Chromosome Constitution Research Articles

Karyotype evaluation of repeated abortions in primary and secondary recurrent pregnancy loss.

To study the contribution of embryo chromosomal abnormalities in primary and secondary recurrent pregnancy loss (RPL) and to analyze the recurrence of chromosomal constitution in miscarriages from the same couple. Retrospective study of abortion karyotypes in RPL families based on the mother's primary or secondary RPL status (563 embryo specimens, 335 samples from primary, and 228 samples from secondary RPL). RPL was defined as two or more consecutive miscarriages. One hundred eight cases of recurrent embryo/fetal loss in 51 families were analyzed to assess the probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in both previous and subsequent pregnancy loss. The karyotypes of abortions were established using standard cytogenetic analysis, as well as interphase fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). The frequency of aberrations was 43.9% in abortions from primary RPL versus 52.6% in secondary RPL (p = 0.041). Women 35years of age or older were the main contributors to this difference. The odds ratio of a subsequent abortion having the same karyotype pattern (normal or abnormal) as the previous one was 6.98 (p = 0.0013). The frequency of abnormalities is higher in abortions from the secondary RPL versus primary RPL group, and this difference is due to the relative deficiency of miscarriages with abnormal karyotypes in older women with primary RPL. The probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in the previous and subsequent abortion is increased significantly compared with chance.

Assessment of risk factor associated with down syndrome

Introduction: A large number of Down syndrome (DS) children are born every year in India and are the leading cause of morbidity and mortality in infants. The aim of the present study was to evaluate the possible risk factors in mothers for having DS. Methodology: A total of 217 DS cases were collected, and lymphocyte culturing was performed to confirm aneuploidy. Mothers having DS children (n = 213) with confirmed trisomy 21 and age-matched controls (n = 220) with normal children were collected. Results: Of 217 cases, 213 had confirmed trisomy 21 in children, and free trisomy 21 was observed in 91.71%, followed by mosaics in 3.68% and Robertsonian translocations in 2.30% of the cases. A double aneuploidy with chromosomal constitution 48, XXY,+21 (0.46%) was also seen. The mean maternal age in cases was 27.34 ± 5.2 years, while in controls, it was 27.75 ± 4.9 years. Logistic regression analysis showed that intake of folic acid (P

Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report.

BackgroundCell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.ObjectiveTo derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.Materials and MethodsHuman ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.ResultsThe pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.ConclusionCryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.

Regulation of the Sox3 Gene in an X0/X0 Mammal without Sry, the Amami Spiny Rat, Tokudaia osimensis

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.

Clinical and Cytogenomic Features of Lymphoblastic Leukemia with Intrachromosomal Amplification of Chromosome 21 (iAMP21) in the Context of Constitutional Ring Chromosome 21

