Mosaic Ratio Research Articles

Confined Placental Mosaicism Detected With Non-Invasive Prenatal Testing: Is There an Association Between Mosaic Ratio and Pregnancy Outcome?

Confined placental mosaicism (CPM) is associated with an increased risk for pregnancy complications, such as fetal growth restriction (FGR), preterm birth and hypertensive disorders. Pregnancies with possible CPM can be identified with non-invasive prenatal testing (NIPT). We performed a retrospective cohort study to investigate whether the mosaic ratio, as calculated with the Veriseq v2 used for NIPT, can predict adverse pregnancy outcomes in cases of CPM. A mosaic ratio for trisomies detected by NIPT and obstetric data such as fetal growth, structural fetal anomalies and birthweight were retrospectively studied in a cohort of patients with CPM diagnosed between February 2021 and October 2023. Structural and sex chromosomal aberrations were not included in this study. Of 122 CPM cases, 52 cases (42.6%) showed adverse perinatal outcomes, including FGR, low birthweight, hypertensive disorders, or preterm birth. A significantly higher mosaic ratio was found in the adverse outcome group compared to those with normal outcome, but a clear-cut threshold could not be set, except potentially for trisomy 16. There is an association between the mosaic ratio and adverse pregnancy outcomes in cases of CPM. However, without a clear-cut threshold, it cannot be used for the individual patient for differentiation between CPM with and without clinical consequences.

Analysis of related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes with mosaic embryo transfers

Objective: To explore the related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes of mosaic embryo transfer in preimplantation genetic testing for aneuploidy (PGT-A) based on next generation sequencing (NGS) technology. Methods: A retrospective study was performed to analyze the clinical data of patients in 745 PGT-A cycles from January 2019 to May 2023 at Chongqing Health Center for Women and Children, including 2 850 blastocysts. The biopsy cells were tested using NGS technology, and the embryos were divided into three groups based on the test results, namely euploid embryos, aneuploid embryos and mosaic embryos. The influence of population characteristics and laboratory-related parameters on the detection rate of mosaic embryo were analyzed, and the pregnancy outcomes of 98 mosaic embryo transfer cycles and 486 euploid embryo transfer cycles were compared during the same period, including clinical pregnancy rate and live birth rate. Results: Among the embryos tested (n=2 850), the number and proportion of euploid embryos, aneuploid embryos and mosaic embryos were 1 489 (52.2%, 1 489/2 850), 917 (32.2%, 917/2 850) and 444 (15.6%, 444/2 850), respectively. Among mosaic embryos, 245 (55.2%, 245/444) were segmental mosaic embryos, 118 (26.6%, 118/444) were whole-chromosome mosaic embryos, and 81 (18.2%, 81/444) were complex mosaic embryos. NGS technology was performed in 4 genetic testing institutions and the detection rate of mosaic embryo fluctuated from 13.5% to 27.0%. The distributions of female age, level of anti-Müllerian hormone, PGT-A indications, ovulation-inducing treatments, gonadotropin (Gn) dosage, Gn days, inner cell mass grade, trophectoderm cell grade, genetic testing institutions and developmental stage of blastocyst were significantly different among the three groups (all P<0.05). Multi-factor analysis showed that the trophectoderm cell grade and genetic testing institutions were significantly related to the detection rate of mosaic embryo; compared with the trophectoderm cell graded as A, the detection rate of mosaic embryo was significantly increased in the trophectoderm cell graded as B-(OR=1.59, 95%CI: 1.04-2.44, P=0.033); compared with genetic testing institution a, the detection rate of mosaic embryo was significantly higher (OR=2.89, 95%CI: 2.10-3.98, P<0.001) in the testing institution c. The clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: 51.0% vs 65.2%, P=0.008; live birth rate: 39.4% vs 53.2%, P=0.017). After adjustment for age, PGT-A indications, trophectoderm cell grade and days of embryo culture in vitro, the clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: OR=0.52, 95%CI: 0.32-0.83, P=0.007; live birth rate: OR=0.50, 95%CI: 0.31-0.83, P=0.007). Conclusions: The trophectoderm cell grade and genetic testing institutions are related to the detection rate of mosaic embryo. Compared with euploid embryos transfer, the clinical pregnancy rate and live birth rate with mosaic embryos transfer are significantly reduced. For infertile couple without euploid embryos, transplantable mosaic embryos could be recommended according to the mosaic ratio and mosaic type in genetic counseling to obtain the optimal pregnancy outcome.

