Factors influencing mosaicism: a retrospective analysis.

RESEARCH QUESTION: What factors affect the incidence of mosaic embryos resulting from assisted reproductive technology? DESIGN: A retrospective analysis of data from preimplantation genetic testing for aneuploidies in 544 couples was conducted using data from an electronic medical record database. RESULTS: Of 1910 embryos studied, 127 (6.6%) were mosaic. In multivariable logistic regression analysis, mosaicism incidence increased in embryos from IVF versus intracytoplasmic sperm injection (ICSI) (odds ratio [OR] 4.560, 95% confidence interval [CI] 2.800-7.424, P < 0.001), and in embryos from abnormal versus normal semen (OR 3.496, 95% CI 2.455-4.979, P < 0.001). Embryos tested using SurePlex 24Sure had lower mosaicism percentages than those tested using MALBAC-NGS and PicoPLEX GenetiSure (OR 2.726, 95% CI 1.532-4.852, P = 0.001; OR 2.389, 95% CI 1.537-3.711, P < 0.001, respectively). CONCLUSIONS: Semen quality, fertilization method and detection system are independent factors associated with embryonic mosaicism.

Genetic mutation analysis of 22 patients with congenital absence of vas deferens: a single-center study†.

Congenital absence of the vas deferens (CAVD), a congenital malformation of the male reproductive system, causes obstructive azoospermia and male infertility. Currently, the cystic fibrosis transmembrane conductance regulator (CFTR) has been recognized as the main pathogenic gene in CAVD, with some other genes, such as adhesion G-protein-coupled receptor G2 (ADGRG2), solute carrier family 9 isoform 3 (SLC9A3), sodium channel epithelial 1 subunit beta (SCNN1B), and carbonic anhydrase 12 (CA12), being candidate genes in the pathogenesis of CAVD. However, the frequency and spectrum of these mutations, as well as the pathogenic mechanisms of CAVD, have not been fully investigated. Here, we sequenced all genes with potentially pathogenic mutations using next-generation sequencing and verified all identified variants by Sanger sequencing. Further bioinformatic analysis was performed to predict the pathogenicity of mutations. We described the distribution of the p.V470M, poly-T, and TG-repeat CFTR polymorphisms and identified novel missense mutations in the CFTR and SLC9A3 genes, respectively. Taken together, we identified mutations in the CFTR, ADGRG2, SLC9A3, SCNN1B, and CA12 genes in 22 patients with CAVD, thus broadening the genetic spectrum of Chinese patients with CAVD.

Pathogenesis of acephalic spermatozoa syndrome caused by SUN5 variant.

Acephalic spermatozoa syndrome (ASS) is a rare teratozoospermia that leads to male infertility. Previous work suggested a genetic origin. Variants of Sad1 and UNC84 domain containing 5 (SUN5) are the main genetic cause of ASS; however, its pathogenesis remains unclear. Here, we performed whole-exome sequencing in 10 unrelated ASS and identified 2 homozygous variants, c.381delA[p.V128Sfs7*] and c.675C>A[p.Y225X], and 1 compound variant, c.88 C > T[p.R30X] and c.381 delA [p.V128Sfs7*], in SUN5 in 4 patients. The c.381delA variant had been identified as pathogenic in previous reports, while c.675C>A and c.88 C > T were two novel variants which could lead to a premature termination codon (PTC) and resulted in loss of SUN5, and may also be pathogenic. SUN5 mRNA and protein were present at very low levels in ASS patients with SUN5 nonsense mutation. Furthermore, the distribution of outer dense fiber protein 1 (ODF1) and Nesprin3 was altered in sperm of ASS patients with SUN5 variants. The co-immunoprecipitation analysis indicated that SUN5 and ODF1, SUN5 and Nesprin3, and ODF1 and Nesprin3 interacted with each other in transfected HEK293T cells. Thus, we propose that SUN5, Nesprin3, and ODF1 may form a 'triplet' structure through interactions at neck of sperm. When gene variants resulted in a loss of SUN5, the 'triplet' structure disappears and then the head-tail junction becomes fragile, leading to the occurrence of ASS.

Sequential interval micro-droplet loading in closed hemi-straw carrier system: A convenient and efficient method for ultra-rapid cryopreservation in extreme oligozoospermia.

