Application research of PGT in blocking the inheritance of novel mutations in the PKHD1 gene in autoso-mal recessive polycystic kidney disease pedigrees / 实用医学杂志

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.

A prediction model of live birth in patients undergoing frozen⁃thawed embryo transfer after TCRA / 安徽医科大学学报

Objective @#To develop a prediction model for live birth based on the basic characters of patients after transcervical resection of adhesions (TCRA) undergoing frozen⁃thawed embryo transfer (F⁃ET) .@*Methods @# A total of 491 cycles who went F ⁃ET after TCRA were included. The cycles were randomly divided into training (n = 347) and validation (n = 144) cohorts at 7 ∶ 3 ratio. Relevant factors of live birth were selected via logistic regression analysis based on the data from patients in the training cohort. Multivariate logistic regression analysis was used to establish a nomogram , and then this predict model was calibrated and verified. @*Results @#Age , body mass index (BMI) , miscarriages and endometrial thickness were significantly related to the live birth. The statistical nomogram was built with the area under the ROC cu2rve for the training cohort was 0. 713 (95% CI: 0. 519 - 0. 815) , Hosmer⁃Lemeshow goodness of fit test were χ = 7. 062 , P = 0. 530 , and the slope of calibration curve was close to 1. AUC of validation cohort was 0. 609 (95% CI: 0. 660 - 0. 561) , presenting a well⁃pleasing goodness⁃of⁃fit and stability in this model.@*Conclusion @#Age , BMI , miscarriages and endometrial thickness can better predict the probability of live birth undergoing F ⁃ET after TCRA.

Influence of ovarian artery blood flow index on pregnancy outcomes of patients undergoing in vitro fertilization-embryo transfer / 实用医学杂志

Objective To investigate the influence of ovarian arterial blood flow index on pregnancy outcomes in patients undergoing in vitro fertilization and embryo transfer (IVF-ET).Methods Totally 136 infertile patients undergoing IVF-ET from February 2014 to April 2016 were divided into pregnant group and non-pregnant group.The monitoring through transvaginal B of two groups for chorionic gonadotropin (HCG) day and ovarian artery resistance index (RI),pulsatility index (PI) and umbilical artery diastolic flow velocity height ratio (S/D) on 2nd day of menstruation (M2) was conducted.The difference of blood flow indexes in two groups was analyzed.Results Compared with those in non-pregnant group,RI,PI and S/D in pregnant group were decreased on M2,and the difference was statistically significant (P ＜ 0.05).RI and PI were also decreased significantly on HCG day,and the difference was statistically significant (P ＜ 0.05),but S/D showed no significant changes (P ＞ 0.05).RI and PI decreased significantly on HCG day in two groups (P ＜ 0.05) when compared with those on M2,but in non-pregnant group,the comparison of S/D on HCG day showed no statistical significance (P ＞ 0.05).Conclusions There is close relationship among RI and PI of ovarian artery and ovarian reserve function.RI and PI are effective indexes to predict outcomes in IVF-ET,and provide scientific basis for the preparation and treatment for IVF-ET.

expression of the autophagy gene beclin-1 mRNA and protein in ectopic and eutopic endometrium of patients with endometriosis

To investigate the expression of Beclin-1 mRNA and protein in eutopic and ectopic endometrium of women with and without endometriosis, and evaluate the association of Beclin-1 protein expression and serum CA125 levels in the endometriosis group due to CA125 being a well-known biomarker of endometriosis. The expression levels [mean +/- SD] of the mRNA and protein of Beclin-1 were examined in uterine endometria from 26 women without endometriosis and in eutopic and ectopic endometria from 26 endometriosis patients through experimental study, as reverse transcription PCR and Western-blotting assays. Serum CA125 levels in the endometriosis and control groups were compared and the correlation between Beclin-1 protein expression and serum CA125 was evaluated in the endometriosis group. Both eutopic [0.12 +/- 0.04, 1.25 +/- 0.42] and ectopic [0.12 +/- 0.05, 1.09 +/- 0.50] endometriotic tissue from 26 women with endometriosis expressed significantly lower levels of Beclin-1 mRNA and protein than endometrium from 26 normal women [0.15 +/- 0.02, 1.67 +/- 0.44] [p<0.05]. Serum CA125 levels were found to be significantly higher in the endometriosis group [p<0.05]. In addition, Beclin-1 protein expression of eutopic endometria in patients with endometriosis was negatively correlated with serum CA125 [r= -0.57, p<0.01]. The present study strongly suggests that Beclin-1 may play a role in the formation and progression of endometriosis