Constitutional ring chromosome 21, r(21)c, is a rare chromosome abnormality associated with variable clinical features that range from mild dysmorphism to severe congenital anomalies and intellectual disability. Recently, r(21)c has been reported to predispose to B-cell acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21), a distinct subgroup of high-risk pediatric B-ALL. Only a few iAMP21-B-ALL cases with r(21)c have been reported to date. The mechanism of leukemogenesis of r(21)c has not been entirely elucidated. Here we report an 11-year-old boy with iAMP21-B-ALL carrying an atypical r(21)c. The patient has a history of attention-deficit/hyperactivity disorder, sensorineural hearing loss s/p cochlear implant, intellectual disability and scoliosis. Three months before admission he developed a soft tissue nodule on the occipital scalp deemed to possibly represent an enlarged lymph node. Subsequently, he presented with spontaneous bruising followed by severe epistaxis. The initial CBC showed a white blood cell count of 4.3K/uL with circulating blasts, absolute neutropenia, profound normocytic normochromic anemia (hemoglobin of 5.2 g/dL), and marked thrombocytopenia (platelets, 12K/uL ). Peripheral blood flow cytometry showed 17.9% phenotypically abnormal B lymphoblasts which were negative for CD45, and positive for CD34, nuclear TdT, CD19, CD22, CD79a, CD10, HLA-DR , as well as CD20 (21% positive). The bone marrow aspirate contained 98% blasts. CNS status was 2a (RBC 0, WBC 0, 8% blasts) and clear after the second lumbar puncture. Fine-needle aspiration of the scalp mass demonstrated B-lymphoblastic leukemia/lymphoma. The patient was treated per COG protocol AALL1131 and was assigned to the very high-risk arm when bone marrow interphase FISH showed iAMP21. The chromosome analysis failed to yield metaphase cells on the diagnostic bone marrow sample. A concurrent genomic microarray showed chromothripsis with multiple non-contiguous losses and high copy gains on 21q involving the RUNX1 gene, as well as mosaic deletions within 7q22.3q36.3, 9p24.3p24.1 (including JAK2), 12q12 (several exons of ARID2), 13q14.2q21.2 (RB1, DLEU1/2/7, miR-15a/miR-16-1 cluster), 14q32.33 (IGH locus), 19p13.2 (several exons of the SMARCA4), and mosaic gains within 3q22.3q29 and Xp22.33p11.3. Day 29 end of induction bone marrow examination was positive for minimal residual disease (MRD) at 0.13% of nucleated mononuclear cells, but FISH was negative for iAMP21. On day 57 of consolidation, the bone marrow was negative for both MRD and iAMP21. However, chromosome analysis on both of these follow-up studies showed an abnormal chromosome 21. Chromosome analysis on peripheral blood lymphocytes confirmed the presence of a constitutional r(21). A subsequent genomic microarray analysis on peripheral blood sample did not show chromothripsis observed in the diagnostic bone marrow sample, but showed a 4.7 Mb terminal deletion and two interstitial deletions (3.0 Mb and 5.5 Mb) on the long arm of chromosome 21. These findings are consistent with a r(21) with interstitial deletions, which is likely responsible for the congenital anomalies reported in this patient. iAMP21 is associated with a poor outcome in B-ALL. Accurate detection of iAMP21 is critical for risk stratification and treatment in B-ALL. The strong association between iAMP21 and r(21)c has been proposed based on previous studies on r(21)c carriers with iAMP21-ALL. Our data further support an increased risk of developing iAMP21-ALL in carriers of constitutional r(21) and demonstrate the value of intensive treatment on iAMP21-B-ALL. The r(21) observed in this patient contains a relatively larger (4.7 Mb) terminal deletion along with two additional interstitial deletions. Due to the scarcity of r(21)c, the pathogenetic mechanisms of this leukemic process is not fully understood, and the clinical significance of loss of additional genetic content is unknown. More case reports are needed to generate more comprehensive clinical and genetic profile for this high risk ALL. Figure 1. Genomic microarray findings on diagnostic bone marrow sample (top) and the follow up peripheral blood sample (bottom). Chromothripsis and amplification were observed only in the diagnostic bone marrow specimen, whereas the peripheral blood sample in remission showed two interstitial and a terminal deletion. Figure 1 Disclosures No relevant conflicts of interest to declare.

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages.

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1-3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.

XIST-Promoter Demethylation as Tissue Biomarker for Testicular Germ Cell Tumors and Spermatogenesis Quality.

Background: The event of X chromosome inactivation induced by XIST, which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of XIST in a series of TGCT tissues, representative cell lines, and testicular parenchyma. Methods: Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated XIST promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses. Results: Seminomas showed a lower amount of methylated XIST fragments as compared to non-seminomas or normal testis (p < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated XIST as compared to non-seminomas. The percentage of demethylated XIST fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen’s score and XIST demethylation was identified (r = 0.75, p < 0.0001). Conclusions: The X chromosome inactivation event and demethylated XIST promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality.

Aneuploid Chromosome Constitutions of the Small Japanese Field Mouse, Apodemus argenteus, in the Western Mountainous Area of Kanagawa Prefecture, Honshu, Japan

Abstract. We studied an occurrence of chromosomal aneuploidy for ten individuals in the small Japanese field mouse, Apodemus argenteus, from the western mountainous area of Kanagawa Prefecture, Honshu, Japan. Eight of the individuals examined showed the standard 2n = 46 and FNa = 48 constitutions as the most frequent karyotype. However, of those eight individuals, seven carried an inter-cellular mosaicism with a few metaphases carrying a small-sized supernumerary element. In addition, one individual showed an aneuploid complement that included a small-sized bi-armed B chromosome (2n = 47). The B chromosome consisted of the C-heterochromatin and was characterized by delayed fluorescence by the quinacrine mustard staining, suggesting that its feature is identical to the B chromosomes previously confirmed in other localities. Moreover, an XO female showing a standard autosomal complement with only one X chromosome (2n = 45, XO) was also recognized. According to the present observations, these chromosomal aneuploidies are retained in wild populations of A. argenteus as constantly occurring phenomena.