Rare mosaic variant of GJA1 in a patient with a neurodevelopmental disorder

GJA1 is the causative gene for oculodentodigital dysplasia (ODDD). A novel de novo GJA1 variant, NM 000165:c263C > T [p.P88L], was identified in a mosaic state in a patient with short stature, seizures, delayed myelination, mild hearing loss, and tooth enamel hypoplasia. Although the patient exhibited severe neurodevelopmental delay, other clinical features of ODDD, including limb anomalies, were mild. This may be due to differences in the mosaic ratios in different organs.

Genetic analysis of an infant death due to a paternally derived FOXF1 somatic-gonadal mosaic variant

To investigate the genetic characteristics and cause of death for an infant with alveolar capillary dysplasia and pulmonary vein misalignment (ACD/MPV). An infant with ACD/MPV diagnosed at the Affiliated Maternity and Child Health Care Hospital of Nantong University in September 2022 was selected as the study subject. Clinical data of the infant were collected. Whole exome sequencing (WES) was carried out to detect genetic variants in the skin tissue, and Sanger sequencing was performed for verifying the candidate variants in the parents. Droplet digital PCR (ddPCR) was used to determine the mosaicism ratio of the variant in different germ layer-derived samples from the father. The infant had died within 2 days after birth due to hypoxemia and respiratory distress. WES revealed that she has harbored a c.433C>T nonsense variant in exon 1 of the FOXF1 gene, which was unreported previously. Sanger sequencing has verified the variant in the infant, with her mother's locus being the wild-type and a minor variant peak noted in her father. ddPCR indicated that the mosaic ratio of the c.433C>T variant in the father's sperm was 27.18%, with the mosaic ratios of the variant in tissues originating from the three germ layers ranging from 11% to 28%. The c.433C>T variant derived from the paternal germline and somatic mosaicism of the FOXF1 gene had probably predisposed to the neonatal death of this infant. ddPCR is an effective method for detecting mosaic variants.

O-291 Clinical Application of Noninvasive PGT for Patients with Recurrent Pregnancy Loss or Repeated Implantation Failure

Abstract Study question Has noninvasive PGT been allied with the patients experiencing recurrent pregnancy loss (RIF) and recurrent implantation failure (RPL)? What are the clinical beneficiaries? Summary answer Embryo selection through noninvasive PGT can reduce the miscarriage rate in patients experiencing RPL and improve the clinical pregnancy rate in patients experiencing RIF. What is known already Chromosomal abnormalities exist in early human embryos, especially in patients with RPL and RIF. Therefore, embryos are usually evaluated through pre-implantation genetic testing for aneuploidy (PGT-A). However, a biopsy is an important concern for undetermined health risks. Meanwhile, the researchers observed genomic DNA contents in the embryo culture medium. Since then, multiple studies have been published using culture medium for analyzing chromosomal ploidy. In 2016, Xu et al. first reported a noninvasive chromosome screening (NICS) assay using a spent blastocyst culture medium. Nevertheless, the clinical application of NICS has not been evaluated in patients experiencing RPL or RIF. Study design, size, duration We designed a retrospective cohort study including 303 subjects from July 2018 to May 2021, according to the records of the Reproductive Centre at the Second Affiliated Hospital of Wenzhou Medical University. Patients experiencing RPL or RIF who received the NICS for aneuploidy were included in the NICS group, while those who underwent conventional morphology embryo transfer during the same period were included in the non-NICS group. Participants/materials, setting, methods We included women with a history of RPL (≥2 pregnancies) or RIF (≥3 implantations), exclusion criteria were antiphospholipid syndrome (APS), diabetes, hypothyroidism, or other severe complications. Routine IVF/ICSI was performed based on sperm quality. The embryos were placed in droplets. Approximately 30 mL of blastocyst medium from each embryo was transferred into cell lysis buffer. Whole-genome amplification was performed using culture media, followed by library preparation using ChromInst (Yikon Genomics; EK100100724 NICS Inst Library Preparation Kit). Main results and the role of chance For the patients experiencing RPL, the miscarriage rate per FET was significantly lower in the NICS group than in the non-NICS group (17.9% vs. 42.6%), whereas the ongoing pregnancy rate (40.7% vs. 25.0%) and live birth rate (38.9% vs. 20.6%) were significantly higher in the NICS group compared to the non-NICS group. Nevertheless, no differences were identified in pregnancy rates per patient between the NICS and non-NICS groups (49.6% vs. 44.9%). For the patients experiencing RIF, the pregnancy rates per FET were significantly higher in the NICS group than in the non- NICS group (46.9% vs. 28.7%), whereas the live birth rate and ongoing pregnancy rate per FET and per patient were no significant difference in the NICS group than in the non-NICS group. Nevertheless, no differences were identified in the miscarriage rate per clinical pregnancy between the NICS and non-NICS groups (23.3% vs. 25.9%). Limitations, reasons for caution As a retrospective study, patients in the NICS groups had different clinical prognoses than those in the non-NICS groups, introducing bias in the study results. Wider implications of the findings Non-invasive PGT can be used not only for aneuploidy detection but also for a comprehensive evaluation of morphology and DNA concentration, mosaic ratio, resolution, etc. The accumulation of clinical outcomes can also be combined with the clinical data of patients as an index to predict the clinical outcomes of embryos. Trial registration number NA

