Analysis of Preimplantation and Clinical Outcomes of Two Cases by Oxford Nanopore Sequencing.

It is challenging to distinguish embryos with a balanced translocation karyotype from a normal karyotype by existing conventional genetic testing methods. However, in germ-cell gamete generation, chromosome exchange and separation through cell meiosis form a different proportion of unbalanced gametes. Adverse birth events may occur, such as repeated miscarriages and fetal birth defects. In this study, the exact breakpoints of structural variation (SV) from two balanced translocation carrier families by using Nanopore long reads sequencing technology were obtained, and haplotype analysis and Sanger verified the accuracy of the detection results, confirming the application value of the Nanopore sequencing technology in the detection of balanced translocation before embryo implantation. Nanopore long-read sequencing was performed to find the precise breakpoint of chromosome-balanced translocation carriers. The breakpoints were subsequently verified by designing primers across the breakpoints and Sanger sequencing. Haplotype linkage analysis of SNPs which can be linked by a read block of families around the breakpoint regions was followed. After frozen (-thawed) embryo transfer (FET), prenatal cytogenetic analysis of amniotic fluid cells confirmed the predicted karyotypes from the transferred embryos. The presence of breakpoints was detected in three embryos of patient 1. No breakpoints were detected in either embryo of patient 2. One balanced translocated embryo from patient 1 and one normal euploid embryo from patient 2 were transplanted back into the patients, and amniotic fluid cells were analyzed for the karyotype of fetuses. The results were entirely consistent with the fetal karyotype. And through late follow-up, both patients successfully had a live birth fetus. The breakpoint location of the balanced chromosome translocation can be accurately found by Nanopore sequencing. The haplotype of carriers can be successfully constructed by Nanopore and sanger sequencing confirmed that the results were accurate. This is very advantageous for preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) detection in the families without proband.

Identification and interruption of inheritance of familial cryptic translocations: A case report.

BACKGROUND: Cryptic translocations can be identified via genetic analysis of aborted tissues or malformed infants, but it is difficult to deduce the parental origins of the translocations. In the absence of such information, it is not easy to distinguish translocations from normal embryos during pre-implantation genetic testing, that seeks to block familial transmission of translocations. METHODS: Here, we present a new method that detects cryptic translocations and blocks familial transmission thereof. Whole-genome, low-coverage mate-pair sequencing (WGLMPS) revealed chromosome breakpoint sequences, and preimplantation genetic haplotyping (PGH) was then used to discard embryos with cryptic translocations. RESULTS: Cryptic translocations were found in all four families, and familial transmission was successfully blocked in one family. CONCLUSION: Whole-genome, low-coverage mate-pair sequencing combined with preimplantation genetic haplotyping methods powerfully and practically identify cryptic translocations and block familial transmissions.

Improved pregnancy outcomes from mosaic embryos with lower mtDNA content: a single-center retrospective study.

RESEARCH QUESTION: The purpose of this study is to investigate whether the mitochondrial DNA (mtDNA) content can reflect the state of mosaic embryos. DESIGN: The study included 1669 blastocysts derived from 394 PGT-A cycles between January 2018 and December 2020, in which preimplantation genetic testing for aneuploidy was performed and mtDNA content was determined. The standard deviation (SD) of whole genomic sequencing data was calculated for quality control. mtDNA content was measured as the proportion of mtDNA to genomic DNA. 1558 blastocysts with SD values less than 4.0 and mtDNA values less than 0.4% were selected for statistical analysis. RESULTS: The mtDNA content of the PGT mosaic group was significantly higher than that of the PGT normal group (P < 0.001). Twenty-six mosaic embryos were transferred, and the results were as follows: 2 out of 26 had undergone a spontaneous miscarriage, 15 were not pregnant, and 9 resulted in a live birth. There were significant differences in the mtDNA content between the miscarriage/non-pregnancy group and the live birth group (**P < 0.01; ***P < 0.001). There was no mosaic embryo with more than 0.157% mtDNA content found in the live birth group. CONCLUSIONS: This study demonstrates that mtDNA analysis has the ability to identify mosaic embryos with high developmental potential. It can be a valuable supplementary index for the selection of mosaic embryos for transfer. Larger studies with a greater sample size will further our understanding of the relationships between metabolic activity and mosaicism.

