Chromosome Constitution Research Articles

The past, present, and future for constitutional ring chromosomes: A report of the international consortium for human ring chromosomes.

SummaryHuman ring chromosomes (RCs) are rare diseases with an estimated newborn incidence of 1/50,000 and an annual occurrence of 2,800 patients globally. Over the past 60 years, banding cytogenetics, fluorescence in situ hybridization (FISH), chromosome microarray analysis (CMA), and whole-genome sequencing (WGS) has been used to detect an RC and further characterize its genomic alterations. Ring syndrome featuring sever growth retardation and variable intellectual disability has been considered as general clinical presentations for all RCs due to the cellular losses from the dynamic mosaicism of RC instability through mitosis. Cytogenomic heterogeneity ranging from simple complete RCs to complex rearranged RCs and variable RC intolerance with different relative frequencies have been observed. Clinical heterogeneity, including chromosome-specific deletion and duplication syndromes, gene-related organ and tissue defects, cancer predisposition to different types of tumors, and reproductive failure, has been reported in the literature. However, the patients with RCs reported in the literature accounted for less than 1% of its occurrence. Current diagnostic practice lacks laboratory standards for analyzing cellular behavior and genomic imbalances of RCs to evaluate the compound effects on patients. Under-representation of clinical cases and lack of comprehensive diagnostic analysis make it a challenge for evidence-based interpretation of clinico-cytogenomic correlations and recommendation of follow-up clinical management. Given recent advancements in genomic technologies and organized efforts by international collaborations and patient advocacy organizations, the prospective of standardized cytogenomic diagnosis and evidence-based clinical management for all patients with RCs could be achieved at an unprecedented global scale.

Precise Identification of Chromosome Constitution and Rearrangements in Wheat-Thinopyrum intermedium Derivatives by ND-FISH and Oligo-FISH Painting.

Thinopyrum intermedium possesses many desirable agronomic traits that make it a valuable genetic source for wheat improvement. The precise identification of individual chromosomes of allohexaploid Th. intermedium is a challenge due to its three sub-genomic constitutions with complex evolutionary ancestries. The non-denaturing fluorescent in situ hybridization (ND-FISH) using tandem-repeat oligos, including Oligo-B11 and Oligo-pDb12H, effectively distinguished the St, J and JS genomes, while Oligo-FISH painting, based on seven oligonucleotide pools derived from collinear regions between barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), was able to identify each linkage group of the Th. intermedium chromosomes. We subsequently established the first karyotype of Th. intermedium with individual chromosome recognition using sequential ND-FISH and Oligo-FISH painting. The chromosome constitutions of 14 wheat–Th. intermedium partial amphiploids and addition lines were characterized. Distinct intergenomic chromosome rearrangements were revealed among Th. intermedium chromosomes in these amphiploids and addition lines. The precisely defined karyotypes of these wheat–Th. intermedium derived lines may be helpful for further study on chromosome evolution, chromatin introgression and wheat breeding programs.

Gonadal tumor risk in pediatric and adolescent phenotypic females with disorders of sex development and Y chromosomal constitution with different genetic etiologies.

ObjectivesThis retrospective study sought to investigate the risk and proportion of gonadal neoplasms in phenotypic female pediatric patients with DSD and the presence of the Y chromosome and different genetic backgrounds in a single Chinese center.Materials and MethodsFrom January 2012 to December 2020, pediatric and adolescent patients with DSD and the presence of the Y chromosome who had unambiguous female genitalia and underwent bilateral gonadectomy or gonadal biopsy were included in this study. Patients’ demographics, karyotype, laboratory test results, gross pathology, and histology of gonadal tissue were all collected. The patients were divided into three groups based on their different genetic backgrounds, and the percentage of gonadal tumors was calculated to assess the risk of gonadal tumor and malignancy by etiology.ResultsA total of 22 patients with DSD and an unambiguous female phenotype with a Y chromosome were recruited. The mean age was 10.91 ± 4.99 years (9 months to 19 years). Gonadal neoplasia was confirmed in six (27.3%) cases by pathological examination of surgical gonadal tissue samples. Among 44 gonadal samples from these 22 patients, the following were identified: five gonadoblastomas, three dysgerminomas, and two Leydig cell tumors. The youngest patient with a tumor was a 2-year-old girl with 46,XY complete gonadal dysgenesis (46,XY CGD or Swyer syndrome) and bilateral gonadoblastoma. Patients with 46,XY complete gonadal dysgenesis (4/6; 66.7%) had the highest tumor occurrence rate. Among 10 patients with Turner syndrome with the presence of the Y chromosome, only one patient was diagnosed with a gonadal tumor. Leydig cell tumor was diagnosed in only one of six patients with 46,XY androgen synthesis/action disorders.ConclusionPediatric patients with 46,XY complete gonadal dysgenesis had a significantly increased risk of developing gonadal tumors and underwent prophylactic gonadectomy as soon as the diagnosis was confirmed, whereas those with Turner syndrome with Y chromosome and 46,XY androgen synthesis/action disorders had a relatively low risk. In view of the limited number of patients, a large multicenter study with close follow-ups is needed to support these conclusions.