Sexual Dimorphism in the Age of Genomics: How, When, Where.

In mammals, sex chromosomes start to program autosomal gene expression and epigenetic patterns very soon after fertilization. Yet whether the resulting sex differences are perpetuated throughout development and how they connect to the sex-specific expression patterns in adult tissues is not known. There is a dearth of information on the timing and continuity of sex biases during development. It is also unclear whether sex-specific selection operates during embryogenesis. On the other hand, there is mounting evidence that all adult tissues exhibit sex-specific expression patterns, some of which are independent of hormonal influence and due to intrinsic regulatory effects of the sex chromosome constitution. There are many diseases with origins during embryogenesis that also exhibit sex biases. Epigenetics has provided us with viable mechanisms to explain how the genome stores the memory of developmental events. We propose that some of these marks can be traced back to the sex chromosomes, which interact with the autosomes and establish sex-specific epigenetic features soon after fertilization. Sex-biased epigenetic marks that linger after reprograming may reveal themselves at the transcriptional level at later developmental stages and possibly, throughout the lifespan. Detailed molecular information on the ontogeny of sex biases would also elucidate the sex-specific selective pressures operating on embryos and how compensatory mechanisms evolved to resolve sexual conflict.

CHROMOSOMAL TRANSLOCATIONS IN MEN WITH AZOOSPERMIA

Male infertility is liable for half of the genetic infertility cases. Robertsonian and Reciprocal translocations are the major chromosomal rearrangements in the infertile population. In this study, we aimed to submit a Robertsonian and two Reciprocal translocations in three couples with a history of male infertility with azoospermia . Chromosomal analysis of the one couple in the male partner appeared with an abnormal karyotype with 45, XY, rob (13,14) chromosomal constitution, while the female partner revealed normal 46, XX karyotype. The other two couple revealed in the male partner with reciprocal translocations, while the female partners showed normal 46, XX karyotype; one of infertile males has karyotype with 46,XY, rcp (19:10), and other infertile male with 46,XY,rcp(6:14) chromosomal constitution. The cytogenetic analysis is mandatory to identify any probable chromosomal anomalies for couples with primer infertility. Couples with repeated abortions should be offered prenatal diagnosis in the case of future pregnancies. Chromosome translocation carriers should be counseled to use advanced technologies such as assisted reproductive technology such as PGD.

Activity and inactivity of moth sex chromosomes in somatic and meiotic cells

Moths and butterflies (Lepidoptera) are the most species-rich group of animals with female heterogamety, females mostly having a WZ, males a ZZ sex chromosome constitution. We studied chromatin conformation, activity, and inactivity of the sex chromosomes in the flour moth Ephestia kuehniella and the silkworm Bombyx mori, using immunostaining with anti-H3K9me2/3, anti-RNA polymerase II, and fluoro-uridine (FU) labelling of nascent transcripts, with conventional widefield fluorescence microscopy and 'spatial structured illumination microscopy' (3D-SIM). The Z chromosome is euchromatic in somatic cells and throughout meiosis. It is transcriptionally active in somatic cells and in the postpachaytene stage of meiosis. The W chromosome in contrast is heterochromatic in somatic cells as well as in meiotic cells at pachytene, but euchromatic and transcriptionally active like all other chromosomes at postpachytene. As the W chromosomes are apparently devoid of protein-coding genes, their transcripts must be non-coding. We found no indication of 'meiotic sex chromosome inactivation' (MSCI) in the two species.

