Sperm and Oocyte Chromosomal Abnormalities.

Gametogenesis, the process of producing gametes, differs significantly between oocytes and sperm. Most oocytes have chromosomal aneuploidies, indicating that chromosomal aberrations in miscarried and newborn infants are of oocyte origin. Conversely, most structural anomalies are of sperm origin. A prolonged meiotic period caused by increasing female age is responsible for an increased number of chromosomal aberrations. Sperm chromosomes are difficult to analyze because they cannot be evaluated using somatic cell chromosome analysis methods. Nevertheless, researchers have developed methods for chromosome analysis of sperm using the fluorescence in situ hybridization method, hamster eggs, and mouse eggs, allowing for the cytogenetic evaluation of individual sperm. Reproductive medicine has allowed men with severe spermatogenic defects or chromosomal abnormalities to have children. However, using these techniques to achieve successful pregnancies results in higher rates of miscarriages and embryos with chromosomal abnormalities. This raises questions regarding which cases should undergo sperm chromosome analysis and how the results should be interpreted. Here, we reviewed clinical trials that have been reported on oocyte and sperm chromosome analyses. Examination of chromosomal abnormalities in gametes is critical in assisted reproductive technology. Therefore, it is necessary to continue to study the mechanism underlying gametic chromosomal abnormalities.

Evaluation of the clinical performance of noninvasive prenatal testing at a Japanese laboratory.

AIM: We aimed to evaluate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of noninvasive prenatal testing (NIPT) in high-risk pregnant women. METHODS: Pregnant women who underwent GeneTech NIPT, the most commonly used NIPT in Japan, between January 2015 and March 2019, at Japan NIPT Consortium medical sites were recruited for this study. The exclusion criteria were as follows: pregnant women with missing survey items, multiple pregnancy/vanishing twins, chromosomal abnormalities in the fetus other than the NIPT target disease, and nonreportable NIPT results. Sensitivity and specificity were calculated from the obtained data, and maternal age-specific PPV and NPV were estimated. RESULTS: Of the 45 504 cases, 44 263 cases fulfilling the study criteria were included. The mean maternal age and gestational weeks at the time of procedure were 38.5 years and 13.1 weeks, respectively. Sensitivities were 99.78% (95% confidence interval [95% CI]: 98.78-99.96), 99.12% (95% CI: 96.83-99.76), and 100% (95% CI: 88.30-100) for trisomies 21, 18, and 13, respectively. Specificities were more than 99.9% for trisomies 21, 18, and 13, respectively. Maternal age-specific PPVs were more than 93%, 77%, and 43% at the age of 35 years for trisomies 21, 18, and 13, respectively. CONCLUSION: The GeneTech NIPT data showed high sensitivity and specificity in the detection of fetal trisomies 21, 18, and 13 in high-risk pregnant women, and maternal age-specific PPVs were obtained. These results could provide more accurate and improved information regarding NIPT for genetic counseling in Japan.

Retrospective details of false-positive and false-negative results in non-invasive prenatal testing for fetal trisomies 21, 18 and 13.

OBJECTIVE: Maternal characteristics and neonatal outcomes associated with cell-free DNA (cfDNA) results were analysed retrospectively to assess the details of false-positive and false-negative results after initial blood sampling in non-invasive prenatal testing (NIPT). STUDY DESIGN: A multicentre retrospective study was performed for women undergoing NIPT who received discordant cfDNA results between April 2013 and March 2018. The NIPT data obtained using massive parallel sequencing were studied in terms of maternal background, fetal fraction, z-scores, invasive procedure results and neonatal outcomes after birth. RESULTS: Of the 56,545 women who participated in this study, 54 false-positive (0.095 %) and three false-negative (0.006 %) cases were found. Seven of the 54 false-positive cases (13.0 %) had vanishing twin on ultrasonography. Among the 18 false-positive cases of trisomy 18, confined placental mosaicism (CPM) was confirmed in three cases (16.7 %), while CPM was present in one of the three false-negative cases of trisomy 21. CONCLUSION: These data suggest that the incidence of women with false-positive or false-negative results is relatively low, that such false results can often be explained, and that vanishing twin and CPM are potential causes of NIPT failure. Genetic counselling with regard to false results is important for clients prior to undergoing NIPT.

