The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm.

STUDY QUESTION: Is the mitochondrial DNA (mtDNA) copy number of cumulus granulosa cells (CGCs) related to the maturation of oocyte cytoplasm? SUMMARY ANSWER: Compared with the mtDNA copy number of CGCs from germinal vesicles (GV), CGCs from Metaphase I (MI) oocytes appear to have a lower mtDNA copy number. WHAT IS KNOWN ALREADY: The growth and development of CGCs and oocyte are synchronised. The interaction between CGCs and the oocyte provides the appropriate balance of energy, which is necessary for mammalian oocyte development. Moreover, in the oocyte-cumulus complex (OCC), mature oocytes with higher mtDNA copy numbers tend to have corresponding CGCs with higher mtDNA copy numbers. STUDY DESIGN, SIZE, DURATION: This is a prospective study of 302 OCCs obtained from 70 women undergoing in vitro fertilisation with intracytoplasmic sperm injection (ICSI) at the Reproductive and Genetic Hospital of CITIC-Xiangya, between 24 February 2018 and 21 December 2019. The CGCs were divided into three groups (GV, MI and MII stages) based on the maturation status of their corresponding oocyte. The sample sizes (n = 302) of CGCs in the three stages were 63 (CGCGV), 70 (CGCMI) and 169 (CGCMII), respectively. Some of the samples (n = 257) was used to quantify the mtDNA copy number, while the rest (n = 45) were used to analyse the expression level of mitochondrial genes. Furthermore, we retrieved 82 immature oocytes from among the 257 OCCs used for mtDNA copy numbers, including 36 GV oocytes and 46 MI oocytes, for analysis of oocyte mtDNA. PARTICIPANTS/MATERIALS, SETTING, METHODS: We selected genes with high consistency of real-time PCR results to accurately measure the mtDNA copy number by testing the efficacy and the reproducibility in whole genome amplification (WGA) samples from a human embryonic stem cell line. The CGCs of each oocyte were individually isolated. The mtDNA copy number and gene expression of the CGCs were assessed using real-time PCR techniques. Mitochondrial DNA copy number of the corresponding immature oocytes was also evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: MT-ND1, MT-CO1 and ß-globin genes were chosen for the assessment of mtDNA content, and mRNA expressions of MT-ND1, MT-CO1, PGC-1α and TFAM were also measured. The genome of 257 CGCs and 82 immature oocytes were amplified according to the multiple displacement amplification (MDA) protocol, and RNA was extracted from 45 CGCs. Compared with CGCGV, CGCMI had a significantly lower mtDNA copy number. In the MT-ND1 assay, the CGCGV: CGCMI was [270 ± 302]: [134 ± 201], P = 0.015. In the MT-CO1 assay, CGCGV: CGCMI was [205 ± 228]: [92 ± 112], P = 0.026. There was no statistical difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [270 ± 302]: [175 ± 223], P = 0.074. In the MT-CO1 assay, CGCGV: CGCMII was [205 ± 228]: [119 ± 192], P = 0.077. No statistical difference of mtDNA copy number was observed between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [134 ± 201]: [175 ± 223], P = 0.422. In the MT-CO1 assay, CGCMI: CGCMII was [92 ± 112]: [119 ± 192], P = 0.478. To verify the reliability of the above results, we further analysed the mtDNA copy number of CGCs of 14 patients with GV, MI and MII oocytes, and the results showed that the mtDNA copy number of CGCMI may be lower. The mtDNA copy number of CGCGV and CGCMI was statistically different in the MT-ND1 assay where CGCGV: CGCMI was [249 ± 173]: [118 ± 113], P = 0.016, but in the MT-CO1 assay, CGCGV: CGCMI was [208 ± 199]: [83 ± 98], P = 0.109. There was no significant difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [249 ± 173]: [185 ± 200], P = 0.096. In the MT-CO1 assay, CGCGV: CGCMII was [208 ± 199]: [114 ± 139], P = 0.096. There was also no significant difference in mtDNA between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [118 ± 113]: [185 ± 200], P = 0.198. In the MT-CO1 assay, CGCMI: CGCMII was [83 ± 98]: [114 ± 139], P = 0.470. Moreover, there were no statistical differences in the expression levels of MT-ND1, MT-CO1, PGC-1α and TFAM between CGCGV, CGCMI and CGCMII (P > 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to the ethical issues, the study did not quantify the mtDNA content of MII oocytes. Thus, whether the change in mtDNA copy number in CGCs is related to the different developmental stages of oocytes has not been further confirmed. Moreover, the sample size was relatively small. WIDER IMPLICATIONS OF THE FINDINGS: The mtDNA copy number of CGCs decreases from the GV phase to the MI phase and stays steady from the MI to MII stage. At different stages of oocyte maturation, the mtDNA of CGCs may undergo self-degradation and replication to meet the energy requirements of the corresponding oocyte and the maturation of the oocyte cytoplasm. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the National Key R&D Program of China (Grant 2018YFC1003100, to L.H.), the science and technology major project of the Ministry of Science and Technology of Hunan Province, China (grant 2017SK1030, to G.L.), the National Natural Science Foundation of China (grant 81873478, to L.H.), and Merck Serono China Research Fund for Fertility Experts (to L.H.). There is no conflict of interest.

