Clinical application of chromosomal microarray for pathogenic genomic imbalance in fetuses with increased nuchal translucency but normal karyotype

Purpose@#To evaluate the additive value of prenatal chromosomal microarray analysis (CMA) in assessing increased nuchal translucency (NT) (≥3.5 mm) with normal karyotype and the possibility of detecting clinically significant genomic imbalance, based on specific indications. @*Materials and Methods@#Invasive samples from 494 pregnancies with NT ≥3.5 mm, obtained from the Research Center of Fertility & Genetics of Hamchoon Women’s Clinic between January 2019 and February 2020, were included in this study and CMA was performed in addition to a standard karyotype. @*Results@#In total, 494 cases were subjected to both karyotype and CMA analyses. Among these, 199 cases of aneuploidy were excluded. CMA was performed on the remaining 295 cases (59.7%), which showed normal (231/295, 78.3%) or non-significant copy number variation (CNV), such as benign CNV or variants of uncertain clinical significance likely benign (53/295, 18.0%). Clinically significant CNVs were detected in 11 cases (11/295, 3.7%). @*Conclusion@#Prenatal CMA resulted in a 3% to 4% higher CNV diagnosis rate in fetuses exhibiting increased NT (≥3.5 mm) without other ultrasound detected anomalies and normal karyotype. Therefore, we suggest using high resolution, non- targeting CMA to provide valuable additional information for prenatal diagnosis. Further, we recommend that a genetics specialist should be consulted to interpret the information appropriately and provide counseling and follow-up services after prenatal CMA.

SNP-Based Fetal DNA Detection in Maternal Serum Using the HID-Ion AmpliSeq™ Identity Panel / 대한법의학회지

Fetal DNA (fDNA) detection in maternal serum is a challenge due to low copy number and the smaller size of fDNA fragments compared to DNA fragments derived from the mother. Massively parallel sequencing (MPS) is a useful technique for fetal genetic analysis that is able to detect and quantify small amounts of DNA. In this study, seven clinical samples of maternal serum potentially containing fDNA were analyzed with a commercial single nucleotide polymorphism (SNP) panel, the HID-Ion AmpliSeq™ Identity Panel, and the results were compared to those from previous studies. Reference profiles for mothers and fetuses were not available, but multiple Y chromosomal SNPs were detected in two samples, indicating that fDNA was present in the serum and thereby validating observations of autosomal SNPs. This suggests that SNP-based MPS can be valuable for fDNA detection, thereby offering an insight into fetal genetic status. This technology could also be used to detect small amounts of DNA in mixed DNA samples for forensic applications.

Expression of the genes for peroxisome proliferator-activated receptor-γ, cyclooxygenase-2, and proinflammatory cytokines in granulosa cells from women with polycystic ovary syndrome / 대한생식의학회지

OBJECTIVE: To identify differences in the expression of the genes for peroxisome proliferator-activated receptor (PPAR)-γ, cyclooxygenase (COX)-2, and the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in granulosa cells (GCs) from polycystic ovary syndrome (PCOS) patients and controls undergoing controlled ovarian stimulation. METHODS: Nine patients with PCOS and six controls were enrolled in this study. On the day of oocyte retrieval, GCs were collected from pooled follicular fluid. Total mRNA was extracted from GCs. Reverse transcription was performed and gene expression levels were quantified by realtime quantitative polymerase chain reaction. RESULTS: There were no significant differences in age, body mass index, and total gonadotropin dose, except for the ratio of luteinizing hormone to follicle-stimulating hormone between the PCOS and control groups. PPAR-γ and COX-2 mRNA was significantly downregulated in the GCs of PCOS women compared with controls (p=0.034 and p=0.018, respectively), but the expression of IL-6 and TNF-α mRNA did not show significant differences. No significant correlation was detected between the expression of these mRNA sequences and clinical characteristics, including the number of retrieved oocytes, oocyte maturity, cleavage, or the good embryo rate. Positive correlations were found among the PPAR-γ, COX-2, IL-6, and TNF-α mRNA levels. CONCLUSION: Our data may provide novel clues regarding ovarian GC dysfunction in PCOS, and indirectly provide evidence that the effect of PPAR-γ agonists in PCOS might result from alterations in the ovarian follicular environment. Further studies with a larger sample size are required to confirm these proposals.