Association between recurrent miscarriages and insulin resistance:a Meta analysis / 中华妇产科杂志

Objective To evaluate the association between recurrent miscarriages and insulin resistance.Methods The case-control studies on the association between recurrent spontaneous abortion and insulin resistance from June 1996 to April 2012 were collected from Medline,Elsevier,Chinese Journal Fulltext Database,Chinese Biological Medicine Database,data base of Wanfang,Springer link and EMBASE.RevMan 5.1 software was used for Meta analysis.Results According to the included criteria,7 clinical trials were finally selected.Total 467 cases with recurrent pregnancy loss were enrolled in study group,while 413 women with no history of abnormal pregnancies were enrolled in control group.No significant difference was found in average age and body mass index between the two groups (P ＞ 0.05).Meta analysis results showed that the level of fasting glucose was no statistical difference between study group and control group (WMD =2.27,95％ CI:-1.11 to 5.65,P ＞0.05); fasting insulin level was higher 2.05 mU/L in study group than that of in control group,the difference was statistically significant (WMD =2.05,95％ CI:1.03 to 3.08,P ＜ 0.01).Case number of study group on Homa-insulin resistance ＞ 4.5 was more than that of control group (OR =3.36,95％ CI:1.72 to 6.57,P ＜ 0.01).Case number of study group on glucose/insulin ratio ＜ 4.5 was more than that of the control group,statistical difference was found (OR =3.37,95％ CI:1.90 to 5.99,P ＜ 0.01).Conclusion Insulin resistance is associated with the susceptibility to recurrent miscarriages,and it may contribute to the occurrence of recurrent miscarriages.

Polymorphism of CYP11A1 gene in Chinese patients with polycystic ovarian syndrome / 中华妇产科杂志

Objective To investigate the association between polymorphism of cytochrome P450 subfamily Ⅺ A polypeptide 1 (CYP11A1) gene and polycystic ovarian syndrome (PCOS) in Chinese population. Methods From May 2005 to Dec.2008,290 PCOS cases treated in the First affiliated hospital of Anhui Medical University matched with 344 reproductive women as controls were enrolled in this study. Genotypes of 7 tagging single nucleotide polymorphisms(tSNP,rs12438594,rs4077582,rs9806234,rsl6968477,rs4887139,rs1843090,rsl 1632698)covering CYP11A1 gene (r~2≥0.8) and 3 microsatellite markers (D15S1547,D16S520,D15S1546) were chosed from the phase II database of Han population in HapMap data.Genotype and frequency of allele were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and haplotype of gene polymorphism were analyzed in 290 PCOS cases and 344 controls.Results Among 7 tSNPs and 3 microsatellite markers,the frequency of rs4077582,D15S1547,D15S1546 and rsl 1632698 between two groups reached statistical difference (P =0.010,0.044,0.018 and 0.026).The allele frequency of rs4O77582,rs4887139,rsl843090,D15S1547 and Dl 6S520 showed significant difference between two groups(P=0.002,0.048,0.030,0.001 and 0.024).Among 5 haplotype of CYP11A1(ACGCA13/6/9AG,ACGTA16/6/11AA,GCACG12/8/8AA,GTACA14/4/7GG,GTGCA13/6/7AG),the frequency of GTACA14/4/7GG and ACGCA13/6/9AG were 7.8% (45/580) and 25.3% (147/580) in PCOS group and 11.9% and 19.6% in control group,which showed statistical difference (P< 0.05 ).There was no significant difference in the level of serum androgen at difference genotype from rs4077582 between two groups (P>0.05).Conclusion The polymorphism of CYP11A1 gene was associated with PCOS,however,the relationship between gene sequence covered by tSNP/microsatellite markers and hyperandrogenism of PCOS should be further investigated.