Cryopreservation of human spermatozoa with low concentration while maintaining adequate post-thawing motility remains a major challenge for male fertility preservation. A convenient and efficient ultra-rapid freezing method for small amounts of human spermatozoa in a closed Hemi-Straw carrier system (CHS) was developed. Spermatozoa from 60 healthy men were involved in a parameter refining test and another 15 extreme oligozoospermic specimens were assigned to a verification test. A commercialized sperm freezing medium, Quinn's Advantage® Sperm Freeze medium (glycerol and sucrose as the cryoprotective agent) was used in the study. The results showed that the highest recovery rates would be obtained via the method of 2 µl single droplet sequential interval loading, by placing the straw at 1 cm above the liquid nitrogen (LN2) surface for 60 s during freezing and 2 cm above the LN2 for 2 min during thawing. This method was applied in cryopreservation for the normozoospermic specimens and compared with a conventional slow freezing method. The results were better than those in the control group in the total motility recovery rate (77.8 ± 11.2% vs 56.6 ± 11.9%, P < 0.01), progressive motility recovery rate (77.6 ± 13.2% vs 47.7 ± 14.6%, P < 0.01), 24 h survival index (60.9 ± 13.4% vs 42.1 ± 14.1%, P < 0.01) and the sperm DNA fragment index (4.2 ± 3.7% vs 5.8 ± 3.7%, P = 0.126). This method was applied to the oligozoospermic specimens. Motile spermatozoa could be found in 12 of 15 cases in the ultra-rapid freezing group, while only in 7 cases in control group. The results indicated that this freezing method was simple, convenient and bio-safe for cryopreservation of severe oligozoospermic specimens.

[Delineation of three structural Y chromosome aberrations combined molecular techniques].

OBJECTIVE: To delineate the structure of Y chromosome aberrations and recombinant mechanisms for three patients. METHODS: Karyotype analysis, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), Y chromosome sequence tagged sites (STS) analysis, human whole genome-wide SNP array were used. RESULTS: The karyotypes of the three patients were 46, X, +mar. As suggested by MLPA analysis, case 1 has increased copy numbers of SRY, ZFY and UTY genes, case 2 had increased copies of SRY and ZFY genes, and deletion of UTY gene, and case 3 had decreased copies for subtelomeric regions of X/Yp and X/Yq. By STSs analysis, case 1 has retained SRY, sY84 and sY86 in the AZFa region, sY1227 in the AZFb region, whilst lost sY1228 in the AZFb region and other STSs in the AZFc region. Its breakpoint was thereby mapped between sY1227 and sY1228. Case 2 has retained SRY and sY1200 in the centromeric region, whilst has deletion of other STSs. Case 3 has retained SRY and STSs in the AZF regions. By SNP array, case 1 had duplicated Yp11.31-p11.2 and deletion of Yq11.22-q11.23 (approximately 5.18 Mb). Case 2 had duplicated Yp11.31-p11.2 and deletion of Yq11.21-q11.23 (approximately 14.644 Mb). Case 3 had single copy number deletion of p22.33 and q28 in the subtelomeric region of X/Yp and X/Yq. By FISH, cases 1 and 2 showed two signals for SRY and DYZ3 but no signal for DYZ1 on their marker chromosomes. Combining above results, the karyotypes of cases 1, 2 and 3 were determined as 46, X, idic(Y) (q11.23), 46, X, idic(Y) (q10) and 46, X, r(Y) (p11q12), respectively. CONCLUSION: Y chromosome aberrations are variable. Combined use of MLPA, STSs, FISH and SNP array is effective for revealing the breakpoints and recombinant mechanisms.

Uncoupling protein 2 expression affects androgen synthesis in polycystic ovary syndrome.

The roles of uncoupling protein-2 (UCP2) on the androgen synthesis of granulosa cells derived from patients with polycystic ovary syndrome (PCOS) and normal subjects were explored. Primary human granulosa cells from 18 patients who received in vitro fertilization (IVF) were examined; nine patients had PCOS with hyperandrogenism. Primary cultures were treated with genipin, a proton leak inhibitor, guanosine diphosphate (GDP), an UCP inhibitor, and triiodothyronine (T3), an inducer of UCP gene expression. Mitochondrial membrane potential was determined using the JC-1 assay. T3 induced P450scc and UCP2 expressions and testosterone synthesis in both normal and PCOS granulosa cells. Their expressions in response to T3 treatments were correlated in the PCOS group. Differences in testosterone synthesis were observed between normal and PCOS cells in response to genipin. Increased mitochondrial membrane potential was observed in response to genipin and GDP; while T3 decreased it. Increased ovarian UCP2 expression in response to T3 treatment in PCOS may alter pregnenolone synthesis by influencing P450scc expression, thus altering testosterone production. Further in vivo studies are necessary to fully elucidate the role of UCP2 in the hyperandrogenism commonly observed in PCOS.