Application of various genetic analysis techniques for detecting two rare cases of 9p duplication mosaicism during prenatal diagnosis.

The identification of genetic mosaicism and the genetic counseling needed following its discovery have been challenging problems in the field of prenatal diagnosis. Herein, we describe the clinical phenotypes and various prenatal diagnostic processes used for two rare cases of 9p duplication mosaicism and review the prior literature in the field to evaluate the merits of different methods for diagnosing mosaic 9p duplication. We recorded ultrasound examinations, reported the screening and diagnosis pathways, and analyzed the mosaic levels of the two cases of 9p duplication using karyotype analysis, chromosomal microarray analysis (CMA), and fluorescence in situ hybridization analysis (FISH). Case 1 had a normal clinical phenotype for tetrasomy 9p mosaicism, and Case 2 showed multiple malformations caused by both trisomy 9 and trisomy 9p mosaicism. Both cases were initially suspected after non-invasive prenatal screening (NIPT) based on cell-free DNA. The mosaic ratio of 9p duplication found via karyotyping was lower than what was discovered by CMA and FISH, in both cases. Contrary to previous findings, the mosaic level of trisomy 9 found by karyotype analysis was greater than what was found by CMA, in terms of complex mosaicism involving trisomy 9 and trisomy 9p, in Case 2. NIPT can indicate 9p duplication mosaicism during prenatal screening. Different strengths and limitations existed in terms of diagnosing mosaic 9p duplication by karyotype analysis, CMA, and FISH. The combined use of various methods may be capable of more accurately determining break-points and mosaic levels of 9p duplication during prenatal diagnosis.

Secular decrease in chromosomal mosaicism in cultured and uncultured peripheral blood cells of six patients with mosaic Down syndrome.