Comprehensive application of multiple molecular diagnostic techniques in pre-implantation genetic testing for monogenic.

BACKGROUND: Pre-implantation genetic testing for monogenic disorders (PGT-M) is an effective approach to reducing the incidence of birth defects by preventing the transmission of inherited diseases to offspring. However, there are still controversies regarding the detection methods and transplantation of embryos. This paper aims to evaluate the effectiveness of different detection technologies applied to PGT-M through a retrospective analysis of clinical detection data. METHODS: The carrier status of pathogenic mutations and chromosomal copy number variants (CNVs) in 892 embryos was characterized using next-generation sequencing (NGS), single-nucleotide polymorphism (SNP) array, and PCR-based detection technologies. Clinical data from PGT-M cases were retrospectively analyzed to assess the effectiveness of these detection methods in identifying genetic abnormalities in embryos. RESULTS: A total of 829 embryos were analyzed, with 63 being unsuccessful. Our study revealed that the success rate of detecting deletional mutations using Gap-PCR 84.9%, which is lower than that of SNP array (98.7%) and NGS (92.5%). However, no significant difference was observed when detecting point mutations using any of the methods. These findings suggest that, when detecting deletional mutations, SNP array and NGS are more suitable choices compared to Gap-PCR. While SNP array may have a lower resolution and success rate (80.5%) in analyzing CNVs compared to NGS (95.5%), it may still be useful for revealing certain abnormal types. CONCLUSION: In conclusion, this study found that SNP analysis is advantageous for identifying polygenic and deletional mutations, whereas NGS is more cost-efficient for detecting common monogenic diseases. Additionally, SNP-based haplotyping and PCR-based direct detection of mutations can be used together to enhance the accuracy and success rates of PGT-M. Our findings offer valuable insights for PGT technicians in choosing suitable detection methods for patients.

Appropriate whole genome amplification and pathogenic loci detection can improve the accuracy of preimplantation genetic diagnosis for deletional α-thalassemia.

Objective: To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Design: Article. Patients: fifty-two deletional α-thalassemia couples. Interventions: Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. Main outcome measures: WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Results: Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional α-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional α-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% ± 3.57% vs. 0.97% ± 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 ± 9 vs. 34 ± 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 ± 0.263 vs. 0.09 ± 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 ± 999124 vs. 3713146 ± 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 ± 1.09 vs. 5.55 ± 1.49, P=0.008. Conclusions: Our findings indicate that MDA is superior to MALBAC for PGT of deletional α-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional α-thalassemia.

Case Report: Christianson Syndrome Caused by SLC9A6 Mutation: From Case to Genotype-Phenotype Analysis.