Abnormal expression of uncoupling protein-2 correlates with CYP11A1 expression in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) may result from hypersensitivity to insulin, which is negatively regulated by uncoupling protein (UCP)-2. Because cholesterol side-chain cleavage enzyme (CYP11A1) is closely linked to PCOS, the expression of UCP-2 and CYP11A1 in ovarian tissues from PCOS patients was examined in the present study. Twelve PCOS patients with hyperandrogenaemia who underwent laparoscopic ovarian wedge resection and 12 age-matched control patients who underwent contralateral ovarian biopsy were enrolled in the study. UCP-2 expression in early stage (primordial, primary and secondary) and late stage (sinus and mature) follicles was examined using immunohistochemistry, whereas UCP-2 and CYP11A1 mRNA and protein levels in ovarian tissue were determined using quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. UCP-2 expression increased significantly with follicular development in both control and PCOS tissue, with expression in early stage follicles from PCOS patients significantly greater than that in controls. In addition, both UCP-2 and CYP11A1mRNA and protein levels, mean fasting blood glucose concentrations and fasting serum insulin levels were significantly higher in PCOS patients compared with the control group. Finally, a significant correlation between UCP-2 and CYP11A1 expression was found in PCOS but not control patients. In conclusion, in PCOS patients, there was a correlation between UCP-2 and CYP11A1 expression, which was significantly higher than in the control group. These changes in UCP-2 and CYP11A1 expression may mediate follicle development in PCOS.

[Generation of transgenic mice by intratesticular injection and electroporation in vivo].

OBJECTIVE: To evaluate the feasibility of establishing transgenic mice by means of seminiferous tubule microinjection and electroporation (EP) in vivo. METHODS: Specific pathogen-free (SPF) male Kunming mice divided into 4 groups were subjected to microinjection of two different transfection solutions labeled with enhance green fluorescent protein (EGFP) into the seminiferous tubule of the testis, and in one of the two groups receiving the identical transfection solutions, EP in vivo was performed. After two weeks, the male mice of each group were mated with SPF female Kunming mice with superovulation treatment, and PCR coupled with Southern blotting was performed for the offspring mice. RESULTS: The results of PCR suggested significant difference in the efficiency of exogenous gene integration between the 4 groups (P<0.01), among which group A achieved the greatest efficiency (45%). Southern blotting did not identify significant difference between the 4 groups (P>0.05), but still suggested the highest efficiency in group A (25%). CONCLUSION: Seminiferous tubule microinjection in conjunction with subsequent EP in vivo can remarkably enhance the integration efficiency of exogenous genes into the host genome, but this new method needs to be further tested for its potential utility in transgenic animal generation.

[Experimental study of in situ electroporation-induced testis injury in specific pathogen-free male Kunming mice].

OBJECTIVE: To observe testis injuries induced by in situ electroporation in specific pathogen-free (SPF) adult male Kunming mice. METHODS: With two sets of parameters of high and low voltages, respectively, in situ electroporation of the testis was performed in vivo by fixing the testis and epididymis of the mice between a pair of rectangular tweezer-type electrodes. Two weeks after electroporation, the mice were killed and the testis and epididymis separated and fixed with 4% paraformaldehyde after recording the weight of the testis. Routine histological sections were prepared and observed under optical microscope after HE staining. The epididymis was transferred into M16 medium for spermatozoa separation, and after dilution of the spermatozoa suspension, the spermatozoa viability was observed under optical microscope. RESULTS: Two weeks after high-voltage electroporation, examination of spermatozoa viability and microscopy of the testis sections revealed irreversible testis injury, and the testis weight was significantly reduced in comparison with that of control mice (P<0.01). Low-voltage electroporation, in contrast, only caused reversible injuries of the testis, and the male mice retained their reproductive capacity after a certain length of recovery period. The testis weight after low-voltage electroporation showed no significant difference from that of the control mice (P>0.05). CONCLUSIONS: Appropriate setting of the parameters for in vivo electroporation may avoid severe impact on the reproductive capacity of the testis in SPF male Kunming mice. This technique also provides a possibility for exogenous gene transfer into the reproductive cells.