As noted in a review paper,1 a large-scale study revealed that approximately 1.1%–3.8% of all children with Down syndrome (DS) are born with mosaic DS.2, 3 DS is diagnosed postnatally by chromosome G-banding analysis of peripheral blood cultures, and most patients do not undergo subsequent repeat analysis. We present secular changes in the mosaic ratio of six patients with mosaic DS. The G-banding, chromosome 21 fluorescence in situ hybridization (chr21 FISH) from peripheral blood cultures and the chr21 FISH analysis of uncultured blood cells showed a drastic reduction in the mosaic ratio in five patients aged between 7 and 23 years. The mosaic ratio of a child aged 3 years and 3 months gradually, but not drastically, decreased over time. However, no decrease in the mosaic ratio was seen in the buccal mucosa or fibroblasts by chr21 FISH in any mosaic patient. Low-invasive chromosomal analyses of 100 cells per patient from different tissues were performed by SRL, Inc. The analyses were as follows (Table 1). (1) T lymphocyte chromosome analyses of cultured peripheral blood samples by PHA (phytohemagglutinin) stimulation, including (1-a) G-banding and (1-b) chr21 FISH analysis using chromosome 21q22.13-q22.2 probes (Vysis, Abbott Molecular); (2) chr21 FISH analysis of uncultured blood cells, including various leukocytes, instead of bone marrow puncture; (3) chr21 FISH analysis of epithelial cells from the buccal mucosa; and (4) chr21 FISH analysis of fibroblasts cultured from a tooth extraction. The six patients (A–F) with mosaic DS (five females and one male) were aged from 3 years and 3 months to 23 years (Table 1). Five patients (B–F) aged between 7 and 23 years showed drastically decreased mosaic ratios (1%–8%) by G-banding, and FISH analysis of the peripheral blood culture and of uncultured blood cells compared with the postnatal result (Table 1, the red font). Patient A showed a gradual decrease in mosaicism on G-banding and FISH of the peripheral blood culture and by FISH analysis of uncultured blood cells at 11 months, 2 years and 9 months, and 3 years and 3 months of age compared with the postnatal result (Table 1, the red font). However, none of the six patients showed a decreased mosaic ratio in the buccal mucosa analysis or tooth-extraction-derived-fibroblast FISH analysis. Previously, two reports documented a decreased mosaic ratio in mosaic DS patients during early childhood, mainly in peripheral blood cultures.4, 5 We studied longitudinal changes in the mosaic ratio of six mosaic DS patients, including young adults. No decrease was observed in the epithelial cells of the buccal mucosa or cultured fibroblasts. The observed secular decrease in the mosaic ratio in cultured and uncultured peripheral blood cells did not appear to be a mechanism of trisomy rescue because six adult patients with standard trisomy 21 and one patient with translocated trisomy 21 showed no decrease in trisomy cell number in cultured and uncultured peripheral blood cells over time (Table S1, the green font). Within the bone marrow, hematopoietic stem cells exist in a hypoxic microenvironment (niche) and are in a quiescent (G0), undifferentiated state.6 In this microenvironment, trisomy cells are considered more detrimental to viability than normal cells, which may explain the secular decrease in chromosomal mosaicism with mosaic DS in this study. Permission has been obtained from the patients' mothers for the participation in this study. Financial support was provided by JSPS KAKENHI (Grant number: JP18K02497). The authors declare no conflict of interest. This study has received ethical approval from Tokyo Kasei University Ethics Committee (Ita h-29-5) and the Tokyo Metropolitan Tobu Medical Center for Children with Developmental Disabilities Ethics Committee (29MoriTobuCe256-1). TABLE S1. Chromosomal analyses of the trisomy ratio of eight patients, seven with standard trisomy 21 and one translocated trisomy 21 (patient H). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Clinical outcomes of subtypes of mosaic single aneuploid embryos after preimplantation genetic testing for aneuploidy.

Mosaic embryos are often characterized by different numbers (single or double or ≥ 3 aneuploidies) or types of chromosomal abnormalities (monosomy or trisomy and involving whole chromosome or chromosome segments). However, due to limitations in the number of samples, the relationship between these abnormalities and clinical outcomes is often not evaluated. This study analyzed chromosomal abnormalities and clinical outcomes in 591 aneuploid mosaic and 3071 euploid embryos from multiple retrospective cohorts as well as from the current authors' unpublished retrospective cohort. Through meta-analysis, it was found that single aneuploid mosaicism reduced implantation and clinical pregnancy rates. In addition, no significant differences were noted between mosaic trisomies and mosaic monosomies in terms of their effects on implantation and clinical pregnancy rates. All subtypes of single aneuploid mosaicism were found to reduce implantation and clinical pregnancy rates for women of over 35years old. Furthermore, it was observed that all subtypes of single aneuploid in higher-level mosaicism reduced implantation and clinical pregnancy rates. Regarding the lower-level group, only segmental mosaicism with segmental chromosome gain reduced both of the above rates. Unexpectedly, the type of chromosome abnormality was more likely to influence miscarriage rates compared with the level of mosaicism. Indeed, monosomy aneuploid mosaic embryos increased miscarriage rates in both lower- and higher-levels mosaic ratio groups, but not other subtypes. Although the mechanism for the above phenomenon remains unknown, it is recommended that attention should still be paid to the increased miscarriage rates caused by monosomy in aneuploid mosaic embryos.

Two infants with mild, atypical clinical features of Kagami-Ogata syndrome caused by epimutation

Kagami-Ogata syndrome (KOS) is an imprinting disorder characterized by polyhydramnios, bell-shaped thorax with coat-hanger appearance (curved ribs), respiratory distress, abdominal wall defects, and distinct facial features, together with intellectual developmental delay with special needs. Abnormal expression of the imprinted genes on chromosome 14q32.2 causes KOS. Epimutation with aberrant hypermethylation of the MEG3/DLK1: intergenic differentially methylated region (MEG3/DLK1:IG-DMR) and the MEG3:TSS-DMR is one of the etiologies of KOS. We report two infants with KOS caused by epimutation presenting with some characteristic clinical features, mild clinical course, and almost normal motor and intellectual development. Methylation analysis for ten DMRs related to major imprinting disorders using pyrosequencing with genomic DNA (gDNA) extracted from leukocytes showed abnormally increased methylation levels of the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR in both patients, but lower than those in patients with paternal uniparental disomy chromosome 14 (upd(14)pat). The methylation levels in the DMRs other than both DMRs were within normal range. We also conducted methylation analysis for the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR with gDNA extracted from nails and buccal cells of both patients. Methylation levels in the MEG3:TSS-DMR, particularly in buccal cells, were closer to normal range compared to those in leukocytes. Microsatellite analysis for chromosome 14 and array comparative hybridization analysis showed no upd(14)pat or microdeletion involving the 14q32.2 imprinted region in either patient. A differential mosaic ratio of cells with aberrant methylation of DMRs at the 14q32.2 imprinted region among tissues (connective tissue, lung, and brain) might have led to their atypical clinical features. Further studies of patients with epimutation should further expand the phenotypic spectrum of KOS.