7. IMPACT OF CHROMOSOMAL MOSAICISM IN IVF OUTCOMES: EXPERIENCE FROM TWO HUNDRED MOSAIC EMBRYOS TRANSFERRED PROSPECTIVELY

Introduction Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. In a recent published study, we have demonstrated that mosaic embryos hold the potential to implant and result in the birth of healthy babies. Therefore, the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF) resulting in only mosaic embryos but no euploid embryos for transfer. However, there is still few data concerning the impact of mosaicism on viability, and the classification of such embryos in relation with their reproductive potential is unclear. Here we investigated if chromosomal constitution of mosaic embryos influences the clinical outcome of in vitro fertilization treatments Material and methods The transfer of mosaic embryos was offered to 200 women for whom IVF had resulted in no euploid embryos between May 2016-May 2018. Trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow growing embryos. The clinical outcome obtained after transfer of mosaic embryos with different chromosomal constitution was compared with each other and with that obtained from a control group of 500 euploid blastocysts. Comprehensive chromosome testing PGT-A was performed using high resolution next generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis mosaic embryos were divided in four groups based on chromosomal constitution: mosaic monosomy (single and double monosomies; MM), mosaic trisomy (single and double trisomies, MT), mosaic complex aneuploidy (more than two different aneuploidies; MC) and mosaic segmental aneuploidy (single and double deletion or insertion >5Mb, MS). Results MM showed significant higher birth rate compared to MT (46% vs 24%,p=0.02), MC (46% vs 23%,p=0.03) and MS (46% vs 22%,p=0.02). No significant difference was observed in clinical outcome between the groups MT, MC and MS. A comparison of the clinical outcomes with control euploid group showed no significant difference between euploid control and MM, while a significant low implantation rate (55.4% vs 37% MT, vs 31% MC, vs 23% MS,p Conclusion The study demonstrated that embryos with MT, MS and MC aneuploidies have lower chances of resulting with birth of healthy babies compared to embryos with MM. More interestingly, no difference has been find in clinical results between MM and euploid control group. These findings should be considered for genetic counseling.

13. CORRELATION BETWEEN ANEUPLOIDY, MOSAICISM AND MORPHOKINETIC DEVELOPMENT IN 1550 BIOPSIED BLASTOCYSTS

Introduction Despite the high number of recent studies, conflicting data are reported, and there is still considerable disagreement regarding which morphokinetic parameters are useful to predict blastocyst formation, implantation potential and ploidy status of the embryo. The first attempt to develop a model predicting embryo implantation used the time of division to 5 cells, the time between division from 3 to 4 cells and the time between division from 2 to 3 cells (Meseguer et al., 2011). More recently, the aneuploidy status of embryos was related to the start of blastulation and the formation of a full blastocyst. The aneuploidy risk classification built proved beneficial in a correlation with live birth when applied to non-biopsied embryos (Campbell et al., 2013). Material and Methods This retrospective study was based on 1550 blastocysts which were subjected to preimplantation genetic testing for aneuploidy (PGT-A) between August 2011 and December 2018 in Istanbul Memorial Hospital (n=659 euploids; n=831 aneuploids; n=60 mosaics). Incubation was performed in time-lapse incubators (EmbryoScope™). All relevant events (fertilization, cleavages, morula and blastocyst formation) were checked daily, and time of cleavage to two-cell embryo (t2) and subsequent divisions t3, t4, t5, t6, t7, t8, t9+, tM, tSB, tB and tEB were recorded in the EmbryoViewer® workstation. PGT-A was done by NGS ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific) or aCGH (Illumina) following trophectoderm biopsy. Mann-Whitney U and Kruskal Wallis tests were used in this study. Results When euploid embryos were compared with aneuploids, the time to reach the blastocyst stage was 3 hours earlier in euploids (tB=104.60h vs. 107.64h; p Conclusions The mosaic embryos’ morphokinetic parameters were similar with the euploid blastocysts. Observed time-lapse differences between euploid and aneuploid blastocysts suggests that the embryos are being affected by the chromosomal constitution throughout the embryo development. Even though the variances at the initial cleavages are as short as half an hour, this increases to a three hour at the blastocyst stage. The study presented above can be used to prioritize the embryos according to the morphokinetic variables to biopsy and diagnose the embryos with highest chance of being euploid thus reducing the costs especially for couples who have more than one blastocyst.