P-551 High concordance of the embryonic cell-free DNA with the inner cell mass: impact of blastocyst quality, patient age and mode of fertilization

Abstract Study question Does the embryonic cell-free DNA (cfDNA) in the culture medium represent the chromosomal content of the inner cell mass (ICM)? Which factors impact concordance rates? Summary answer There is high ploidy concordance between ICM biopsies and embryonic cfDNA. This value is independent of female age, insemination technique and embryo quality. What is known already The existence of embryonic cfDNA in spent blastocyst medium (SBM) has been confirmed in recent studies, opening a new era of possibilities for non-invasive preimplantation genetic testing for aneuploidy (niPGT-A). High concordance rates of cfDNA with trophectoderm (TE) biopsies and with whole blastocysts have been reported. However, the compartment(s) from where this DNA originates remain unclear. Both TE and ICM are potential sources, but, at the moment, the origin of this cfDNA is unknown as well as the mechanisms underlying its secretion into the medium. Study design, size, duration We carried out a prospective study to investigate the concordance of cfDNA with the corresponding TE and ICM biopsies. 141 day-6/7 blastocysts were donated for research after written informed consent signature for the project approved by the Ethics Committee. Embryos underwent TE biopsy and SBM collection in the same PGT-A cycle. ICM biopsy in thawed blastocysts was performed after TE biopsy diagnosis. cfDNA, TE and ICM biopsies were analyzed from January 2019 to November 2021. Participants/materials, setting, methods Embryos were cultured in routine conditions up to day 4. They were then washed and transferred to a new 10μl culture medium droplet. On day 6, SBM was collected and frozen at -20 °C; and blastocyst biopsy and vitrification were performed. Subsequently, blastocysts were thawed and ICM biopsy was conducted. All samples were analyzed by NGS (Ion ReproSeq PGS kit, ThermoFisher Scientific) and the results were analyzed with customized algorithms for TE, ICM and cfDNA. Main results and the role of chance In combination, the three sample types (cfDNA, ICM and TE) were informative in 81.6% of the blastocysts (115/141). Considering the ICM as the reference, ploidy concordance (i.e. being both euploid or aneuploid) for cfDNA was 86.1% (99/115) and for TE was 89.6% (103/115), without statistical difference. False positive rates were similar for cfDNA and for TE biopsies (6.1% and 9.6%, respectively), and false negative rates were not significantly different, but higher in cfDNA (7.8%) than in TE (0.9%), due to potential contamination with maternal DNA. Ploidy concordance between embryo cfDNA and TE biopsies was 89.6% (103/115). When the results were stratified by female age (≤37 or > 37 years), insemination technique (ICSI or IVF), blastocyst expansion degree (expanded, hatching or fully hatched), and ICM/TE quality (A or B), the informativity of the cfDNA was very similar between the different groups and ranged from 83.7% to 100%. Nevertheless, there were subtle differences for ICM-cfDNA ploidy concordance. It was slightly increased for the older female age group (88.3% vs 83.6% female age ≤37) as well as for ICSI (89.7% vs 82.5% in IVF) and for ICM quality B (88.4% vs 80.0% for ICM A). None of those differences reached statistical significance. Limitations, reasons for caution When stratifying according to the different criteria, the sample size analyzed was too small to draw strong conclusions. Therefore, more studies, with bigger sample size, are needed to replicate the results. Wider implications of the findings The embryonic cfDNA released to the culture medium provides information of the overall blastocyst chromosomal constitution, as suggested by the high ploidy concordance rates reported between ICM and cfDNA. This supports the use of niPGT-A as an alternative to other invasive aneuploidy detection methods that require a biopsy. Trial registration number ClinicalTrials.gov. ID NCT03520933

P-396 Is biochemical pregnancy loss (BPL) associated with embryo or endometrium? A multicentre retrospective study with +7000 cases