Prenatal screening and diagnosis for a fetus with mosaic sex chromosome abnormality

To carry out prenatal screening and diagnosis for a woman with advanced maternal age. Non-invasive prenatal testing (NIPT) was carried out to determine the risk of fetal chromosome aneuploidy. Aminiocentesis was proceeded for fetal chromosomal karyotyping and copy number variation sequencing (CNV-seq). The fetus was subjected to systematic ultrasound screening in the second trimester. NIPT has indicated there was a loss of fetal sex chromosome. Karotyping of the amniocyte showed a mosaic sex chromosome abnormality 45,X[53]/46,X,+mar[7]. The result of fetal DNA CNV-seq was seq[GRCh37]del(Yq11.1q12) chrY: g.13 104 553-28 819 361del, seq[GRCh37]del(Yp11.32p11.2) chrY: g.10 001-9 873 915del (mosaic ratio: 30%). Ultrasonography discovered that the fetus had renal dysplasia and male external genitalia. The karyotypes of the couple were both normal. Multiple genetic tests should be carried out for fetus with a high risk for chromosome aneuploidies signaled by NIPT. It is difficult to predict the post-natal phenotype for fetuses with mosaic sex chromosomal aneuploidies. The couple should be carefully counseled upon genetic counseling.

INFERTILITY DIAGNOSIS AND MOSAICISM RATE

Laboratory interventions or environmental exposures are suspected to be the primary drivers of mosaicism detected by preimplantation genetic testing (PGT).1 The contributing role of female infertility influencing the ratio of mosaicism has not previously been described.

Whole Exome Sequencing Facilitated the Identification of a Mosaic Small Supernumerary Marker Chromosome (sSMC).

Small supernumerary marker chromosomes (sSMCs) are a group of rare chromosomal anomalies, which pose challenges in the clinical practice of prenatal diagnosis and genetic counseling. This study enrolled an extended family with an underage male patient displaying infantile seizures, intellectual disability, and retarded speech and psychomotor function. A series of multiplatform genetic detections was conducted to explore the diagnostic variation. Whole exome sequencing (WES) and chromosomal microarray analysis (CMA) indicated a mosaic sSMC derived from the pericentromeric region of chromosome 8 in the patient, which was confirmed using cytogenetic methods. The proband and his mother, who carried this mosaic variant, exhibited strong phenotypic variability. We also ruled out the pathogenicity of a KDM5C variant by extended validation. Our results emphasized the capacity of WES to detect mosaic SMCs and the importance of mosaic ratios in the appearance and severity of symptomatic phenotypes.

Impact of confined placental mosaicism on non-invasive prenatal testing and pregnancy outcomes

To assess the impact of confined placental mosaicism (CPM) on non-invasive prenatal testing (NIPT) and pregnancy outcomes. Copy number variation sequencing (CNV-seq) and single nucleotide polymorphism array (SNP-array) were carried out on placental specimen sampled from eight pregnancies with confirmed false-positive NIPT results. The impact of CPM on NIPT and pregnancy outcomes were analyzed based on the laboratory tests and clinical characteristics. Five of the eight cases with false-positive NIPT results were proven to be CPM involving trisomy 9, 13, 21, 22, and X, respectively. The mosaic ratios for different placental regions have varied from 4% to 80%. Two fetuses with confirmed CPM showed fetal growth restriction (FGR) and additional ultrasound abnormalities, 1 fetus showed only FGR. The remaining two fetuses showed normal growth. NIPT is highly sensitive to CPM, whilst CPM is an important cause for false-positive NIPT result. CPM may be associated with FGR. Investigation of the presence of CPM is important for both pre- and post-test genetic counseling and management of the pregnancy.