9. ASSESSMENT OF ANEUPLOIDY AND MOSAICISM CONCORDANCE BETWEEN DIFFERENT TROPHECTODERM BIOPSY SITES AND THE INNER CELL MASS EVALUATED WITH NEXT-GENERATION SEQUENCING

Introduction It is recognized that the biopsy of 6-8 TE cells allows for high-resolution next-generation sequencing. However, concerns have been raised as to whether one biopsy sample can be representative of the whole blastocyst regarding aneuploidy and mosaicism. A recent study estimated the TE confined mosaicism rate at 14% and to be due to discordant chromosomal constitution of ICM and TE (Chuang et al., 2018). Another study showed that one clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but not for cases with only segmental aneuploidy (Victor et al., 2019). Material and Methods 101 clinical biopsies were taken from a site opposite to the ICM (TE1) and the second sample was taken from thawed blastocysts previously diagnosed as being aneuploid with the informed consent of the patients (TE2). Five aneuploid blastocysts were also biopsied from within the ICM. Both samples were analyzed by the same platform, ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific) next generation sequencing (NGS) to assess ploidy and mosaicism concordance. Blastocysts of good or top-quality belonging to 78 patients were subjected to TE biopsy for PGT for aneuploidy (PGT-A) with the clinical indications of advanced maternal age, repeated implantation failures or a previous history of abnormal fetal karyotype. Blastocysts were then vitrified as to our routine practice. Samples were analyzed by NGS and blastocysts diagnosed as being aneuploid were thawed, re-biopsied and re-analyzed by NGS. Results A total of 101 TE biopsies with whole chromosome aneuploidies were compared with 96 TE biopsy samples taken at another site of the blastocyst and 5 biopsies of the corresponding ICM. 76 samples out of 101 (75.2%) were found to be fully concordant. 25 diagnoses (24.7%) differ between TE1 and TE2: 13 mosaicisms and 10 segmental aneuploidies were either found in TE1 and not in TE2 or vice versa, without altering the overall aneuploid diagnosis. For four blastocysts, whole chromosome aneuploidies differ between TE1 and TE2, the first diagnosis indicating a chaotic aneuploidy and the second one a simple monosomy/ trisomy or vice versa. Regarding the five ICM samples, they were entirely concordant for full chromosome aneuploidies with their corresponding TE samples. However, in one sample, the 30% mosaicism of chromosomes 1, 13 and 16 was not shared by the ICM. Conclusions The high rate of concordance in two separate TE biopsies and ICM observed in this study validates the reliability of PGT-A by NGS. Blastocysts with segmental aneuploidies only may be re-evaluated if no euploids are found, as results suggest that segmental aneuploidies may not be shared among all TE cells. The study involved blastocysts initially diagnosed as being aneuploid, therefore the findings cannot be extrapolated to euploid or euploid-mosaic embryos.

Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications

To study whether embryonic cell-free DNA (cfDNA) in spent blastocyst media is representative of the chromosomal constitution of a blastocyst. Pilot prospective blinded study. Invitro fertilization center and genetics laboratory. A total of 115 trophectoderm (TE) biopsies and spent blastocyst media (SBM) from 46 patients with ages ranging from 32 to 46years, whose indications for preimplantation genetic testing of aneuploidy (PGT-A) were advanced maternal age, recurrent miscarriage, or recurrent implantation failure. Spent blastocyst media collection and TE biopsy. Concordance rates, sensitivity, and specificity between TE biopsies and SBM. Clinical outcomes in cases with euploid TE biopsies and euploid SBM compared with cases with euploid TE and aneuploid SBM. In general, the total concordance rate for ploidy and sex was 78.7%, and sensitivity and specificity were 94.5% and 71.7%, respectively. A significant increase for all parameters was observed for day 6/7 samples compared with day 5 samples, with day 6/7 samples showing total concordance for ploidy and sex of 84%, and sensitivity and specificity of 95.2% and 82.1%, respectively. Ongoing implantation rates in euploid TE/euploid SBM showed a threefold increase compared with euploid TE/aneuploid SBM (52.9% vs. 16.7%, respectively), without reaching significant differences. Interestingly, no miscarriages were observed when TE and SBM were euploidy concordant. These results offer a better understanding of the dynamics of cfDNA during embryo development and despite more basic research being needed, they are reassuring to consider in the future this noninvasive approach as an alternative to TE biopsy for PGT-A.