Prenatal diagnosis of an unbalanced translocation between chromosome Y and chromosome 15 in a female fetus

We report the prenatal diagnosis of an unbalanced translocation between chromosome Y and chromosome 15 in a female fetus. Cytogenetic analysis of parental chromosomes revealed that the mother had a normal 46,XX karyotype, whereas the father exhibited a 46,XY,der(15)t(Y;15) karyotype. We performed cytogenetic analysis of the father's family as a result of the father and confirmed the same karyotype in his mother and brother. Fluorescence in situ hybridization and quantitative fluorescent-polymerase chain reaction analysis identified the breakpoint and demonstrated the absence of the SRY gene in female members. Thus, the proband inherited this translocation from the father and grandmother. This makes the prediction of the fetal phenotype possible through assessing the grandmother. Therefore, we suggest that conventional cytogenetic and molecular cytogenetic methods, in combination with family history, provide informative results for prenatal diagnosis and prenatal genetic counseling.

Detection of Adeno-associated Virus from Semen Suffering with Male Factor Infertility and Having Their Conception Partners with Recurrent Miscarriages

Adeno-associated virus (AAV) and human papillomavirus (HPV) DNAs were found in abnormal quality semen, early abortus and female genital tissues. It was suggested that they might cause male infertility and miscarriages. This study was performed to determine the detection rate of these viruses in the semen and to assess the relationship between the presence of virus and male factor infertility and recurrent miscarriages. Sixty-three of 99 recruited male were included in this study according to the completeness of follow-up and the sample availability. Fourteen male with normal reproductive capacity were allocated to control group, 15 male with abnormal results in semen analysis were grouped as male factor infertility (MF) group, and 34 male whose spouses have had history of repeated spontaneous abortions were designated as repeated miscarriage (RM) group. AAV and HPV were detected in semen by polymerase chain reaction. The detection rate of AAV in the MF infertility group and RM group was 60.0% and 50.0%, respectively, while 14.3% in the control group (p < 0.05). However, the differences in the detection rate of HPV were not statistically significant among groups. These results suggest that AAV could be related to repeated miscarriages and male infertility.

Outcomes of preimplantation genetic diagnosis using either zona drilling with acidified Tyrode's solution or partial zona dissection / 대한생식의학회지

OBJECTIVE: To review the outcomes of preimplantation genetic diagnosis (PGD) using zona drilling with acid Tyrode's solution (chemical zona pellucida drilling, chemical ZD) and those of partial zona dissection (PZD). METHODS: Clinical outcomes of seventy-one couples undergoing 85 PGD cycles from January 2005 to December 2010 were included. Blastocyst formation and the hatching rate, clinical pregnancy rate, ongoing pregnancy rate, implantation rate, and fetal gender ratio of the PZD and chemical ZD groups were compared. RESULTS: Application of PZD resulted in a significantly higher rate of clinical pregnancy (40.7% vs. 15.4%, p=0.022), ongoing pregnancy (35.6% vs. 11.5%, p=0.023), and implantation (18.1% vs. 5.7%, p=0.007) compared with chemical ZD. Among non-transferred embryos, the rate of blastocyst formation on day 5 (49.1% vs. 39.5%, p=0.016) and hatching on day 6 (47.2% vs. 26.5%, p<0.001) were also significantly higher in the PZD group. CONCLUSION: The mechanical zona dissection method showed better outcomes than chemical ZD in terms of the blastocyst development and pregnancy rate. In this study, the fact that chemical ZD was conducted in different period from mechanical method should be considered in interpreting the result.

Application of Sperm Selection Using Hyaluronic Acid Binding in Intracytoplasmic Sperm Injection Cycles: A Sibling Oocyte Study

The purpose of this study was to investigate whether sperm selection by hyaluronic acid (HA) binding could improve fertilization rate and embryo quality in intracytoplasmic sperm injection (ICSI) cycles. Two hundred nineteen oocytes obtained from eighteen women were injected with either HA-bound (n = 107) or conventionally selected spermatozoa (n = 112) in a randomized way. All of the participants were infertile couples who had normal sperm parameters but low fertilization rate in previous in vitro fertilization (IVF) cycle (n = 5) or experienced multiple IVF failures (n = 13). Lower fertilization (75.7% vs 83.0%) and cleavage rate on day 2 (72.9% vs 83.0%) was observed in oocytes injected with HA-bound spermatozoa than the conventional group, but the difference was not significant. Significantly lower cleavage rate was observed on day 3 in HA group (56.0% vs 69.6%, P = 0.038). Blastocyst formation rate and the number of transferred embryos were similar in both groups. In multiple IVF failure patients, significantly reduced fertilization rate (71.8% vs 85.3%, P = 0.046) and cleavage rate on day 2 (70.4% vs 85.3%, P = 0.029) and day 3 (53.5% vs 77.3%, P = 0.002) were noticed in HA group. Five women achieved pregnancy continuing more than 12 weeks after transfer (27.8%). Success of ICSI was not related with the number of embryos fertilized by HA-bound spermatozoa. Application of ICSI by sperm selection using HA binding is not helpful in couples with repeated poor fertilization or implantation despite normal sperm parameters.