Effect of immunosuppressants on a mouse model of osteogenesis imperfecta type V harboring a heterozygous Ifitm5 c.-14C\u2009>\u2009T mutation

Osteogenesis imperfecta (OI) type V is an autosomal dominant disorder caused by the c.-14C > T mutation in the interferon-induced transmembrane protein 5 gene (IFITM5), however, its onset mechanism remains unclear. In this study, heterozygous c.-14C > T mutant mice were developed to investigate the effect of immunosuppressants (FK506 and rapamycin) on OI type V. Among the mosaic mice generated by Crispr/Cas9-based technology, mice with less than 40% mosaic ratio of c.-14C > T mutation survived, whereas those with more than 48% mosaic ratio exhibited lethal skeletal abnormalities with one exception. All heterozygous mutants obtained by mating mosaic mice with wild-type mice exhibited a perinatal lethal phenotype due to severe skeletal abnormalities. Administration of FK506, a calcineurin inhibitor, in the heterozygous fetuses improved bone mineral content (BMC) of the neonates, although it did not save the neonates from the lethal effects of the mutation, whereas rapamycin, an mTOR inhibitor, reduced BMC, suggesting that mTOR signaling is involved in the bone mineralization of heterozygous mutants. These findings could clarify certain aspects of the onset mechanism of OI type V and enable development of therapeutics for this condition.

Low-level parental somatic mosaic SNVs in exomes from a large cohort of trios with diverse suspected Mendelian conditions

PurposeThe goal of this study was to assess the scale of low-level parental mosaicism in exome sequencing (ES) databases. MethodsWe analyzed approximately 2000 family trio ES data sets from the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) and Baylor Genetics (BG). Among apparent de novo single-nucleotide variants identified in the affected probands, we selected rare unique variants with variant allele fraction (VAF) between 30% and 70% in the probands and lower than 10% in one of the parents. ResultsOf 102 candidate mosaic variants validated using amplicon-based next-generation sequencing, droplet digital polymerase chain reaction, or blocker displacement amplification, 27 (26.4%) were confirmed to be low- (VAF between 1% and 10%) or very low (VAF <1%) level mosaic. Detection precision in parental samples with two or more alternate reads was 63.6% (BHCMG) and 43.6% (BG). In nine investigated individuals, we observed variability of mosaic ratios among blood, saliva, fibroblast, buccal, hair, and urine samples. ConclusionOur computational pipeline enables robust discrimination between true and false positive candidate mosaic variants and efficient detection of low-level mosaicism in ES samples. We confirm that the presence of two or more alternate reads in the parental sample is a reliable predictor of low-level parental somatic mosaicism.

Prenatal diagnosis of Pallister-Killian syndrome using cord blood samples

BackgroundPallister-Killian syndrome (PKS) (OMIM:#601803) is a rare sporadic genetic disorder characterized by multi-malformations which is caused by the presence of the extra isochromosome 12p. PKS is featured by the tissue-limited mosaicism of the isochromosome 12p [i(12p)]. There were a wide spectrum of prenatal ultrasound findings of PKS, which made it difficult to be found in first or second trimester. Polyhydramnios, diaphragmatic hernia, and rhizomelic limb shortening were the most common prenatal ultrasound abnormalities in PKS. This study retrospectively analyzed the ultrasound findings and molecular cytogenetic results of four PKS fetuses diagnosed by using cord blood samples.ResultsThe ultrasound anomalies of four PKS fetuses are described as follows: fetal macrosomia, cerebral ventriculomegaly, increased NT thickness, rhizomelic limbs shortening, polyhydramnios. Biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL) measurements were above the mean in three fetuses,while one fetus showed rhizomelic limbs shortening. Combined with this study and previous literature, polyhydramnios was the most frequent anomaly observed in prenatal ultrasound examination of PKS, which accounted for 48% (94/194). Fetal macrosomia was present in 15% (29/194), cerebral ventriculomegaly in 13% (25/194), thickened nuchal fold in 9% (18/194), rhizomelic limbs shortening in 26% (51/194). I(12p) was found in the karyotype analysis of cultured cord blood lymphocytes and the mosaic ratios ranged from 2 to 5%. Single nucleotide polymorphisms array (SNP-array) results suggested that the whole short arm of chromosome 12 was duplicated with 2~3 copies. Fluorescence in situ hybridization (FISH) was performed to confirm the results of karyotype and SNP-array.ConclusionsIn case non-specific indicators such as fetal macrosomia, polyhydramnios and rhizomelic limbs shortening are observed meanwhile in prenatal ultrasound, targeted detection of PKS should be considered. In the prenatal diagnosis of PKS, the combination of SNP-array and FISH with conventional karyotype are the key to seek i(12p) and for precise diagnosis.