Abstract Study question Is biochemical pregnancy loss rate (BPLR) associated with alterations within the embryo or the endometrium? Summary answer The BPLR was similar in frozen embryo transfers among groups: euploid embryo (PGT-A group), adjusted by endometrial receptivity assay (ERA group), ERA+ PGT-A group, and controls. What is known already The etiology of BPL is not well known, but the most likely causes hypothesized seem to be either a low-quality embryo or impaired endometrial receptivity. Different studies showed that factors such as age, ovarian stimulation, the oocyte and embryo quality and endometrium receptivity may have something to do with the occurrence of biochemical pregnancy loss post-assisted reproduction treatments. However, other studies have shown that BPLR does not increase as a function of age or seems independent of embryo’s development stage or embryonic chromosomal constitution. No studies have evaluated nor controlled specifically the uterine factor with tools as ERA. Study design, size, duration Retrospective cohort study between January 2013 and January 2021, on patients undergoing 7491 frozen embryo transfers, comparing BPLR per pregnancy among groups of patients with optimized embryo and endometrial quality (ERA+ PGT-A, n = 169), optimized embryo quality (PGT-A, n = 2670), optimized endometrium quality (ERA=61), and controls (neither ERA nor PGT-A, n = 4591) from own oocytes, from two University-affiliated centers. We defined BPL as a pregnancy diagnosed only by serum beta-hCG>10UI/L without resulting in a clinical pregnancy. Participants/materials, setting, methods BPLR and other variables including age, body mass index, number of previous IVF, days of endometrial preparation, type of cycle (natural or with hormonal replacement), endometrial thickness, fresh sperm concentration and fresh sperm volume were evaluated among groups using chi-squared test, for univariate analysis. Main results and the role of chance Mean women’s age was 35.95 years 95%CI (35.86 ,36.04), BMI 22.52kg/m2 95%CI (22.45, 22.59), endometrial preparation length 17.96 days 95%CI (17.78, 18.14), 29.8% of them on natural cycle, and 70.25% with hormonal therapy replacement. Donor sperm was used on 7.8% of the treatments, and embryo quality was comparable between groups of study, with an overall A rating of the embryos 24.9%, B 50.95%, C 23.37% based on Spanish ASEBIR society criteria. Mean sperm characteristics were: volume 2.79 ml 95%CI (2.74-2.85), concentration 38.78 mill/ml 95%CI (33.77-39.39), on the raw ejaculate, and after preparation, concentration varied to 7.80mill/ml 95%CI(6.76-7.39) On the univariate analysis, overall BPLR was 8.06% 95%CI (7.45-8.70). Control group presented 7.65% 95%CI (6.89-8.46), with ERA group (optimized endometrial factor) showing 8.33% 95%CI (2.76-18.39), PGT-A group (optimized embryonic factor) 8.82% 95%CI (7.77-8.96), and PGT-A+ERA 7.14% 95%CI (3.75-12.14) without statistically significant differences. Limitations, reasons for caution As this was a retrospective cohort study, we are exposed to several biases that have been tried to control by means of inclusion/exclusion criteria and proper statistical adjustment. Subsequently, results should be interpreted with caution, and a clear cause-effect relationship cannot be assumed. Wider implications of the findings Our results indicate that with PGT-A, ERA test or both it is not possible to modify the BPLR. A different mechanism no associated with chromosomal structure or transcriptomic expression of the endometrium could be studied as cause of BPL. More studies are needed for explore this reproductive issue. Trial registration number not applicable

P-537 PRIMARY SEX RATIO IS DECREASED IN EUPLOID EMBRYOS OF CONSANGUINE COUPLES AFTER IVF/ICSI WITH PGT-A

Abstract Study question What is the primary sex ratio outcome of embryos from consanguine couples as per PGT-A analysis during IVF/ICSI treatments? Summary answer The primary sex ratio (PSR) (males-to-females) is decreased in euploid blastocysts from consanguine couples. What is known already In the general population, the sex ratio (males-to-females) tends to be 1:1 (approximately 101 males to 100 females). In epidemiological studies secondary sex-ratio (ratio at the time of birth) of inbred-offspring seems to be equilibrated. However, it is well known that there is an increased incidence of autosomal recessive diseases for the offspring, compared to non-consanguineous couples. Studies have found elevated autosomal inheritances as compared to sex-linked inheritance. Assessing the impact of consanguinity on developed embryos might contribute to the understanding of the events leading to genetic diseases. Study design, size, duration This analysis includes data from 5135 blastocysts after preimplantation genetic testing for aneuploidy (PGT-A) with NGS. Embryos were obtained from 1836 IVF/ICSI cycles of infertile couples, at ART Fertility Clinics UAE, from November 2016 to December 2020. Consanguinity was defined when couple were first-degree or second-degree cousins. Participants/materials, setting, methods A total of 1138 blastocysts from consanguine couples, and 3997 from non-consanguine couples were included in the analysis. All blastocysts presented normal sexual chromosome constitution with or without autosomal aneuploidies. Mosaic and non-informative embryos were excluded. Trophectoderm biopsy was performed on day 5 for PGT-A using Next Generation Sequencing (NGS) platform. Primary sex ratio (PSR) was observed for CG and NCG couples. Ethical approval was obtained from the Research Ethics Committee (REFA023b). Main results and the role of chance In consanguine couples the age of female and male partner was 30.7±5.5 and 35.9 ±5.3 years old, respectively; while non consanguine couples were older (32.2±5.8 and 37.6±7.3 years old, respectively) (p < 0.001). Expanded blastocysts deriving from consanguine couples had 52.3% of XX versus 47.7% of XY constitution of their trophectoderm biopsied cells, presenting thus a significant decrease in primary sex ratio (PSR: 0.91, p = 0.03). In non-consanguine couples, about 51.2% of trophectoderm biopsied cells had a XX constitution compared to 48.8% of XY constitution, presenting a PSR of 0.95 (NS). The significant decreased PSR in consanguine couples was only related to normal euploid embryos and not to abnormal embryos (PSR of abnormal embryos = 0.98; NS). Euploid embryos from couples presenting consanguinity generated 53.4% of blastocysts of XX constitution versus 46.6% of XY constitution with a PSR at 0.87 (p = 0.01). Euploid embryos from non-consanguine couples presented a PSR at 0.96 (p=NS) (50.9% blastocysts of XX constitution versus 49.1% with XY constitution). Limitations, reasons for caution Differences in PSR between consanguine and non-consanguine couples did not reach significance level, justified by the lower number of embryos derived from consanguinity compared to non-consanguinity couples. Moreover 85% of the population included derives from Arabian Peninsula, therefore these results should not be transposed to other consanguine populations. Wider implications of the findings It is known that consanguine couples derive embryos with increased abnormalities in comparison to the general population. The fact that euploid embryos from consanguine couples seems to present a higher female constitution might incite us to investigate to what extent does heterozygosity for x-linked loci contribute to embryo survival. Trial registration number not applicable

