Genomic aspects in reproductive medicine.

Infertility is a complex disease characterized by extreme genetic heterogeneity, compounded by various environmental factors. While there are exceptions, individual genetic and genomic variations related to infertility are typically rare, often family-specific, and may serve as susceptibility factors rather than direct causes of the disease. Consequently, identifying the cause of infertility and developing prevention and treatment strategies based on these factors remain challenging tasks, even in the modern genomic era. In this review, we first examine the genetic and genomic variations associated with infertility, and subsequently summarize the concepts and methods of preimplantation genetic testing in light of advances in genome analysis technology.

Clinical evaluation of noninvasive prenatal testing for sex chromosome aneuploidies in 9,176 Korean pregnant women: a single-center retrospective study.

BACKGROUND: To evaluate the clinical significance of noninvasive prenatal testing (NIPT) for detecting fetal sex chromosome aneuploidies (SCAs) in Korean pregnant women. METHODS: We retrospectively analyzed NIPT data from 9,176 women with singleton pregnancies referred to the CHA Biotech genome diagnostics center. Cell-free fetal DNA (cffDNA) was extracted from maternal peripheral blood, and high-throughput massively parallel sequencing was conducted. Subsequently, the positive NIPT results for SCA were validated via karyotype and chromosomal microarray analyses. RESULTS: Overall, 46 cases were SCA positive after NIPT, including 20, 12, 8, and 6 for Turner, triple X, Klinefelter, and Jacob syndromes, respectively. Among 37 women with invasive prenatal diagnosis, 19 had true positive NIPT results. The overall positive predictive value (PPV) of NIPT for detecting SCAs was 51.35%. The PPV was 18.75% for Turner, 88.89% for triple X, 71.43% for Klinefelter, and 60.00% for Jacob's syndromes. NIPT accuracy for detecting sex chromosome trisomies was higher than that for sex chromosome monosomy (P = 0.002). No significant correlation was observed between fetal SCA incidence and maternal age (P = 0.914), except for the borderline significance of Jacob's syndrome (P = 0.048). No significant differences were observed when comparing NIPT and karyotyping validation for fetal SCA according to pregnancy characteristics. CONCLUSION: Our data suggest that NIPT can reliably screen for SCAs, and it performed better in predicting sex chromosome trisomies compared with monosomy X. No correlation was observed between maternal age and fetal SCA incidence, and no association was observed between different pregnancy characteristics. The accuracy of these findings requires improvements; however, our study provides an important reference for clinical genetic counseling and further management. Larger scale studies, considering confounding factors, are required for accurate evaluation.

Cell-Free Fetal DNA Screening Analysis in Korean Pregnant Women: Six Years of Experience and a Retrospective Study of 9327 Patients Analyzed from 2017 to 2022.

Cell-free DNA (cfDNA) screening for normal fetal aneuploidy has been widely adopted worldwide due to its convenience, non-invasiveness, and high positive predictive rate. We retrospectively evaluated 9327 Korean women with single pregnancies who underwent a non-invasive prenatal test (NIPT) to investigate how various factors such as maternal weight, age, and the method of conception affect the fetal fraction (FF). The average FF was 9.15 ± 3.31%, which decreased significantly as the maternal body mass index (BMI) increased (p < 0.001). The highly obese group showed a 'no-call' rate of 8.01%, which is higher than that of the normal weight group (0.33%). The FF was 8.74 ± 3.20% when mothers were in their 40s, and lower than that when in their 30s (9.23 ± 3.34, p < 0.001) and in the natural pregnancy group (9.31% ± 3.33). The FF of male fetuses was observed to be approximately 2.76% higher on average than that of female fetuses. As the gestational age increased, there was no significant increase in the fraction of fetuses up to 21 weeks compared to that at 10-12 weeks, and a significant increase was observed in the case of 21 weeks or more. The FFs in the NIPT high-risk result group compared to that in the low-risk group were not significantly different (p = 0.62). In conclusion, BMI was the factor most associated with the fetal fraction. Although the NIPT is a highly prevalent method in prenatal analysis, factors affecting the fetal fraction should be thoroughly analyzed to obtain more accurate results.

A novel variant of NPPC causes abnormal post-translational cleavage: A candidate gene for premature ovarian insufficiency.

OBJECTIVE: Premature ovarian insufficiency (POI) is a clinical disease that is diagnosed by the loss of ovarian function before the age of 40. Despite recent progress in molecular diagnosis, the genetic etiology of POI is not well established. The aim of this study is to reveal pathogenic genetic variants involved in POI. STUDY DESIGN AND MAIN OUTCOME MEASURES: To reveal pathogenic genetic variants involved in POI, whole exome sequencing was performed in nonconsanguineous family members with POI. Constitutional variants were filtered against population databases and a missense mutation of natriuretic peptide C (NPPC) (c.131A>G, p.Q44R) was selected as a convincing candidate mutation among 14 heterozygous mutant alleles in 13 genes. RESULTS: The wild-type NPPC and mutant NPPC (NPPC131A>G) were expressed in HeLa cells, and cells expressing NPPC131A>G secreted unique peptides. The ProP 1.0 Server, a neural network prediction tool, predicted the presence of a cleavage site at the substituted arginine residue (p.Q44R) of NPPC. The molecular weight of predicted cleaved peptides processed from mutant NPPC precursor corresponded to that of the actual mutant peptide. The cGMP synthetic activity of NPR2-expressing cells was significantly decreased by interaction with the mutant NPPC peptide compared with wild-type NPPC. CONCLUSIONS: The peptide generated by a rare mutation of NPPC might influence paracrine C-type natriuretic peptide (CNP)-mediated preantral follicle development and/or sustain meiotic arrest in oocytes. We therefore suggest that a mutation of the NPPC gene is involved in the pathogenesis of POI.