Christianson syndrome (CS) is an X-linked neurodevelopmental syndrome characterized by microcephaly, epilepsy, ataxia, and severe generalized developmental delay. Pathogenic mutations in the SLC9A6 gene, which encodes the Na+/H+ exchanger protein member 6 (NHE6), are associated with CS and autism spectrum disorder in males. In this study, whole exome sequencing (WES) and Sanger sequencing revealed a novel de novo frameshift variant c.1548_1549insT of SLC9A6 in a 14-month-old boy with early-onset seizures. According to The American College of Medical Genetics and Genomics (ACMG)/the Association for Molecular Pathology (AMP) guidelines, the variant was classified as pathogenic. The proband presented with several core symptoms of typical epilepsy, including microcephaly, motor delay, distal muscle weakness, micrognathia, occasional unprovoked laughter, swallowing and speech difficulties. Electroencephalography (EEG) showed spikes-slow waves in frontal pole, frontal, anterior temporal and frontal midline point areas. Gesell development schedules (GDS) indicated generalized developmental delay. We also summarized all the reported variants and analyzed the correlation of genotype and phenotype of CS. Our study extends the mutation spectrum of the SLC9A6 gene, and it might imply that the phenotypes of CS are not correlated with SLC9A6 genotypes.

A novel splicing mutation in F8 causes various aberrant transcripts in a hemophilia A patient and identifies a new transcript from healthy individuals.

: Hemophilia A is an X-linked hemorrhagic disorder caused by deficiency or dysfunction of the coagulation factor VIII (FVIII), and a great variety of mutations in the factor VIII gene (F8) are identified. We aimed to identify the genetic defects of the F8 gene in a Chinese patient with moderate hemophilia A. We have identified a novel intronic variant in the hemophilia A patient by DNA sequence analysis, cDNA sequencing, and TA clone sequencing. An intronic variant, c.5816-1G>A, was identified and the cDNA sequencing confirmed the pathogenicity of the transition. TA clone sequencing showed that the splicing mutation produced two aberrant premRNA skipping exons (18 and exon 18 + 19, respectively). These aberrant mRNA forms maintain the reading frame and are predicted to code for deleted FVIII isoforms and the shorter abnormal transcript accounted for one-eighth of the total mRNA. There was a new unreported transcript with E22 spliced out in healthy individuals and our patient, whose specific functions need to be determined in further studies. Our study widens the mutation spectrum of the F8 gene. In addition, the study findings could provide the opportunity to reveal alternative splicing patterns.

Next-generation sequencing analysis of embryos from mosaic patients undergoing in vitro fertilization and preimplantation genetic testing.

OBJECTIVE: To investigate the effects of parental mosaicism on their preimplantation embryos. DESIGN: Case series. SETTING: An institute for reproductive and stem cell engineering. PATIENT(S): Sixty-eight mosaic couples. INTERVENTION(S): Assisted reproduction with preimplantation genetic testing (PGT). MAIN OUTCOME MEASURE(S): Karyotypes, embryo-related chromosomal abnormalities, and PGT results. RESULT(S): A total of 209 embryos were obtained from 68 mosaic couples, and 153 (73.21%) of 209 of the total embryos were obtained from 55 mosaic couples with abnormal sex chromosome numbers. Of these 153 embryos, 2 (1.31%) had an abnormal copy number of X chromosome, 1 had mosaicism with 46,XN,+X(mosaic, 40%), 1 (0.65%) had an extra Y chromosome, 3 (1.96%) exhibited both X chromosomal and autosomal abnormalities, and 4 (2.61%) exhibited de novo X chromosome structural abnormalities. A total of 56 (26.79%) of 209 embryos were obtained from mosaic couples (n = 13) with abnormal autosomal structures. Notably, of these 56 embryos, 5 (8.93%) had a 16q21-q24.3 copy number abnormality related to the parental karyotype, with a fragile site at 16q22; 5 (7.14%) exhibited 46,XX,dup(8p23.1-8p11.21) and 46,XY,del(8p22-8p11.21), which were related to the parental karyotype; and 10 (17.86%) were de novo chromosome abnormalities. CONCLUSION(S): Our data demonstrate that the risk of embryo-related chromosome abnormalities in mosaic patients with abnormal sex chromosomes is very low. Therefore, PGT may not need to be recommended for mosaic patients with abnormal copy numbers of sex chromosomes, especially for patients with financial difficulties. By contrast, the mosaic patients with structural abnormalities of autosomes may have a relatively high risk of abnormal embryos with an unbalanced segment of the involved chromosomes. Thus, PGT is highly recommended for mosaic patients with autosomal structure abnormalities, especially those with a fragile site at 16q22.