P-189 Association between iDAScore v1.0, senior embryologists’ grading and euploidy in 546 blastocysts obtained during 189 PGT-A cycles

Abstract Study question Is (intelligent data analysis) iDAScore v1.0 associated with euploidy at the blastocyst stage? Summary answer iDAScore v1.0 significantly correlated with euploidy (maternal age-adjusted OR:1.3 and AUC:0.72). Euploid blastocysts were ranked highest in ca.70% of the cohorts with both diagnoses. What is known already With machine learning and artificial intelligence (AI) implementation in IVF, several studies have been published mostly aimed at providing standardized and reproducible tools for gamete/embryo assessment and selection. Several of the proposed models might not be generally applicable due to their development on only a single center, small sample size and poor representation of the numerous clinical scenarios. Furthermore, the evidence has been rarely confirmed prospectively and/or in multicenter studies. Lately, the EmbryoScope+ has incorporated the iDAScore v1.0. This algorithm scores the chance of embryo implantation based on the video of blastocyst development and with no need for timing annotations. Study design, size, duration Interim analysis of a prospective study. Between April-December 2021, 189 preimplantation-genetic-testing (PGT) cycles (maternal age:38.4±4yr) with ≥1 blastocyst (N = 546 blastocysts, mean±SD:2.9±1.8, range:1-13) were included. We aimed at blindly analyzing the correlation between iDAScore v1.0 and (i) blastocyst quality estimated by senior embryologists, (ii) day of blastocyst full-expansion, (iii) chromosomal constitution diagnosed by NGS on a trophectoderm biopsy, (iv) the blastocyst to prioritize for transfer within cohorts with ≥2 blastocysts. Participants/materials, setting, methods Undisturbed culture was conducted in the EmbryoScope+. Assisted hatching was not performed and only fully-expanded blastocysts were biopsied. Morphology was assessed by 2 senior embryologists based on Gardner criteria. Average iDAScores were reported for the following groups: (i)excellent (AA)/good (AB,BA)/average (BB,AC,CA)/poor-quality (CC,BC,CB) blastocysts, (ii)day5/6/7 blastocysts, (iii)euploid/aneuploid/complex aneuploid blastocysts. Lastly, we reported how often the highest iDAScore corresponded to the highest ranked morphology (N = 143 cycles with ≥2 blastocysts) and/or euploid blastocysts (N = 79 cycles with both diagnoses). Main results and the role of chance In the study period, 546 blastocysts (iDAScore: 6.9±2.0, 2-9.7) were biopsied. The iDAScore was significantly different (Kruskal-Wallis<0.01) across blastocysts graded excellent (N = 256,46.9%; 8.1±1.3, 2.5-9.7), good (N = 97,17.7%; 6.9±1.6, 2.3-9.5), average (N = 75,13.9%; 5.8±1.4, 2.9-8.7) and poor (N = 118,21.5%; 4.8±1.6, 2-8.8). A significant difference (Kruskal-Wallis<0.01) was also found for the day of full-expansion (day5: N = 184,33.9%, 8.8±0.8, 4.3-9.7; day6: N = 324,59.1%, 6.0±1.6, 2.2-9.1; day7: N = 38,6.9%, 4.6±1.6, 2-7.8). Euploid blastocysts (N = 178,32.6%) had a significantly higher (Kruskal-Wallis<0.01) iDAScore (7.5±1.7, 2.4-9.6) than both simple (N = 209,38.3%, 6.7±2.1, 2.1-9.7) and complex aneuploid blastocysts (N = 159,29.1%, 6.3±2.0, 2-9.4). The logistic regression adjusted for maternal age highlighted a multivariate-OR 1.3, 95%CI 1.18-1.45, adjusted-p<0.01 for the association between iDAScore v1.0 and euploidy. The Receiver-Operating-Characteristic (ROC) curves outlined similar performance in predicting euploidy among the models encompassing iDAScore v1.0 adjusted for maternal age (AUC: 0.72, 95%CI 0.67-0.76, p < 0.01) or blastocyst quality (defined by senior embryologists) plus day of biopsy also adjusted for maternal age (AUC: 0.73, 95%CI 0.69-0.78, p < 0.01). iDAScore v1.0 and embryologists ranked the same blastocyst highest in 123 of 143 cycles with ≥2 blastocysts (86%). The highest ranked blastocyst according to iDAScore was a euploid blastocyst in 54 of the 79 cycles (68%) containing both euploid and aneuploid blastocysts. Limitations, reasons for caution The main purpose of iDAScore v1.0, for which the algorithm was trained, is implantation prediction of untested blastocysts. Thus, once the sample size of this blinded prospective study will be large enough, we will also examine the association between iDAScore v1.0 and the implantation of euploid blastocysts. Wider implications of the findings The similar predictivity on euploidy reported between subjective senior embryologists’ grading and objective AI-powered iDAscores is promising in view of IVF automation and standardization. This is especially relevant since iDAScore v1.0 has not been trained yet to specifically predict euploidy, and its future versions could be fine-tuned accordingly. Trial registration number Not Applicable