42. RETROSPECTIVE PGT-A ANALYSIS FOR MULTIPLE DISPLACEMENT AMPLIFICATION (MDA) PRODUCTS OF EMBRYOS CORRESPONDING TO 21 SPONTANEOUS ABORTIONS BY NGS

Introduction Preimplantation genetic testing for aneuploidy (PGT-A) is widely used to identify aneuploidy embryos, thereby reducing miscarriage rate and improving the clinical outcome of in vitro fertilization (IVF). But there are many reasons for miscarriage. Not all miscarriages are caused by embryonic aneuploidy. Moreover, PGT-A has its inherent defects, the results of PGT-A may not fully represent the true karyotype of embryos. Many problems still exist in this technology: 1. Biases caused by whole genome amplification (WGA) or high-throughput detection techniques; 2.Embryo mosaicism. Therefore, it remains unclear what percentage of spontaneous abortion can be prevented by PGT-A. Since 2015, our center has adopted multiple displacement amplification (MDA) as the first step in all PGT cycles. So a complete library of MDA products has been well-established. Material & methods We collected PGT cycles performed for monogenic diseases (PGT-M) at our center from January 2015 to December 2017. Cycles in which the blastocyst biopsy and single blastocyst transplantation were applied, and spontaneous miscarriage appeared during early pregnancy, and MDA products of the embryos were frozen were considered eligible. Exclusion criteria: cycles in which PGT-A had been conducted and the embryo was already selected according to the PGT-A results. All MDA products were performed PGT-A using next generation sequencing (NGS) by Illumina NextSeq ® 550 platform. The software determined the ploidy status with 0.01X read coverage. Results A total of 21 MDA products were detected, and 4 of them were unable to evaluate due to amplification failure or poor test data. The PGT-A results of the remaining 17 embryos showed that 47.1% (8/17) embryos were euploidy, 11.6% (2/17) embryos were segmental chromosomal aneuploidy (46,XN,-(1)(q23.3-q44)(85.03Mb) and 46,XN,+(13)(q11-q34)(95.75Mb)), 27.8% (5/18) of embryos were whole chromosomal aneuploidy mosaicism (the proportion of mosaicism range from 21 to 47%) and 2 cases were trisomy (trisomy 21 and trisomy 22). 6 patients performed karyotype analysis of abortion tissues, among which PGT-A results of 4 cases (3 cases were normal, one was trisomy 22) were consistent with the karyotype of abortion tissues. However, 2 cases showed aneuploidy mosaicism (46,XN,+(mosaic)(19)(30%) and 46,XN,-(mosaic)(6)(44%)) by PGT-A, while the karyotype of their abortion tissues were normal. Conclusions Our study showed that PGT-A could prevent a proportion of spontaneous abortion. However, embryos with low ratio of mosaicism may not be the cause of miscarriage. In fact, spontaneous abortion of these cases was caused by other factors. How high the proportion of mosaicism may lead to miscarriage still requires further research to confirm.

Determination of urinary podocin and podocalyxin levels by liquid chromatography–mass spectrometry

Noninvasive prenatal screening (NIPS) has higher sensitivity and specificity compared to traditional prenatal screening. Nevertheless, the discordant results between the NIPS and prenatal diagnosis were occasionally reported. In current study, we investigated the genetic basis of a T18 fetus with a discordant trisomy 5 (T5) positive and trisomy 18 (T18) negative NIPS result.NIPS was used to detect fetal DNA in maternal circulating plasma based on semiconductor sequencing platform. The aneuploidies of the fetus and different part of placental tissues were investigated by copy number variation sequencing (CNV-seq) and chromosome microarray analysis (CMA).The positive result of T5 was detected for the pregnant woman in NIPS, while T18 was found in the fetal karyotyping analysis after amniocentesis. Furthermore, placental mosaicism of T5 and T18 was found by CNV-seq and CMA, which revealed the mosaic ratio of T5 was gradually increased from umbilical cord to the placenta center, while that of T18 was gradually decreased.For the reason of cell-free fetal DNA (cff DNA) in the maternal circulation originates from trophoblast cells of placenta, the level of placental mosaicism could cause false negative NIPS result in multiple aneuploidies. The present study proved that a discordant T5 positive and T18 negative NIPS result was caused by placental mosaicism. This study highlights placental mosaicism as a significant risk factor for discordant NIPS results. The result will be helpful for genetic counseling and clinical management of such pregnant woman.