Reactive Oxygen Species Secreted by Leukocytes in Semen Induce Self-Expression of Interleukin-6 and Affect Sperm Quality.

Reproductive tract inflammation is considered an important cause of male infertility. Increased leukocytes in semen can produce many reactive oxygen species (ROS), which affect sperm function. The aim of this study is to identify the main source of ROS in seminal plasma and to assess the effect of ROS on leukocytes. Semen samples (n = 20) with leukocyte concentration >1 × 106 were collected from a male infertility clinic. This study mainly compares the sperm function parameters of the normal group and the semen white blood cell group >1 × 106. The results identified that ROS in semen was closely related to sperm function parameters, and CD45+ leucocytes were the main source of ROS. Compared with the control group, the concentration of IL-2, IL-4, IL-6, IFN-γ, and TNF-α was higher in the experimental group. Leukocytes in semen may regulate the secretion of ROS through the mammalian target of rapamycin (mTOR) pathway. A considerable amount of ROS can upregulate the expression of IL-6 in leukocytes via the nuclear factor kappa-B (NF-kB) pathway.

[Correlation between differential expression of microRNA and quality of embryos].

OBJECTIVE: To explore the correlation between microRNA (miRNA) differential expression and quality of embryo. METHODS: The miRNA expression profiles of 8 blastocysts were detected by a TaqMan microRNA array, and miRNAs with a stable expression were selected. Additional blastocysts were selected, and the candidate miRNA was detected by real-time PCR. Meanwhile, chromosomal abnormalities of the embryos were detected by using next-generation sequencing, and the results were compared. RESULTS: The expression of mir-720, mir-372, mir-886-3p and mir-512-3p was higher than that of miR-145, which suggested that mir-720, mir-372, mir-886-3p and mir-512-3p are related to early embryo development. The expression of miR-145 and mir-886-3p were significantly lower in the normal chromosome group. With the threshold values of above 9 and 3 for the relative expression of miR-145 and mir-886-3p, respectively, there was no embryo without a chromosomal abnormality. CONCLUSION: There is a correlation between the expression level of specific miRNA and chromosomal abnormalities of embryos, which may be used as a novel biomarker for embryo selection.

A novel compound heterozygous mutation of the SMARCAL1 gene leading to mild Schimke immune-osseous dysplasia: a case report.

BACKGROUND: Schimke immune-osseous dysplasia (SIOD, OMIM 242900) is characterized by spondyloepiphyseal dysplasia, T-cell deficiency, renal dysfunction and special facial features. SMARCAL1 gene mutations are determined in approximately 50% of patients diagnosed with SIOD. CASE PRESENTATION: The case presented here is that of a 6-year-old boy who was born at 33 weeks to healthy, non-consanguineous Chinese parents. He presented with short stature (95 cm; <3rd percentile) and proteinuria. Initially suspected of having IgM nephropathy, the patient was finally diagnosed with mild Schimke immune-osseous dysplasia. One novel mutation (p.R817H) and one well-known mutation (p.R645C) was identified in the SMARCAL1 gene. CONCLUSION: This report describes a clinical and genetic diagnostic model of mild SIOD. It also highlights the importance of molecular testing or clinical diagnosis and the guidance it provides in disease prognosis.

Polymorphisms of protamine genes contribute to male infertility susceptibility in the Chinese Han population.

Protamine (PRM) plays important roles in the packaging of DNA within the sperm nucleus. To investigate the role of PRM1/2 and transition protein 1 (TNP1) polymorphisms in male infertility, 636 infertile men and 442 healthy individuals were recruited into this case-controlled study of the Chinese Han population, using MassARRAY technology to analyze genotypes. Our analysis showed that there were no significant differences between controls and infertile cases among the five single nucleotide polymorphisms identified in PRM1, PRM2 and TNP1 [rs737008 (G/A), rs2301365 (C/A), rs2070923 (C/A), rs1646022 (C/G) and rs62180545 (A/G)]. However, we found that the PRM1 and PRM2 haplotypes GCTGC, TCGCA and TCGCC exhibited significant protective effects against male infertility compared to fertile men, while TCGGA, GCTCC and TCGGC represented significant risk factors for spermatogenesis. Our data showed that rs737008 and rs2301365 in PRM1, and rs1646022 in PRM2, were significantly associated with male infertility and that gene-gene interaction played a role in male infertility. A linkage disequilibrium plot for the five SNPs showed that rs737008 was strongly linked with both rs2301365 and rs2070923. These findings are likely to help improve our understanding of the etiology of male infertility. Further studies should include a larger number of genes and SNPs, particularly growing critical genes; such studies will help us to unravel the effect of individual genetic factors upon male infertility.