Chromosomal mosaicism in human blastocysts: a cytogenetic comparison of trophectoderm and inner cell mass after next-generation sequencing

Research questionWhat is the incidence of chromosomal mosaicism in human blastocysts and can a single trophectoderm (TE) biopsy accurately predict the chromosomal constitution of the inner cell mass (ICM)? DesignObservational study in 46 surplus cryopreserved preimplantation embryos of unknown chromosomal constitution. For each embryo, a TE biopsy was performed and the ICM was collected separately. Both samples underwent next-generation sequencing (NGS) for cytogenetic analysis and were classified as chromosomally normal, abnormal or mosaic. Mosaic samples were classified as low or high mosaic, based on the majority dominance of either normal or abnormal cells in the biopsied sample. Findings within each embryo were compared. ResultsChromosomal mosaicism was detected in 59% (n = 27/46) of the embryos, with a cytogenetic concordance rate between TE and corresponding ICM of 48% (n = 22/46). Concordance was higher from a clinical perspective: in 86% of embryos with a high-mosaic or abnormal TE, the ICM was also high-mosaic or abnormal. In 88% of the blastocysts with a normal or low-mosaic TE biopsy, a normal or low-mosaic ICM was observed. ConclusionDespite the low cytogenetic concordance rate due to chromosomal mosaicism present in blastocysts, it was found that a single TE biopsy could correctly predict whether the ICM consists of mostly normal or abnormal cells in the majority of cases.

B‐cell acute lymphoblastic leukemia with iAMP21 in a patient with Down syndrome due to a constitutional isodicentric chromosome 21

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is associated with various specific cytogenetic and molecular markers that have significant influence on treatment and prognosis. A subset of children has a much higher risk of developing B-ALL due to constitutional genetic alterations such as trisomy 21 (Down's syndrome). In these patients, B-ALL is often associated with specific genomic profiles leading to leukemic transformation. In rare cases, constitutional structural chromosomal abnormalities involving chromosome 21, such as the der(15;21) Robertsonian translocation and a ring 21 chromosome, have been associated with intrachromosomal amplification of chromosome 21 (iAMP21) B-ALL. Here, we report the development of B-ALL in a child with Down's syndrome who carries a constitutional isodicentric chromosome 21 [idic(21)], described previously by Putra et al., 2017. This idic(21) appeared to be unstable during mitosis, leading to somatic rearrangements consistent with iAMP21 amplification, resulting in the development of leukemia. In this case, a single constitutional structural chromosome 21 rearrangement resulted in a B-ALL with Down syndrome-associated genomic lesions as well as genomic lesions not common to the Down syndrome subtype of B-ALL. Our findings highlight the need for counseling of individuals with constitutional structural chromosome 21 rearrangements regarding their risks of developing a B-ALL.

Male and female blastocysts: any difference other than the sex?