Inconsistency between non-invasive prenatal testing (NIPT) and conventional prenatal diagnosis due to confined placental and fetal mosaicism: Two case reports.

We aimed to identify the causes of inconsistent results between non-invasive prenatal testing (NIPT) and invasive testing methods for trisomy 21. In the first case, NIPT was performed at 11 weeks of pregnancy, and the result showed a high risk of trisomy 21 [fetal fraction (FF) = 6.98%, 21 chromosome Z-score = 3.6]. The patient underwent quantitative fluorescent (QF)-PCR and karyotyping at 14 + 0 weeks of pregnancy through CVS showing mosaicism of 47, XX, + 21[11] and 46, XX [39] in karyotyping. The patient underwent amniocentesis at 15 + 6 weeks, showing a normal pattern in QF-PCR and 46, XX karyotyping in long term culture. The second case underwent NIPT at 16 + 5 weeks of pregnancy (FF = 7.52%, 21 chromosome Z-score = 2.503). She underwent an invasive test at 19 weeks through amniotic fluid sampling. As a result, trisomy 21 was detected by QF-PCR, and mosaicism of XX, +21[22]/46, XX [4] was identified by karyotyping. Despite significant advances in fetal chromosome analysis using NIPT, invasive testing is still needed as placenta-derived DNA does not reflect 100% fetal genetic information. Placental mosaicism can be detected by NIPT, but more research is needed to increase its sensitivity. Therefore, if the NIPT result is positive, an invasive test can confirm the result, and continuous monitoring is required even if the NIPT result is negative.

Changes in Methylation Patterns of Tumor Suppressor Genes during Extended Human Embryonic Stem Cell Cultures.

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

A transport system based on a quantum dot-modified nanotracer is genetically and developmentally stable in pregnant mice.

The use of nanoscale materials (NMs) could cause problems such as cytotoxicity, genomic aberration, and effects on human health, but the impacts of NM exposure during pregnancy remain uncharacterized in the context of clinical applications. It was sought to determine whether nanomaterials pass through the maternal-fetal junction at any stage of pregnancy. Quantum dots (QDs) coated with heparinized Pluronic 127 nanogels and polyethyleneimine (PEI) were administered to pregnant mice. The biodistribution of QDs, as well as their biological impacts on maternal and fetal health, was evaluated. Encapsulation of QDs with a nanogel coating produces a petal-like nanotracer (PNt), which could serve as a nano-carrier of genes or drugs. PNts were injected through the tail vein and accumulated in the liver, kidneys, and lungs. QD accumulation in reproductive organs (uterus, placenta, and fetus) differed among phases of pregnancy. In phase I (7 days of pregnancy), the QDs did not accumulate in the placenta or fetus, but by phase III (19 days) they had accumulated at high levels in both tissues. Karyotype analysis revealed that the PNt-treated pups did not have genetic abnormalities when dams were treated at any phase of pregnancy. PNts have the potential to serve as carriers of therapeutic agents for the treatment of the mother or fetus and these results have a significant impact on the development and application of QD-based NPs in pregnancy.

Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs).

Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become increasingly important in the context of stem cell therapy. Methods: Functional nanoparticles (Sunflower typed nanoparticles; SF-NPs) were fabricated by coating heparin pluronic F127 gels with quantum dot nanoparticles (QDs), and then bound the SOX9 gene to the QD nanogels. The resultant nanoparticles were transferred into stem cells, and the effect on genetic stability was monitored. To determinate gene delivery efficacy and long-term genomic stability of cells transfected with QD nanogels, hMSCs were transfected with nanogels at passage 4 (T1; Transfected cells 1) and then sub-cultured to passage of (T4). Following transplantation of transfected T1-T4 cells, the cells were monitored by in vivo imaging. The genetic stability of cells treated with nanoparticles was confirmed by chromosomal analysis, copy number variation (CNV) analysis, and mRNA profiling. Results: After 21 days of pellet culture after sub-culture from T1 to T4, hMSCs treated with QD nanogels complexed with SOX9 plasmid DNA (pDNA) significantly increased expression of specific extracellular matrix (ECM) polysaccharides and glycoproteins, as determined by Safranin O and Alcian blue staining. Moreover, the T4 hMSCs expressed higher levels of specific proteins, including collagen type II (COLII) and SOX9, than P4 hMSCs, with no evidence of DNA damage or genomic malfunction. Microarray analysis confirmed expression of genes specific to matured chondrocytes. Stem cells that internalized nanoparticles at the early stage retained genetic stability, even after passage. In in vivo studies in rats, neuronal cartilage formation was observed in damaged lesions 6 weeks after transplantation of T1 and T4 cells. The degree of differentiation into chondrocytes in the cartilage defect area, as determined by mRNA and protein expression of COLII and SOX9, was higher in rats treated with SF-NPs. Conclusion: The QD nanogels used in this study, did not affect genome integrity during long-term subculture, and are thus suitable for multiple theranostic applications.