A gradual change of chromosome mosaicism from placenta to fetus leading to T18 false negative result by NIPS

BackgroundNoninvasive prenatal screening (NIPS) has higher sensitivity and specificity compared to traditional prenatal screening. Nevertheless, the discordant results between the NIPS and prenatal diagnosis were occasionally reported. In current study, we investigated the genetic basis of a T18 fetus with a discordant trisomy 5 (T5) positive and trisomy 18 (T18) negative NIPS result. MethodsNIPS was used to detect fetal DNA in maternal circulating plasma based on semiconductor sequencing platform. The aneuploidies of the fetus and different part of placental tissues were investigated by copy number variation sequencing (CNV-seq) and chromosome microarray analysis (CMA). ResultsThe positive result of T5 was detected for the pregnant woman in NIPS, while T18 was found in the fetal karyotyping analysis after amniocentesis. Furthermore, placental mosaicism of T5 and T18 was found by CNV-seq and CMA, which revealed the mosaic ratio of T5 was gradually increased from umbilical cord to the placenta center, while that of T18 was gradually decreased. ConclusionFor the reason of cell-free fetal DNA (cff DNA) in the maternal circulation originates from trophoblast cells of placenta, the level of placental mosaicism could cause false negative NIPS result in multiple aneuploidies. The present study proved that a discordant T5 positive and T18 negative NIPS result was caused by placental mosaicism. This study highlights placental mosaicism as a significant risk factor for discordant NIPS results. The result will be helpful for genetic counseling and clinical management of such pregnant woman.

Male factor & mosaicism: assessing the contribution of abnormal semen parameters to rate of mosaicism in next generation sequencing (NGS) preimplantation genetic screening (PGS) intracytoplasmic sperm injection (ICSI) cycles

Advances in preimplantation genetic screening (PGS) have led to practices changes in assisted reproductive technology (ART), enabling fertility centers to transfer single embryos while maintaining excellent ongoing pregnancy rates, reducing miscarriage rates, and dramatically reducing ART-associated multiple pregnancies [1,2]. The introduction of next generation sequencing (NGS) has allowed PGS laboratories to assess for embryo mosaicism – although the true incidence and reproductive potential of mosaic embryos is controversial. Severe male factor is known to increase risk of aneuploidy [3]. However, the influence of male factor infertility on mosaicism is unknown at this time. While aneuploidy is mostly due to maternal meiotic errors, mosaicism is likely caused by post-fertilization mitotic errors [4] and abnormal sperm may contribute to an embryo's risk of being mosaic. We sought to examine the influence of abnormal semen parameters on mosaicism in NGS PGS-tested embryos. The purpose of this study was to evaluate rate of mosaic embryo formation in NGS PGS cycles according to semen abnormality. Secondary aims were to assess sex ratio and subtypes of mosaicism in NGS PGS cycles according to semen abnormality. We performed a retrospective review of women undergoing PGS at a single academic fertility center from July 1, 2015 to September 1, 2017. Only intracytoplasmic sperm injection (ICSI) PGS cycles were included. In all cycles, PGS was performed via trophectoderm biopsy on day 5 or 6 and analyzed using NGS at a single reference lab. Patient demographics, fertility testing, cycle characteristics and PGS outcomes were collected. PGS outcomes were compared between oligospermic and non-oligospermic cohorts, between asthenospermic and non-asthenospermic cohorts, and between oligoasthenospermic and normospermic cohorts. 228 ART-ICSI cycles with PGS testing were included for analysis: 130 normospermic, 32 oligospermic, 47 asthenospermic, and 12 oligoasthenospermic cycles, resulting in 374 normospermic, 106 oligospermic, 162 asthenospermic, and 38 oligoasthenospermic biopsied embryos. Baseline characteristics were similar between cohorts with the exception of semen parameters (Tables 1-3). PGS cycles with oligospermia demonstrated a higher proportion of mosaic embryos (36/100, 34%) versus PGS cycles without oligospermia (115/617, 18.6%), p<0.001. PGS cycles with oligoasethenospermia demonstrated a higher proportion of mosaic embryos (15/38, 39.5% versus 88/384, 23.5%, p<0.001) compared to PGS cycles without oligoasthenospermia. Additional PGS outcomes were similar between cohorts (Tables 1-3). Oligoasthenospermia and oligospermia, but not asthenospermia, may increase the risk of mosaic embryos following ART-ICSI cycles with NGS PGS.