Concurrent chemoradiotherapy for locally advanced squamous cell carcinoma of the cervix in a uterus didelphys with vaginal septum.

In November 2011, a 61-year-old woman was diagnosed with squamous cell carcinoma (SCC) of the cervix in a uterus didelphys with vaginal septum. The patient was diagnosed with Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stage IIB because of infiltration to the left parametrium without infiltration to the pelvic wall. The patient was treated with external-beam radiotherapy (EBRT) and brachytherapy (BT), using concomitant chemotherapy with cisplatin. A total of 50 Gy were delivered (2 Gy/fraction/day) to the pelvis, with a central shield after 40 Gy. The patient was treated four times with BT (6 Gy × 4 fractions), with tandem and ovoid applicators inserted once to the left side; tandem to the left side and ovoid bilaterally were inserted twice; and tandem to the right side and ovoid bilaterally were inserted once. Six years and 8 months after the start of treatment, the patient had had no relapse or severe late adverse effects. For accurate diagnosis and optimal treatment of the uterus didelphys, careful interview and pelvic examination at initial diagnosis of a patient are very important.

Current status of non-invasive prenatal testing in Japan.

AIM: The purpose of this study was to report the 3-year experience of a nationwide demonstration project to introduce non-invasive prenatal testing (NIPT) of maternal plasma for aneuploidy, and review the current status of NIPT in Japan. METHODS: Tests were conducted to detect aneuploidy in high-risk pregnant women, and adequate genetic counseling was provided. The clinical data, test results, and pregnancy outcomes were recorded. We discuss the problems of NIPT on the basis of published reports and meta-analyses. RESULTS: From April 2013 to March 2016, 30 613 tests were conducted at 55 medical sites participating in a multicenter clinical study. Among the 30 613 women tested, 554 were positive (1.81%) and 30 021 were negative (98.1%) for aneuploidy. Of the 289, 128, and 44 women who tested positive for trisomies 21, 18, and 13, respectively, and underwent definitive testing, 279 (96.5%), 106 (82.8%), and 28 (63.6%) were determined to have a true-positive result. For the 13 481 women with negative result and whose progress could be traced, two had a false-negative result (0.02%). The tests were performed on the condition that a standard level of genetic counseling be provided at hospitals. CONCLUSION: Here, we report on the 3-year nationwide experience with NIPT in Japan. It is important to establish a genetic counseling system to enable women to make informed decisions regarding prenatal testing. Moreover, a welfare system is warranted to support women who decide to give birth to and raise children with chromosomal diseases.

No correlation between the number of fetal nucleated cells and the amount of cell-free fetal DNA in maternal circulation either before or after delivery.

OBJECTIVES: We have determined the number of fetal nucleated cells and the concentration of cell-free fetal DNA in parallel in the same maternal blood samples either before or after delivery, and studied the relationship between these two. METHODS: Venous blood samples were taken at four points around delivery from ten women who had singleton male fetus with informed consent. The number of fetal nucleated cells having a Y chromosome specific signal treated by two-color fluorescence in situ hybridization technique was counted using maternal whole blood. The concentration of sex-determining region Y gene sequence was determined using real-time quantitative PCR. RESULTS: The number of fetal nucleated cells decreased after delivery, and some fetal cells were present 1 month after delivery. While cell-free fetal DNA decreased rapidly after delivery and became undetectable 1 day after delivery in eight out of ten cases. The number of fetal nucleated cells did not correlate with the concentration of cell-free fetal DNA in maternal circulation. CONCLUSION: The present study demonstrates that cell-free fetal DNA disappears very rapidly after delivery and fetal nucleated cells remain longer in maternal circulation, and that there is no correlation between these two either before or after delivery.

Increased plasma mRNAs of placenta-specific 1 (PLAC1) and glial cells-missing 1 (GCM1) in mothers with pre-eclampsia.