The Recurrent Pregnancy Loss Associated with a Female Carrier of a Structural Chromosome Rearrangement

Inversion, one of the balanced rearrangements, usually does not lead to phenotypic abnormalities; all genetic information exists in the proper amount, merely in a different order or in an abnormal location. However, offspring of an inversion carrier is at risk of chromosomal imbalance because an inversion loop can be formed during crossing-over of the paternal and the maternal chromosomes in meiosis. We report a 38-year-old woman with inversion and balanced translocation and her fetus with unusual rearrangement causing chromosomal imbalance. We performed conventional cytogenetic analysis, MLPA, and subtelomeric FISH in the cells of the embryo. The results showed that the distal portion of chromosome 13q was added to the terminal portion of chromosome 9p during crossing-over. Therefore, the final karyotype of the fetus was 46,XY,rec(9)t(9;13)(p22;q32)inv(9)(p12q13)mat, confirmed using molecular-cytogenetic analyzing tools.

The recent trend of prenatal screening

Twenty years have passed since a prenatal screening for Down syndrome and neural tube defect was applied to obstetric field. The Quad test (AFP, hCG, uE3, Inhibin-A) of the second trimester and the combination test (PAPP-A, hCG, NT) of the first trimester became popular now. The recent trend of prenatal screening is to combine these two screening tests together in order to increase a detection rate of Down syndrome. Three types of screening methods are introduced as follows; integrated test, sequential test and contingent test. In addition to combination of each test, an incorporation of characteristic ultrasound findings of Down syndrome is suggested for its risk calculation. The absence of fetal nasal bone would be a very useful marker especially in the first trimester screening test. According to a change of way calculating risk of Down syndrome, obstetrician's role will be more increased not by passive participation, but by active participation using ultrasound in risk calculation.

Unbalanced translocation der(8)t(8:13)(p23.3;q32.1)dn identified by array CGH and subtelomeric FISH in a patient with mental retardation

Molecular cytogenetics allows the identification of unknown chromosome rearrangements, which is clinically useful in patients with mental retardation and/or development delay. We report on a 31-year- old woman with severe mental retardation, behavior development delay, and verbal performance delay. Conventional cytogenetic analysis showed a 46,XX,add(8)(p23.3) karyotype. To determine the origin of this unbalanced translocation, we performed array CGH and subtelomeric FISH. The results showed that the distal region of chromosome 8p was added to the terminal of chromosome 13q. This was confirmed the final result of 46,XX,der(8)t(8:13)(p23.3;q32.1)dn.

A Case of a 46,XX Male with SRY Gene

46,XX male is a rare sex constitution characterized by the development of bilateral testis in persons who lack a Y chromosome. Manifestations of 46,XX males are usually hypogonadism, gynecomastia, azoospermia, and hyalinations of seminiferous tubules. The incidence of XX male reversal is approximately 1 in 20,000 male neonates. The SRYgene is located at the short arm of the Y chromosome(Yp11.31) and codes for testis determining factor in humans. Here, the patient, who presented with a normal male phenotype, was referred for azoospermia. Conventional cytogenetic analysis showed a 46,XX karyotype. Quantitative fluorescent polymerase chain reaction(QF-PCR) and Multiplex PCR studies identified SRY gene. And, Fluorescence In Situ Hybridization(FISH) confirmed the SRY gene on the distal short arm of chromosome X. We identified the SRY gene on the distal short arm of chromosome X by molecular cytogenetic and molecular analyses. Therefore, molecular-cytogenetics and molecular studies were proved to be clinically useful adjunctive tool to conventional prenatal cytogenetic analysis.

Genetic counselling in prenatal genetic testing

No abstract available.

Diagnosis of factor VIII gene inversion by PCR in Korean patients with hemophilia A and its application to carrier detection / 대한산부인과학회지

OBJECTIVE: To establish PCR (polymerase chain reaction) method for detecting factor VIII gene inversion (intron 22) causing hemophilia A, and to apply it to carrier detection of hemophilia A. DESIGN: A laboratory analysis MATERIALS AND METHODS: An inversion pattern of the factor VIII gene was analyzed in 130 unrelated Korean patients with hemophilia A and 26 female subjects using PCR. RESULTS: PCR analysis of the factor VIII gene for intron 22 inversion revealed that 91 patients (70%) were negative for the inversion, yielding 12 kb band by PQ primer. And all the other 39 (30%) patients who showed no amplification by PQ primer were positive for the inversion, yielding 11kb band by AQ primer. Among 113 patients with severe hemophilia A, 39 (35%) patients were positive for the inversion. Carrier detection for intron 22 inversion in 26 female subjects was performed, and revealed that 22 cases were carriers and 4 cases were normal female. CONCLUSION: This result suggests that PCR analysis of the inversion within the factor VIII gene is useful in the carrier detection of hemophilia A as well as in identifying hemophilia A patients with intron 22 inversion, in the Korean population.