Is there any imbalance in the sex ratio at the blastocyst stage of human embryos? And what is the sex ratio in euploid, transferred, implanted blastocysts and at birth? Embryos from 646 women undergoing 921 preimplantation genetic testing for aneuploidy (PGT-A) cycles from September 2017 to February 2020 were included. Data from the chromosomal constitution of 2637 biopsied blastocysts were retrospectively analysed. Trophectoderm samples were analysed by next-generation sequencing. Embryos were categorized as euploid, mosaic or aneuploid. A total of 548 blastocysts diagnosed as euploid were warmed and transferred in a subsequent single-embryo transfer cycle. The blastocyst sex ratio was skewed in favour of male sex with 53.1% (1401/2637) of blastocysts diagnosed as male and 46.9% (1236/2637) as female (sex ratio 1.13, 95% confidence interval [CI] 1.05-1.22). Following biopsy and PGT-A, 41.2% (1086/2637) of blastocysts were classified as euploid, 7.7% (202/2637) as mosaic and 51.2% (1349/2637) as aneuploid. More chromosome euploidy was observed among female than male blastocysts (adjusted odds ratio 1.29, 95% CI 1.08-1.55) after adjusting for female age, male age and gonadotrophin dose. Euploid blastocysts were comparable between the sexes (sex ratio 0.99, 95% CI 0.88-1.11). No significant differences were observed between the sexes in implantation (sex ratio 0.86, 95% CI 0.68-1.08), miscarriage (sex ratio 1, 95% CI 0.51-1.97) or live birth rate (sex ratio 0.85, 95% CI 0.66-1.08). More male than female embryos develop to the blastocyst stage. Male blastocysts exhibit a higher aneuploidy rate. The capacity to implant and lead to a live birth is similar between the sexes.

Factors associated with fetal karyotype in spontaneous abortion: a case-case study

BackgroundMost embryos that spontaneously abort during early pregnancy are found to have chromosomal abnormalities. The purpose of this study is to explore the factors involved in chromosome aberrations during embryogenesis.MethodsA case-case study was performed to compare the risk factors for spontaneous abortion with and without embryo chromosome aberration. A total of 160 cases of spontaneous abortion were enrolled from a tertiary general hospital in Kunming. KaryoLite BACs-on-Beads (KL-BoBs) and fluorescence in situ hybridization (FISH) were employed to determine chromosomal constitution of abortion chorion villus samples. Maternal serum levels of homocysteine (Hcy) were detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Information about clinical background and environmental exposure was collected through a self-designed questionnaire. To identify the inherited chromosomal abnormalities, couples with chromosomal abnormalities in abortus were recalled for karyotyping.ResultsThe overall rate of chromosomal abnormalities was 62.5% (100/160, KL-BoBs combined with FISH) including 51.9% (83/160) aneuploidies, 6.3% (10/160) polyploidies, and 4.4% (7/160) structural abnormalities. Only one case of structural abnormality was found to be inherited from maternal balanced translocation. Compared to abortus with normal karyotype, abortus with abnormal karyotype showed a positive association with parental age and elevated maternal serum homocysteine (Hcy) level, but negative association with previous miscarriage and perceived noise.ConclusionsEmbryonic chromosomal aberrations accounted for the majority of spontaneous abortion cases. A combination of internal and external factors may induce spontaneous abortion through fetal chromosomal aberrations or other pathogenic mechanisms.

Ploidy Testing of Blastocoel Fluid for Screening May Be Technically Challenging and More Invasive Than That of Spent Cell Culture Media.

BackgroundRecent studies have demonstrated that both blastocoel fluid (BF) and spent cell culture media (SCM) have potential as materials for non-invasive or less-invasive pre-implantation genetic analysis. BF may allow more opportunity to obtain cell-free DNA from the inner cell mass (ICM), and it has a lower risk of containing contaminant DNA from cumulus cells, sperm and culture media. There are no data regarding the ICM as a gold standard to evaluate the chromosome constitution of BF or SCM for embryo liquid biopsy.MethodsTwo hundred eighteen donated human blastocysts were warmed and cultured in blastocyst culture media for 18–24 h. The corresponding SCM was collected, and only clear ICM was biopsied in blastocysts; otherwise, the whole blastocyst (WB) was biopsied. Quantitative PCR was performed to determine the DNA levels in the SCM and BF before and after amplification. ChromInst was used to amplify BF/SCM and blastocyst DNA before sequencing. Chromosomal copy number variation (CNV) was investigated to evaluate the chromosome constitution.ResultsIn total, 212 blastocysts were available for SCM and BF collection. The technical success rates (next-generation sequencing data) were 100 and 69.8% (148/212) for SCM and BF, respectively. Among the 148 blastocysts with both SCM and BF data, 101 were euploid and 47 were aneuploid based on ICM (n = 89) or WB (n = 59) analysis as the gold standard. Among all blastocysts, SCM was comparable to BF [specificity: 80.2 versus 61.4% (P = 0.005, χ2 test); sensitivity: 91.5 versus 87.2% (P = 0.738, χ2 test); negative predictive value (NPV): 95.3 versus 91.2% (P = 0.487, χ2 test); positive predictive value (PPV): 68.3% versus 51.3% (P = 0.042, χ2 test)]. The SCM and BF samples were 83.8% (124/148) and 69.6% (103/148) concordant with the corresponding ICM/WB samples when only two categories, euploid or aneuploid/mosaic, were grouped to calculate the concordance.ConclusionsCompared with BF, SCM has superior diagnostic performance, and it is non-invasive for embryos.Clinical Trial Registration[http://www.chictr.org.cn], identifier [ChiCTR-BPD-17014087].