In this study we have investigated whether quantitative analysis of placental mRNAs in maternal plasma provides a way to monitor placental status. We measured plasma concentrations of human chorionic gonadotropin beta-subunit (betahCG) and human placental lactogen (hPL) mRNAs as previously reported mRNAs and pregnancy associated plasma protein A (PAPP-A), placenta-specific 1 (PLAC1) and glial cells-missing 1 (GCM1) mRNAs, which have not been measured during the course of normal pregnancy. Firstly, peripheral blood was obtained at various times from healthy pregnant women to clarify the time course of placental mRNAs. Secondly, blood was obtained from women with pre-eclampsia and gestational age-matched controls to examine whether placental mRNAs change in pre-eclampsia. Plasma was separated from these samples for extraction of RNA, followed by reverse transcription polymerse chain reaction analysis. Median concentrations of PLAC1 and GCM1 mRNA in plasma of pre-eclamptic subjects respectively were 1625 and 2141 copies/ml, significantly higher than 195 and 881 copies/ml, the values for controls (Mann-Whitney test, p<0.001). No significant difference was seen in hPL, betahCG, or PAPP-A mRNA concentration between pre-eclamptic and control groups. Plasma PLAC1 and GCM1 mRNAs appear promising as noninvasively measurable molecular markers for pre-eclampsia.

Functional disomy for Xq22-q23 in a girl with complex rearrangements of chromosomes 3 and X.

A 5-year-old girl with developmental and growth retardation is reported with complex chromosome rearrangements consisting of a partial Xq deletion and an abnormal chromosome 3 with multiple breakpoints. GTG-banding, and multiplex and conventional FISH studies showed that a 6.6-Mb Xq22-q23 segment was inserted into 3q, in addition to three intrachromosomal insertions in chromosome 3. Her karyotype was thus interpreted as 46,X,der(X)(Xpter-->Xq22::Xq23-->Xqter),der(3)(3pter-->3p26::3p12-->3q25.3::3p12-->3p26::Xq22-->Xq23::3q25.3-->3qter). Replication R-banding study showed that the der(X) was inactivated in all blood lymphocytes analyzed. Methylation-specific PCR at the androgen receptor gene (HUMARA) locus at Xq11-q12 showed a skewed inactivation pattern with the active/inactive X chromosome ratio of 92/8. These data indicated the presence, in the majority of cells, of a functioning Xq22-q23 segment in both the normal X and the der(3) chromosomes. Her growth retardation, developmental delay, and other minor anomalies were most likely caused by dosage effects of the genes in the functionally disomic Xq22-q23 region. Despite the presence of two active copies of the proteolipid protein 1 gene (PLP1), she did not show the symptoms of Pelizaeus-Merzbacher disease, a subset of which has been known to be caused by the duplication of PLP1.

Chromosomal FISH analysis of unfertilized human oocytes and polar bodies.

The incidence of chromosomal aneuploidy was studied in 208 unfertilized metaphase II human oocytes from an in vitro fertilization program by fluorescence in situ hybridization using probes for chromosomes 18, 21, and X. Chromosome spreads were prepared by a gradual fixation-air-drying method. We obtained analyzable results from 183 oocytes and 93 polar bodies; 167 oocytes (91%) were normal, 11 (6%) were diploid, and 5 (3%) were aneuploid. Of the five aneuploid oocytes, four involved chromosome 21, and one involved the X chromosome. In this study, oocyte aneuploidy caused by both nondisjunction of bivalent chromosomes and predivision of univalent chromosomes was observed. The aneuploidy rate (9.8%) in the oocytes from women aged >==35 years was significantly higher than that (0.7%) in those aged 23 to 34 years ( P = 0.0017).

Fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum.

Fetal DNA in maternal plasma and serum has been shown to be a useful material for fetal gender determination and for screening tests for abnormal pregnancies except during early gestational ages. Maternal serum samples were obtained from 81 pregnant women during the 5th-10th weeks of gestation. Fetal gender was determined by conventional polymerase chain reaction (PCR) to detect a Y-chromosomal sequence (DYS14) in maternal serum during early gestation and confirmed by examination of the newborns after delivery. Real-time quantitative analyses of the SRY and beta-globin genes were also performed in order to determine fetal gender and to quantify fetal DNA concentration in maternal serum during early gestation. When using conventional PCR, the total sensitivity of identifying a male fetus was 95%, but its sensitivity after the 7th week was 100%, whereas in real-time quantitative PCR, the total sensitivity after the 5th week was 100%. Quantitative analyses of the SRY gene revealed that the mean concentration of fetal DNA in maternal serum was 30.55 copies/ml, that fetal DNA concentration showed a tendency to increase with the progression of pregnancy, and that it had a wide normal range. Thus, we could confidently determine fetal gender by using maternal serum samples taken as early as the 7th week.