An Inherited Balanced Translocation Between Chromosomes 4 and 6 in Recurrent Pregnancy Loss: A Case Report

Recurrent pregnancy loss is an important reproductive issue with a heterogeneous etiology where two or more consecutive abortions occur before 20 weeks of gestation. Approximately 15% of all clinically recognized pregnancies result in miscarriage with an incidence of 1 in 300 cases. Couples, who experience repeated pregnancy loss before three months of gestation, mostly have fetuses with chromosomal aneuploids. A non-consanguineous couple with a married life of 4 years was referred to the Institute with a clinical history of three first trimester abortions. Karyotype analysis revealed a balanced autosomal translocation between chromosomes 4 and 6 with 46, XX, t (4;6)(q35; q22) karyotype in the female and normal 46, XY in the male partner. Therefore, the siblings and the couple were suggested for extended genetic counseling. Interestingly, similar translocation was seen in her father and three sisters, whereas her mother and elder sister showed a normal chromosomal constitution, indicating the paternal inheritance.

Assessment of the association between ACYP2 and laryngeal squamous cell carcinoma risk in Chinese males.

BackgroundLaryngeal squamous cell carcinoma (LSCC) is one of the most prevalent malignant neoplasms of the upper respiratory tract. Studies have confirmed that an unstable chromosome constitution promotes the progress of laryngeal tumorigenesis, and ACYP2 has been confirmed as a telomere length‐related gene. However, to date, the association between ACYP2 polymorphisms and LSCC susceptibility has not been investigated.MethodsWe performed this study to explore the effect of 11 single‐nucleotide polymorphisms (SNPs) in ACYP2 on LSCC susceptibility in Chinese Han males. Unconditional logistic regression analysis adjusted for age was used to calculate the odds ratios and 95% confidence intervals.ResultsBased on allele and genotype models, our results showed that rs1682111 variant was significantly associated with a decreased LSCC susceptibility (p < 0.05). On the contrary, polymorphisms of rs10439478, rs11125529, rs12615793, rs843711, rs11896604, and rs17045754 were significantly associated with an increased LSCC risk (p < 0.05). The results of haplotype analysis indicated that haplotypes “TTCTCG” and “TTCTAA” in block 1 and “TG” in block 2 showed a risk factor for the development of LCSS (p = 0.009, p < 0.001, and p = 0.001, respectively). The results of Genotype‐Tissue Expression analysis indicate that these significant SNPs were known to be associated with ACYP2 expression.ConclusionOur data demonstrated that ACYP2 polymorphisms may exert effects on LSCC susceptibility in Chinese Han males.

Cytological variations and long terminal repeat (LTR) retrotransposon diversities among diploids and B-chromosome aneuploids in Lilium amabile Palibin.

B chromosomes are supernumerary chromosomes found in numerous plant species, including in the genus Lilium. Lilium amabile, an endemic Korean Lilium species, carries B chromosomes which are highly variable in terms of numbers and shape among the accessions collected throughout the Korea. Class 1 retrotransposons are highly abundant in the genome of Lilium species, but their biological functions are still obscure. Lilium species were known to hold high diversities derived from retrotransposons. In this study, genetic diversities among the L. amabile accessions were analyzed to better understand relationships between genetic variations and cytological diversities. Chromosomes were prepared from 95 L. amabile accessions for cytological identification. Genetic variations were analyzed by inter-retrotransposon amplified polymorphism (IRAP), and genetic differentiation was evaluated via Tajima's D neutrality and FST analyses. Population structure and phylogenetic analyses were also carried out. The L. amabile accessions were classified into 11 cytotypes by the chromosome constitutions. Genetic diversity measured by IRAP analysis revealed high genetic diversity among the accessions. In the joint analysis of cytological variation with genetical variation, IRAP diversity was not related to the cytological diversities of diploid and aneuploids among L. amabile accessions, and genetic differentiation was not obvious. Moreover, the geographical distribution of L. amabile was not related to either IRAP diversity or cytological diversity. The B chromosome-carrying aneuploids occurred randomly among diploids throughout Korea, and IRAP diversification predated L. amabile dispersion in Korea without genetic differentiation.

Chromosome Arm Locations of Barley Sucrose Transporter Gene in Transgenic Winter Wheat Lines.

