A case report of a normal fertile woman with 46,XX/46,XY somatic chimerism reveals a critical role for germ cells in sex determination.

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction.

Next-generation sequence-based preimplantation genetic testing for monogenic disease resulting from maternal mosaicism.

BACKGROUND: Mosaicism poses challenges for genetic counseling and preimplantation genetic testing for monogenic disorders (PGT-M). NGS-based PGT-M has been extensively used to prevent the transmission of monogenic defects, but it has not been evaluated in the application of PGT-M resulting from mosaicism. METHODS: Four women suspected of mosaicism were confirmed by ultra-deep sequencing. Blastocyst trophectoderm cells and polar bodies were collected for whole genome amplification, followed by pathogenic variants detection and haplotype analysis based on NGS. The embryos free of the monogenic disorders were transplantable. RESULTS: Ultra-deep sequencing confirmed that the four women harbored somatic mosaic variants, with the proportion of variant cells at 1.12%, 9.0%, 27.60%, and 91.03%, respectively. A total of 25 blastocysts were biopsied and detected during four PGT cycles and 5 polar bodies were involved in one cycle additionally. For each couple, a wild-type embryo was successfully transplanted and confirmed by prenatal diagnosis, resulting in the birth of four healthy infants. CONCLUSIONS: Mosaic variants could be effectively evaluated via ultra-deep sequencing, and could be prevented the transmission by PGT. Our work suggested that an NGS-based PGT approach, involving pathogenic variants detection combined with haplotype analysis, is crucial for accurate PGT-M with mosaicism.

Neonatal outcomes of live births after blastocyst biopsy in preimplantation genetic testing cycles: a follow-up of 1,721 children.

OBJECTIVE: To investigate whether blastocyst biopsy in preimplantation genetic testing (PGT) increases the risk of adverse neonatal outcomes. DESIGN: Retrospective cohort study. SETTING: University-affiliated center. PATIENTS: Live births after blastocyst biopsy combined with frozen ET (PGT group) and frozen blastocyst transfer after in vitro fertilization or intracytoplasmic sperm injection (control group). INTERVENTION(S): Blastocyst biopsy. MAIN OUTCOME MEASURE(S): Gestational age (GA), birth weight (BW), and rates of preterm birth (PB), very preterm birth (VPB), extreme preterm birth (EPB), low birth weight (LBW), very low birth weight (VLBW), and macrosomia. RESULT(S): No significant differences were observed in the sex ratio, GA, PB, VPB, EPB, BW, or rates of LBW, VLBW, and macrosomia between the PGT and control groups for either singletons or twins. However, the cesarean section rate of the PGT group was significantly higher than that of the control group for twins (adjusted odds ratio, 2.383 [1.079, 5.259]). Regarding fluorescence in situ hybridization-PGT neonates, neonatal outcomes, including GA, BW, and rates of PB, VPB, LBW, and VLBW, did not differ between the different groups of biopsied cells (≥10 group and <10 group) for either the grade B or grade C trophectoderm score subgroups; however, in the grade B trophectoderm score subgroup, the rate of boy babies in the ≥10 group was significantly higher than that in the <10 group (83.3% vs. 40.9%). The association between the number of biopsied cells and GA/BW was not statistically significant. CONCLUSION(S): Blastocyst biopsy may not add additional risk to neonatal outcomes when compared with a control group.

Expanded carrier screening and preimplantation genetic diagnosis in a couple who delivered a baby affected with congenital factor VII deficiency.