The effect of the adenomyosis on the outcome of in vitro fertilization-embryo transfer in woman with endometriosis / 中国基层医药

Objective To evaluate the influence of adenomyosis on IVF-ET outcomes in infertile patients with endometriosis.Methods 122 cases were divided into two groups:group A(research group)contained 32 patients in infertile with both adenomyosis and endometriosis;group B(control group) contained 90 patients with endometriosis.The main data measured were response to COH,outcome of IVF-ET.Results Both groups had no significant difference in Gn dosage,number of oocytes obtained,number of fertilization and number of cleavage(all p＞0.05),The clinical pregnancy rate of group A was 21.9%,which was lower than that of group B(30.0%),but there were no statistical difference(P＞0.05).Conclusion Adenomyosis had no adverse effects on IVF-ET outcomes in infertile women with proven endometriosis.

Effect of double activation on the development of frozen-thawed oocytes after intracytoplasmic sperm injection / 中华妇产科杂志

Objective To investigate the influence on developmental potential of frozen-thawed rabbit oocytes with double assisted activation followed by intracytoplasmic sperm injection (ICSI). Methods A total of rabbit oocytes were collected and thawed after vitrification cryopreservation. Among all oocytes were cultured for 1 hour followed by ICSI. 156 Survived oocytes were divided into 5 groups randomly. I0634 single activation: 30 oocytes were added with calcium ionomycin ( I0634 ) at 5 μmol/L for 5 minutes;SrCl2single activation: 26 oocytes were added with strontium chloride at 10 mmol/L for 10 minutes;10634 double activation: 33 oocytes were activated by I0634 twice;SrCl2 double activation: 28 oocytes were activated by strontium chloride twice. Control group: 39 oocytes were not added with any activators. The rate of fertilization, cleavage and blastocysts formation were observed and compared between various groups. Result The rates of fertilization, cleavage and blastocysts formation were in group of SrCl2 single activation were higher than those of I0634 single activation group without statistical difference (54% vs. 33%, 27% vs. 17%, 8% vs. 3%, P <0.05 ). However, those above rates in double activation by I0634 were higher significantly than those of single I0634 activation (82% vs. 33%, 55% vs. 17%, 15% vs. 3%, P < 0.05). The rates of fertilization (61%) was higher and the rate of cleavage (21%) and blastocysts formation (7%) were lower in group of SrCl2 double activation in comparison with group of SrCl2 single activation without reaching statistical difference (P<0.05 ). Notably, the rates of fertilization, cleavage and blastocysts formation in I0634 double activation group were higher than those in group of SrCl2 double activation with statistical difference (82% vs. 61%, 55% vs. 21%, 15% vs. 7%, P<0.05). Conclusion It might enhance the potential of fertilization of oocytes and early embryo development treated by double activation following ICSI, however, those activated oocytes demonstrate rapid cleavage.

Effect of culture time on the spindle and chromosome configurations of human thawed oocytes with slow-cooHng and raid-thawing protocol / 中华妇产科杂志

0bjectlve To examine the metaphase II spindle and chromosome configurations of human oocytes cultured for different times after thawing.MethodsUsing slow.cooling and raid—thawing protocol combined with 0.3 mol/L sucrose and 1.5 mol/L 1,2-propanedio 1(1,2-PROH)to cryoprotect human mature oocytes(n=102),the 64 survival oocytes without abnormal zona pellucida and cytoskeletal were randomly assigned to three groups after thawing:group A:culture 1 hour(n=20),group B:culture 3 hour(n=22),group C:culture 5 hours(n=22),the flesh oocytes served as control group(n=18).Immunocytochefifical staining and fluorescence microscopy were used to assess the morphology of the metaphase II spindle and chromosome.Results(1)The normal spindle rates of groups A,B and C were 10％(2/20),46％(10/22)and 41％(9/22)respectively,significantly decreased compared with control group(83％,15/18;P<0.05).The rates of absent spindle in group A(45％,9/20)was significantly higher than control group(6％,1/18;P<0.01).Also,the rates of absent spindle in group A was higher than groups B(14％,3/20)and C(14％,3/20;P<0.05).However,no significant differences were observed in groups B and C(P>0.05).(2)A significant increase in abnormal chromosome rate was observed in group A(30％,6/20)compared to groups B(68％,15/22),C(64％,14/22)and control group(78％,14/18;尸<0.05).No differences in chromosome morphology were observed in groups B,C and control group(P>0.05).Conclusions The cryoproteetant protocol leads to a deleterious effect on the organization of the meiotic spindle and chromosome at MI stage.The 3—5 hours post—thawing incubation could permit restoration of the meiotic spindles and chromosome.