Three transgenic HOSUT lines of winter wheat, HOSUT12, HOSUT20, and HOSUT24, each harbor a single copy of the cDNA for the barley sucrose transporter gene HvSUT1 (SUT), which was fused to the barley endosperm-specific Hordein B1 promoter (HO; the HOSUT transgene). Previously, flow cytometry combined with PCR analysis demonstrated that the HOSUT transgene had been integrated into different wheat chromosomes: 7A, 5D, and 4A in HOSUT12, HOSUT20, and HOSUT24, respectively. In order to confirm the chromosomal location of the HOSUT transgene by a cytological approach using wheat aneuploid stocks, we crossed corresponding nullisomic-tetrasomic lines with the three HOSUT lines, namely nullisomic 7A-tetrasomic 7B with HOSUT12, nullisomic 5D-tetrasomic 5B with HOSUT20, and nullisomic 4A-tetrasomic 4B with HOSUT24. We examined the resulting chromosomal constitutions and the presence of the HOSUT transgene in the F2 progeny by means of chromosome banding and PCR. The chromosome banding patterns of the critical chromosomes in the original HOSUT lines showed no difference from those of the corresponding wild type chromosomes. The presence or absence of the critical chromosomes completely corresponded to the presence or absence of the HOSUT transgene in the F2 plants. Investigating telocentric chromosomes occurred in the F2 progeny, which were derived from the respective critical HOSUT chromosomes, we found that the HOSUT transgene was individually integrated on the long arms of chromosomes 4A, 7A, and 5D in the three HOSUT lines. Thus, in this study we verified the chromosomal locations of the transgene, which had previously been determined by flow cytometry, and moreover revealed the chromosome-arm locations of the HOSUT transgene in the HOSUT lines.

Evaluation of chromosomal constitution in choronic villi samples of aborted fetuses after embryo transfers of euploid blastocysts

Introduction Preimplantation genetic testing of aneuploidies (PGT-A) using Next Generation Sequencing (NGS) platforms decreases significantly the risk of abortion from aneuploidy reason, nevertheless some fetuses are unfortunately aborted despite PGT-A from another sakes. NGS platforms provide high dynamic range and are able to detect copy number variations starting at 20% mosaicism. Material of trophectoderm biopsy includes several trophectoderm cells and it is quite often discussed whether such sample is representative enough to reflect the inner cell mass chromosomal status. The aim of this retrospective study is to reveal a representativeness and a significance of a trophectoderm biopsy sample evaluation with respect to the chromosomal constitution during the further embryonic development and assignments of concordance between trophectoderm biopsy day 5-6 and the finding in chorionic villi of aborted fetuses. Material and methods 59 chorionic villi samples were obtained from aborted fetuses during the surgical curettage for incomplete spontaneous or missed early abortions during a period January 2016 till March 2018. All samples were cleaned, washed and stored in a phosphate buffer and after DNA isolation the chromosome copy number was evaluated using 24sure array (Illumina). 42 samples were obtained from fetuses conceived after embryo transfers, 10 embryos were transferred without PGT-A and 32 were analyzed using PGT-A (VeriSeq™ PGS Kit by Illumina) before an embryo transfer. In all study cases a single embryo transfer was performed. All 32 PGT-A embryos were originally diagnosed as euploid. 17 samples were derived from spontaneously conceived embryos. The samples were divided into 3 groups. Group A consists of 10 non-PGT-A samples, group B of 17 samples from spontaneously conceived fetuses and group C of 32 samples from fetuses after PGT-A. Results In total, 23,7 % (14/59) chorionic villi samples were diagnosed as aneuploid. In the group A (non-PGT-A fetuses) 50% (5/10) were aneuploid, in the group B (spontaneous conceptions) 47% (8/17) samples were aneuploid while in the group C (PGT-A fetuses) only 3% (1/32) were aneuploid. Euploid status was concordant in 31 out of 32 PGT-A analyzed embryos (97%). Conclusions Our retrospective study showed that 50% of aborted fetuses after IVF with no PGT-A were aneuploid. This corresponds very well with the aneuploidy rate observed for products of spontaneous conception in infertile couples (47%). Chorionic villi samples from aborted fetuses derived from previously negatively tested blastocysts were aneuploid in 3% of cases (1/32). The concordance between an euploid status of trophectoderm samples and chorionic villi tissue from aborted fetuses is 97%. This confirms the figures referred previously in another studies comparing evaluation of trophectoderm and inner cell mass of the embryo and fully supports routine implementation of PGT-A into assisted reproductive techniques. The slight discordance is possible to be explained by unequal distribution of aneuploid/mosaic cell lines within the trophectoderm tissue at the blastocyst stage when the biopsy is performed.