Effects of different concentrations of amino acids in the culture medium on preimplantation mouse embryo development in vitro.

OBJECTIVE: To evaluate the effects of amino acids (AA) on the development of in vitro cultured preimplantation embryos of Kunming mice, and define the optimal AA concentration for embryo culture. METHODS: Totally 630 zygotes were collected from the oviducts of superovulated female Kunming mice, which were cultured in protein-free potassium simplex optimized medium (mKSOM) supplemented with Eagle's essential amino acids and Eagle's non-essential amino acids of different concentrations (mKSOM, mKSOM+1/16AA, mKSOM+1/8AA, mKSOM+1/4AA, mKSOM+1/2AA, mKSOM+AA, and mKSOM+2AA). RESULTS: The embryos cultured with the amino acids showed higher development rate to both 8-cell embryo stage and blastocyst stage than those cultured without amino acids. The correlation of amino acid concentration with 8-cell and blastocyst development rates conformed to the cubic model, with the highest development rate to both of the two stages observed at half of the amino acid concentration. CONCLUSION: Amino acids can promote the development of preimplantation Kunming mouse embryos, but excessively high concentration of amino acids impair embryo development possibly because of metabolic and osmotic pressure changes of the embryos as well as toxicity of ammonium resulting from the metabolism of amino acids.

[Effects of modification of the nutritional components in potassium simplex optimized medium on preimplantation development and cleavage of mouse embryos in vitro].

OBJECTIVE: To solve the problem known as 2-cell block in in vitro culture of preimplantation embryos, modification of the nutritional components in potassium simplex optimized medium (KSOM) was attempted and the preimplantation development and cleavage of mouse embryos in such medium observed. METHOD: One-cell mouse embryos collected from the oviduct of the superovulated mice were cultured in microdrops of different culture media for 5 days [144 h after human chorionic gonadotrophin (hCG) injection], and the percentages of embryos developed to pre-blastocyst, unhatched blastocyst, partially hatched blastocyst and completely hatched blastocyst were recorded at deferent time points (96, 120 and 144 h post-hCG) and total cell number of blastocysts at 120 h post-hCG counted. With these indices as the evaluation criteria, we evaluated the modifications of the concentrations of glucose (0.2, 5.56 mmol/l), bovine serum albumin (BSA, 1 and 4 mg/ml), and amino acids (1/2X NEAA and EAA, or not) in KSOM for their effect on the development of Kunming mouse zygotes in vitro. RESULTS: Increasing the concentration of glucose and BSA was not shown to have significant effects on the rate of blastocyst formation at 120 h post-hCG or the total cell counts in KSOM without amino acids. After supplementation of the medium with amino acids, the rate of partially and completely hatched blastocyts was significantly increased in KSOM or KSOM supplemented with high concentrations of glucose and BSA (KSOMGB). Glucose at the high concentration of 5.56 mmol/L did not inhibit the development of mouse zygotes to hatched blastocysts cultured in KSOM supplemented with amino acids. CONCLUSION: Increasing the concentration of glucose and BSA and amino acids in KSOM can promote the preimplantation development of mouse zygotes in in vitro culture.

[Expression of human tissue-type plasminogen activator in cow mammary gland].

OBJECTIVE: To construct an expression vector for highly efficient expression of tissue-type plasminogen activator (t-PA) confined in the mammary gland of cow to develop a cow mammary gland bioreactor. METHODS: RT-Touch down-PCR was employed to amplify human tissue-type plasminogen activator (t-PA) cDNA, which was digested with the restriction enzymes and subsequently cloned into the vector pSP72 for constructing specific fusion gene only expressed in the mammary gland. The fusion gene was then transferred into the mouse zygote and the mammary gland tissue of mice and cows. RESULTS: t-PA was detected in the milks of mice and cows after the transgenic manipulation with microinjection and mammary gland injection of the fusion gene. CONCLUSIONS: The vector we constructed can effectively induce t-PA expression in the mammary gland, which is not influenced by different transgenic methods. The expression level of t-PA, however, is significantly higher in the milk of cows than in the milk of mice, suggesting the species-specific difference in milk protein regulating system possibly is due to different factors and regulatory systems.