BACKGROUND: Preimplantation genetic diagnosis (PGD) is a powerful tool for preventing the transmission of Mendelian disorders from generation to generation. However, PGD only can identify monogenically inherited diseases, but not other potential monogenic pathologies. We aimed to use PGD to deliver a healthy baby without congenital FVII deficiency or other common Mendelian diseases in a couple in which both individuals carried a deleterious mutation in the F7 gene. METHODS: After both members of the couple were confirmed to be carriers of the F7 gene mutation by Sanger sequencing, expanded carrier screening (ECS) for 623 recessive inheritance diseases was performed to detect pathological mutations in other genes. PGD and preimplantational genetic screening (PGS) were employed to exclude monogenic disorders and aneuploidy for their embryos. RESULTS: ECS using targeted capture sequencing technology revealed that the couple carried the heterozygous disease-causative mutations c.3659C > T (p.Thr1220Ile) and c.3209G > A (p.Arg1070Gln) in the CFTR gene. After PGD and PGS, one of their embryos that was free of congenital FVII deficiency, cystic fibrosis (CF) and aneuploidy was transferred, resulting in the birth of a healthy 3200 g male infant. CONCLUSION: We successfully implemented PGD for congenital FVII deficiency and PGD after ECS to exclude CF for the first time to the best of our knowledge. Our work significantly improved the reproductive outcome for the couple and provides a clear example of the use of ECS combined with PGD to avoid the delivery of offspring affected not only by identified monogenically inherited diseases but also by other potential monogenic pathologies and aneuploidy.

[Proteomic identification of necrozoospermia-related proteins].

OBJECTIVE: To compare the proteomic differences between normal and necrozoospermic human spermatozoa. METHODS: We isolated proteins from 30 ejaculates of 10 healthy donors and 3 specimens of a necrozoospermia patient by two-dimensional gel electrophoresis (2-DE), analyzed their 2-DE maps and identified some differentially expressed proteins by mass spectrometry. RESULTS: A total of (905 +/- 57) and (792 +/- 28) proteins were isolated from the spermatozoal maps of the normal and necrozoospermic men, respectively, and 178 found to be differentially expressed in that of the necrozoospermia patient. The 6 missing protein spots in the necrozoospermic map were identified as sperm protein (human, accession No. 060904), zinc finger protein 174 (AW-1), F-actin capping protein alpha-3 subunit, testis-specific inhibitor of apoptosis, death domain receptor 3 soluble form (fragment) and peptide similar to the activator of CREM in the testis. CONCLUSION: Six missing protein spots were identified in the necrozoospermic spermatozoal map which may be associated with the development of necrozoospermia.

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors.

AIM: To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis (2-DE) reference map of human spermatozoal proteins in a pH range of 3.5-9.0. METHODS: In order to reveal more protein spots, immobilized pH gradient strips (24 cm) of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient (IPG) strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. RESULTS: The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip (1306 spots). CONCLUSION: The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.

Newly expressed proteins of mouse embryonic fibroblasts irradiated to be inactive.

It has been found that post-radiation mouse embryonic fibroblasts can well maintain the pluripotency in human embryonic stem cells. However, the molecular mechanism remains unclear. In the present study, the new protein expression profile of post-radiation mouse embryonic fibroblasts was analyzed by immobilized pH gradient 2-dimensional polyacrylamide gel electrophoresis. Image analysis following silver staining revealed (969+/-57) vs. (1085+/-107) spots from post-radiation mouse embryonic fibroblasts and pre-radiation ones, respectively. Some newly expressed proteins, which were only abundantly present after irradiation, were subjected to peptide mass fingerprint analysis and identified using MALDI-TOF-MS, SWISS-PROT database, and RT-PCR. Several of those proteins were preliminarily identified to participate in cytokine secretion, cell signal transduction, transcriptional regulation, and apoptosis, etc., which suggested that inactive post-radiation mouse embryonic fibroblasts expressed some new proteins that may underlie the molecular mechanisms to maintain the pluripotency in human embryonic stem cells.

[Diagnosing achondroplasia by single cell nested-PCR].

OBJECTIVE: To research on the reliability of diagnosing achondroplasia (ACH) on single cell level and to provide a basis for preimplantation genetic diagnosis(PGD). METHODS: The high-frequency mutation region G380R of fibroblast growth factor receptor 3(FGFR3) gene was amplified by nested-PCR with single lymphocyte and single blastomere. The products of PCR were digested by restriction enzyme Bfm I, then the digested products were detected by 10% polyacrylamida gel electrophoresis(PAGE). RESULTS: The amplification success rate, allele dropout rate and correct diagnosis rate of single lymphocyte's PCR were 90.4%, 8.2% and 91.8%,respectively. The amplification success rate of single blastomere was 75.4%. CONCLUSION: The diagnosis of ACH by single cell nested-PCR is comparatively stable and reliable.