Development of medical genetics training program and certification process for medical geneticist as a specialist in Korea

PURPOSE: This study was undertaken to provide prerequisites for accreditation of medical genetics training program and certification process for medical genetics professionals as clinical specialist and set up guidelines on curriculum of medical genetics training program in Korea. METHODS: Six ad hoc committees for clinical geneticist, clinical cytogeneticist, clinical molecular geneticist, clinical biochemical geneticist, medical genetics technologists and genetic counselors were organized for reviewing current status in Korea as well as foreign countries. Each committee is composed of 6-8 members. They summarized their opinions according to the structured questionnaire inquiring the ways of accrediting training program, qualification of program director, trainee requirements, contents of curriculum, duration of training program, certification process, estimation of numbers of each specialist needed in next 5 years in Korea. RESULTS: Both prerequisites for the accreditation of medical geneticist training institutions and qualification of program director are suggested. Candidacy of trainees requires MD with board of medical specialty, or PhD degree with professional experiences in related field except clinical genetics program which only accepts MD with board of medical specialty, and Non-MD genetic counselor and medical technologists with degrees of BS or MS. General duration of fellowship will be 2-3 years depending on the categories they are enrolled into. Contents of curriculum for each speciality training are described. For the certification of each category, the candidacy should submit a log book detailing the cases they experienced during the fellowship, prove that they successfully completed course work and clinical experiences in the accredited program, and pass the written examination. CONCLUSION: As medical genetics becomes more important in daily routine clinical practice, the accreditation of medical genetics training program and certification of personnel are urgently needed. In this regard, the study will be providing guidelines and prerequisites for accreditation of medical genetics training program and certification process for medical genetics professionals as clinical specialist.

Rapid detection of aneuploidy using FISH in uncultured amniocytes for prenatal diagnosis : 8-year experience

PURPOSE: FISH is suggested as a useful tool for rapid detection of specific aneuploidy in uncultured amniocytes abnormality in interphase nucleus. In this study, we are going to share our experience using FISH in prenatal diagnosis and suggest the criteria for the diagnosis of aneuploidy by analyzing the results of FISH test. METHODS: From January, 1999 to May, 2006, 8,613 tests in amniotic fluids obtained from 7,893 pregnant women were performed by using FISH for prenatal diagnosis of trisomy 21, trisomy 18 and trisomy 13. The indications of chromosome study were a screen positive for Down syndrome or Edwards syndrome in maternal serum marker screening test and an advanced maternal age (> or =35 years old). RESULTS: We have the 8,502 informative results from 8,613 tests (98.7%) which is submitted our criteria and the sensitivity is 98.2%. CONCLUSION: FISH on uncultured amniocytes is a rapid, clinically useful tool for prenatal diagnosis, with informative specimens being highly accurate. But the limitation of FISH is both expensive and labor-intensive.

Factor VIII Gene Mutations in Korean Patients with Hemophilia A / 대한산부인과학회잡지

OBJECTIVE: To identify mutations within the factor VIII gene in Korean patients with severe hemophilia A. DESIGN: A laboratory analysis. METHODS: We systematically sequenced the promoter, all exons and splice junctions of factor VIII gene in 23 unrelated Korean patients with severe hemophilia A. Patients with factor VIII gene inversion were excluded. RESULTS: Twelve patients (52.2%) showed a point mutation, among which 6 were nonsense mutations and the other 6 were missense mutations. A large deletion was found in 6 (26.1%) patients, a small deletion in 2 (8.7%), a small insertion in one patient. Two patients had compound mutations: one patient had two missense mutations, and the other had a missense mutation and 4 bp insertion. Ten (43.5%) out of 23 mutations found are novel. CONCLUSION: Mutations within the factor VIII gene found in Korean patients with moderate to severe hemophilia A are diverse as expected. And we have found 10 novel mutations. Our results can help understanding the molecular pathology of hemophilia A.

In Situ Culture and Harvest of Amniocytes using coverslip processing method

Genetic amniocenteses were performed in a series of 127 patients as a routine study. Samples from the patients were cultured by in situ method, flask method or both according to the state of amniotic fluid. The overall success rate of culture was 97.6% and no culture failure was observed in the flask method. It took 5 days first of all and 8.15 days average from set-up to harvest and there were 7.2 colonies per dish in in situ method. Therefore, it is suggested that in situ method which decreased the mean culture days and made clonal analyses possible, is a clinically available and even more reliable method in parallel with flask method in prenatal diagnosis.

Chromosome Analysis in Clinical Samples by Chromosome Diagnostic System Using Fluorescence in Situ Hybridization / 대한불임학회지

Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Zl, DXZI, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.