Asynapsis and unreduced gamete formation in a Trifolium interspecific hybrid

BackgroundUnreduced gametes, a driving force in the widespread polyploidization and speciation of flowering plants, occur relatively frequently in interspecific or intergeneric hybrids. Studies of the mechanisms leading to 2n gamete formation, mainly in the wheat tribe Triticeae have shown that unreductional meiosis is often associated with chromosome asynapsis during the first meiotic division. The present study explored the mechanisms of meiotic nonreduction leading to functional unreduced gametes in an interspecific Trifolium (clover) hybrid with three sub-genomes from T. ambiguum and one sub-genome from T. occidentale.ResultsUnreductional meiosis leading to 2n gametes occurred when there was a high frequency of asynapsis during the first meiotic division. In this hybrid, approximately 39% of chromosomes were unpaired at metaphase I. Within the same cell at anaphase I, sister chromatids of univalents underwent precocious separation and formed laggard chromatids whereas paired chromosomes segregated without separation of sister chromatids as in normal meiosis. This asynchrony was frequently accompanied by incomplete or no movement of chromosomes toward the poles and restitution leading to unreduced chromosome constitutions. Reductional meiosis was restored in progeny where asynapsis frequencies were low. Two progeny plants with approximately 5 and 7% of unpaired chromosomes at metaphase I showed full restoration of reductional meiosis.ConclusionsThe study revealed that formation of 2n gametes occurred when asynapsis (univalent) frequency at meiosis I was high, and that normal gamete production was restored in the next generation when asynapsis frequencies were low. Asynapsis-dependent 2n gamete formation, previously supported by evidence largely from wheat and its relatives and grasshopper, is also applicable to hybrids from the dicotyledonous plant genus Trifolium. The present results align well with those from these widely divergent organisms and strongly suggest common molecular mechanisms involved in unreduced gamete formation.

Unusual Mammalian Sex Determination Systems: A Cabinet of Curiosities.

Therian mammals have among the oldest and most conserved sex-determining systems known to date. Any deviation from the standard XX/XY mammalian sex chromosome constitution usually leads to sterility or poor fertility, due to the high differentiation and specialization of the X and Y chromosomes. Nevertheless, a handful of rodents harbor so-called unusual sex-determining systems. While in some species, fertile XY females are found, some others have completely lost their Y chromosome. These atypical species have fascinated researchers for over 60 years, and constitute unique natural models for the study of fundamental processes involved in sex determination in mammals and vertebrates. In this article, we review current knowledge of these species, discuss their similarities and differences, and attempt to expose how the study of their exceptional sex-determining systems can further our understanding of general processes involved in sex chromosome and sex determination evolution.

Case Report: The Emerging Role of Ring Chromosome 22 in Phelan-McDermid Syndrome With Atypical Teratoid/Rhabdoid Tumor: The First Child Treated With Growth Hormone.

Atypical teratoid/rhabdoid tumors (AT/RTs) in the rhabdoid tumor predisposition syndromes are most often caused by germline mutations of the SMARCB1 gene located in chromosome 22q11.2. Although rarely, it can also result from the constitutional ring chromosome 22 (r22): during mitosis the ring chromosome may lead to an increased rate of somatic mutations, resulting in rhabdoid tumor predispositions when the tumor-suppressor gene SMARCB1 is involved. Individuals with r22 may present similar features as those with Phelan-McDermid syndrome (PMDS) due to 22q13.3 deletion, including the SHANK3 gene. Despite several reports on AT/RT in children with r22 and/or PMDS have been published, the role of constitutional r22 as new oncogenic mechanism for AT/RT is still under investigation. There is not a lot of data available on therapeutic and prognostic implications of r22 in AT/RT and PMDS. Herein, we present the first case of a child with constitutional r22, PMDS and AT/RT of the brain, who is a long term survivor and is been treated with growth hormone. We also describe an unexpected adverse reaction to midazolam.