Application of next­generation sequencing for molecular diagnosis in a large family with osteogenesis imperfecta type I.

Increased bone fragility and low bone mass are common features of osteogenesis imperfecta (OI), which is associated with connective tissue. Its type is distinguished by clinical phenotypes and molecular genetics. Although fifteen types (I­XV) of OI have been identified at present, the majority of patients are diagnosed as OI type I­IV. Type I collagen is responsible for OI type I­IV, consists of α1 (I) and α2 (I) chains and is encoded by COL1A1 and COL1A2. To identify the pathogenic gene of a large Chinese family with OI type I and explain genetic heterogeneity of the patients, next­generation sequencing (NGS) was conducted in a female with OI type I and her affected niece and daughter to search for the mutation. Subsequently, it was confirmed in other family members by Sanger sequencing. Analysis of COL1A1 gene identified a splicing mutation (c.471+1G>A, also termed IVS5+1G>A) that converted the 5' end of intron 5 from GT to AT. The current study aimed to investigate why there are different phenotypes with the same mutation observed within the same OI pedigree, and the results suggested that there may be environmental factors involved. The present study provided genetic counseling and prenatal diagnosis for the family members, however additionally provided insight into the phenotype­genotype association in OI.

The susceptibility of FSHB -211G > T and FSHR G-29A, 919A > G, 2039A > G polymorphisms to men infertility: an association study and meta-analysis.

BACKGROUND: Male infertility is a complex disorder caused by genetic, developmental, endocrine, or environmental factors as well as unknown etiology. Polymorphisms in the follicle stimulating hormone beta subunit (FSHB) (rs10835638, c.-211G > T) and follicle stimulating hormone receptor (FSHR) (rs1394205, c.-29G > A; rs6165, c.919A > G; rs6166, c.2039 A > G) genes might disturb normal spermatogenesis and affect male reproductive ability. METHODS: To further ascertain the aforementioned effects, we conducted a case-control study of 255 infertile men and 340 fertile controls from South China using the Mass ARRAY method, which was analyzed by the t-tests and logistic regression analysis using SPSS for Windows 14.0. In addition, a meta-analysis was performed by combining our results with previous reports using STATA 12.0. RESULTS: In the FSHB or FSHR gene single nucleotide polymorphism (SNP) evaluation, no statistically-significant difference was found in the frequency of allelic variants or in genotype distribution between cases and controls. However, a significant association for the comparison of GAA (P: 0.022, OR: 0.63, 95%CI: 0.43-0.94) was seen between the oligozoospermia and controls in haplotype analysis of rs1394205/rs6165/rs6166. In the meta-analysis, rs6165G allele and rs6166 GG genotype were associated with increased risk of the male infertility. CONCLUSIONS: This study suggested that FSHR GAA haplotype would exert protective effects against male sterility, which indicated that the combination of three SNP genotypes of FSHR was predicted to have a much stronger impact than either one alone. Then in the meta-analysis, a significant association was seen between FSHR rs6165, rs6166 polymorphisms and male infertility. In terms of male infertility with multifactorial etiology, further studies with larger sample sizes and different ethnic backgrounds or other risk factors are warranted to clarify the potential role of FSHB and FSHR polymorphisms in the pathogenesis of male infertility.

The P20R mutation of αB-crystallin diminishes its anti-apoptotic activity in human lens epithelial cells.

αB-crystallin acts as an anti-apoptosis protein in human lens epithelial (HLE) cells. We recently identified a missense mutation in αB-crystallin that changes proline 20 to an arginine (P20R) in a Chinese family with autosomal dominant congenital posterior polar cataract. The impact of the P20R mutation on the anti-apoptosis function remains unclear. To explore the anti-apoptotic activity of αB-crystallin wild type (αB-wt) and its P20R mutant under oxidative stress, HLE cells were transfected with αB-wt and αB-P20R constructs and expression was measured by western blotting. Flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL) staining were performed to investigate apoptosis. We found that αB-wt performed a dominant role in inhibiting stress-induced apoptosis, but this function was impeded in cells expressing αB-P20R. The P20R mutant of αB-crystallin exhibits diminished anti-apoptotic activity compared with the native protein.

