, and more attention should be paid to AL in PCOS patients to reduce stress.

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 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.