[Single nucleotide polymorphism of the TP53 gene is not correlated with male infertility].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphism (SNP) rs1042522 of the tumor protein p53 (TP53) gene with the risk of male infertility. METHODS: This casecontrol study included 380 male patients with idiopathic infertility and 398 normal fertile men as controls from the Nanjing area. We genotyped the SNP rs1042522 of the TP53 gene by Sequence Mass Array and analyzed the correlation of the SNP with male infertility using the logistic regression model. RESULTS: Compared with the normal controls, the patients with idiopathic infertility showed significantly decreased sperm concentration (ï¼»77.34±49.24ï¼½ vs ï¼»13.13±24.96ï¼½ ×106/ml), percentage of progressively motile sperm (ï¼»42.55±9.57ï¼½ vs ï¼»10.38±5.57ï¼½%), serum testosterone level (ï¼»14.07±5.36ï¼½ vs ï¼»11.89±4.50ï¼½ nmol/L), and folliclestimulating hormone level (ï¼»16.80±18.20ï¼½ vs ï¼»4.55±7.17ï¼½ U/L) (P < 0.05) but no statistically significant differences in other parameters. No correlation was observed between the SNP frequencies and male infertility and similar results were found in the subgroups of the cases. CONCLUSIONS: SNP rs1042522 of the TP53 gene is not significantly correlated with the risk of male infertility.

[Nucleotide polymorphism rs4880 of the SOD2 gene and the risk of male infertility].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphism (SNP) rs4880 of the superoxide dismutase 2 (SOD2) gene with the risk of male infertility. METHODS: This casecontrol study included 519 male patients with idiopathic infertility (aged 19－40 ï¼»28.93±4.93ï¼½ years) in the case group and 338 fertile men (aged 19－40 ï¼»28.40±4.25ï¼½ years) in the control group. We collected the clinical data, genotyped the SNP rs4880 of the SOD2 gene by Sequenom Mass Array, and analyzed the association of different genotypes with male infertility using the logistic regression model. RESULTS: Statically significant differences were observed between the case and control groups in the level of folliclestimulating hormone (FSH) (ï¼»4.72±2.51ï¼½ vs ï¼»15.65±17.24ï¼½ U/L, P< 0.01), the percentage of progressively mobile sperm (ï¼»9.12±13.5ï¼½ vs ï¼»41.95±9.03ï¼½%, P< 0.01), and sperm concentration (ï¼»12.95±24.38ï¼½ vs ï¼»72.88±45.60ï¼½ ×106/ml, P< 0.01), but not in other parameters. No correlation was found between male infertility and the heterozygous genotype TC (OR = 0.90, 95% CI: 0.65－1.25, P = 0.516) or the homozygous genotype CC (OR=1.49, 95% CI: 0.38－5.81, P = 0.566) as compared with the wild genotype TT, and similar results were obtained in the analysis of the subgroups. CONCLUSIONS: The SNP rs4880 of the SOD2 gene was not correlated with male infertility, which, however, is to be supported by further studies with larger samples from more areas.

A novel missense mutation of ADAR1 gene in a Chinese family leading to dyschromatosis symmetrica hereditaria and literature review.

Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant pigmentary genodermatosis, which is characterized by a mixture of hyperpigmented and hypopigmented macules on the dorsal of the hands and feet, and on the face presented like freckle. Identification of RNA-specific adenosine deaminase 1 (ADAR1) gene results in DSH. This study was mainly to explore the pathogenic mutation of ADAR1 gene and provide genetics counselling and prenatal diagnostic testing for childbearing individuals.Mutational analysis of ADAR1 gene was performed by polymerase chain reaction (PCR) and electrophoretic separation of PCR products by 1.5% agarose gel electrophoresis. The coding exons and intron/exon flanking regions followed by bidirectional sequencing was performed on all participants. In this study, we found that a 28 year-old male patient harbouring a deleterious substitution of Leu1052Pro in the ADAR1 gene in a typical DSH family. His mother suffered from the DSH also owns the same mutation. This mutation, however, is not identified in the unaffected members in this family and those 200 normal controls. In summary, this new mutation Leu1052Pro reported here is pathogenic and detrimental for DSH. Our finding not only enriches mutation database and contributes to dissecting further the correlation between mutation position and phenotypical features of DSH, but also provides genetics counselling and prenatal diagnostic testing for childbearing couple.

[CYP1A1 rs4646422 gene polymorphisms not correlated with male infertility in Chinese Han population].

OBJECTIVE: To determine the correlation of the CYP1A1 (rs4646422) gene polymorphisms with male infertility in the Chinese Han population. METHODS: Using the Mass ARRAY iPLEX GOLD technique, we conducted a case-control study on theCYPlA1 (rs4646422) gene polymorphisms in 636 infertile males aged 21-49 years (case group) and 442 normal healthy men aged 23-47 years (control group) of the Chinese Han population. We analyzed the genotypes and allele frequencies in the two groups ofsubjects with the SPSS 20.0 software. RESULTS: Compared with the wild homozygous genotype GG, the heterozygous genotype AG (OR = 1.06, 95% CI 0.81-1.38) and homozygous genotype AA (OR = 1.11, 95% CI 0.56-2.21) showed no correlation with male infertility, nor did the mutant allele A (OR = 1.06, 95% CI 0.85-1.32) in comparison with the wild allele G. CONCLUSION: The CYP1A1 (rs4646422) gene polymorphisms might not be correlated with male infertility in the Chinese Han population.

Predictive value of GGN and CAG repeat polymorphisms of androgen receptors in testicular cancer: a meta-analysis.

The risk of testicular cancer (TC) is markedly increased in subjects with androgen insensitivity, and previous studies have proposed that GGN and CAG repeats in androgen receptors (AR) could be related to the risk of TC. To evaluate the association between the length of GGN and CAG repeats in AR and TC, a meta-analysis involving 3255 TC cases and 2804 controls was performed. The results suggested that long GGN repeats are associated with an increased risk of TC compared with those < 23 [odds ratio (OR) = 1.22, 95% confidence interval (CI) = 1.05-1.41]; similarly, a subgroup analysis revealed that this association occurred in studies with case sizes > 200, and in the mid-latitude, and seminoma subgroups. The subgroup analysis based on populations, high-latitude, and seminomas/non-seminomas suggested that AR CAG repeat polymorphisms with > 25 and < 21 + > 25 repeats might confer a protective effect to the patients with TC (in the high-latitude subgroup analysis, for > 25 vs. 21-25: OR = 0.54, 95% CI = 0.41-0.70). In contrast, an increased risk of TC was observed for AR CAG repeat polymorphisms with > 25 and < 21 + > 25 repeats in the mid-latitude subgroup (for > 25 vs. 21-25: OR = 1.65, 95% CI = 1.09-2.50). In addition, no associations between the remaining subgroups and male infertility were observed. In short, this meta-analysis suggested that AR GGN and CAG repeat polymorphisms may be involved in the etiology of TC.

[Single nucleotide polymorphisms of the N-acetyltransferase 2 gene not correlated with male infertility].

Objective: To investigate the correlation of the single nucleotide polymorphisms（SNPs） rs1799930 and rs1799931 of the N-acetyltransferase 2 gene（ NAT2） with the risk of male infertility in Nanjing area. Methods: We made a case-control study of 636 cases of male idiopathic infertility and 442 normal fertile men as controls. We genotyped the two SNPs by Sequenom Mass Array, analyzed the correlation of different genotypes with male infertility using the logistic regression model, and determined the association of the linkage effect of the two SNPs with male infertility by haplotype analysis. Results: Statistically significant differences were found between the case and control groups in sperm concentration（[32. 32 ± 45. 49] vs [72. 77 ± 45. 21] × 106/ ml, P < 0. 01）,the percentage of progressively motile sperm（[15. 29 ± 5. 06] vs [42. 02 ± 9. 04]%,P < 0. 01）,and the level of follicle-stimulating hormone（[14. 69 ± 12. 37] vs [4. 72 ± 2. 51] U / L,P < 0. 01), but not in other parameters. No correlation was observed between the frequencies of the two SNPs or alleles in different models and male infertility. Haplotype analysis suggested a linkage effect within rs1799930 and rs1799931（D' = 0. 998,r2= 0. 05） but no evident correlation between male infertility and genotype combination. Conclusion: The SNPs rs1799930 and rs1799931 of the NAT2 gene were not found to be correlated with the risk of idiopathic infertility in men.