Atypical genotype in a typical phenotype

Introduction: Usually, onset of thelarche heralds puberty. Delayed puberty is worrisome and needs medical attention. Our patient in her late adolescence presented with primary amenorrhea, whose evaluation left us surprised. Case report: An eighteen-year-old scholastically backward girl, presented with complaints of not attaining menarche. Physical examination included a height of 156 cm(10th-25thcentile),weight 51 kg(50th centile), wide carrying angle, multiple nevi and a broad chest, however no other Turner stigmata was noted. Her sexual maturity rating (SMR) was A2P2B1 Laboratory investigations revealed increased gonadotropins (FSH:77mIU/ml; LH:25.4mIU/ml), low estradiol (14 pg/ml) and vitamin-D deficiency (21ng/ml). Ultrasonography of abdomen-pelvis showed small infantile uterus with streak ovaries. Karyotype (50 metaphases) demonstrated mosaicism [47,XXX (29)/45,X(19)/46,XX(2)]. Hormone replacement therapy and vitamin D replacement was initiated. Conclusion: 30-40% of the Turner syndrome are mosaics, the most common being 45,X/46,XX. Mosaicism is the presence of 2 or more cell lines with different chromosomal constitutions. The cell lines are derived due mostly to postzygotic mitotic nondisjunction. X/XX/XXX can present with or without classical turner stigmata. Trisomy X has a spectrum of presentation from normal menses and fertility to recurrent abortions and primary/secondary amenorrhea (primary ovarian insufficiency). Varied clinical phenotype due to three cell lines in a Turner mosaic makes this case unique.

EMBRYONIC MOSAICISM IN PREIMPLANTATION GENETIC TESTING (PGT): EVALUATION OF VARIABLES THAT COULD AFFECT ITS FREQUENCY

Next-generation sequencing (NGS) for PGT has improved the resolution for detecting mosaicism, which creates challenging embryo transfer decisions. Currently, there is a growing bulk of reports about mosaic embryo transfers and their clinical outcomes. We consider that the evidence is still limited regarding the factors that could be related to the risk of mosaicism and the clinical outcome after mosaic transfer. This study sought to understand if mosaic embryos frequency are related to different variables and to report their clinical outcomes. This single-center retrospective study included 2174 blastocysts tested between 2016 and 2020 by NGS (Veriseq, Illumina). DNA obtained from trophectoderm biopsies was tested in our laboratory and embryos were classified in euploid, aneuploid, or mosaic. The mosaic embryos were divided into groups based on mosaic levels: low grade (20-40%), high grade (41-80%), and chromosomal constitution (single, double, or complex mosaic aneuploidy). The frequency of mosaic was analyzed according to female age (<30, 30-34, 35-39, >40 years), PGT indication (advanced maternal age, recurrent miscarriages, repeated IVF failure, patient request, translocations, others), day of the biopsy (5, 6 or 7), male factor, and embryo quality: high (3-6AA and 4-6AB, 3-6BA), mid (any BB, 1-3AB, 1-2AA and 1-2BA) and low (any AC, CA, BC, CB or CC). A Chi-square test was applied for statistical analysis. The distributions of the mosaic groups according to the variables studied were determined. In addition, the reproductive outcomes of the mosaic embryos transferred to date were analyzed regarding positive serum βhCG, ongoing pregnancy, and live births. 891 (41%) out of the 2,174 embryos were euploid, 702 (32%) aneuploid, 521 (24%) mosaic, and in 60 cases (3%) the amplification failed. When the embryonic mosaic distributions were analyzed according to the study variables, significant differences (p <0.05) were found for the age of the oocyte and the indication of the study. When we divided mosaic embryos into low grade, high grade or complex, we could observe that they were distributed across all the variables studied and low grade mosaics were the most frequent ones in each variable. Regarding the reproductive outcomes, in 50% of the 22 transferred mosaic embryos, there was a positive serum βhCG, 23% had an ongoing pregnancy and this resulted in a live birth. On the other hand, considering the 391 euploid embryo transfers in our center, 56% had a positive serum βhCG, 42% an ongoing pregnancy, and 33% resulted in a live birth. Our results suggested that the frequency of mosaics could be associated with female age and indication for PGT and that the lower grade embryos are the most frequent. However, the identification of mosaic embryos, mainly the low-grade ones, could be due to methodological artifacts, therefore a prospective study including more samples will validate these results.

EMBRYO CELL-FREE DNA HIGHLY REPRESENTS THE CHROMOSOMAL CONSTITUTION OF BOTH THE INNER CELL MASS AND THE TROPHECTODERM OF HUMAN BLASTOCYSTS

Recent studies have shown the existence of embryo cell-free DNA (cfDNA) in spent blastocyst medium (SBM), opening a new era of possibilities for non-invasive preimplantation genetic testing (niPGT-A). High concordance rates of cfDNA with trophectoderm (TE) biopsies and with whole blastocysts have been reported. The objective of this study was to evaluate the concordance rate of cfDNA in SBM with the chromosomal constitution of the inner cell mass (ICM) and TE biopsies.

Degenerated, Undifferentiated, Rearranged, Lost: High Variability of Sex Chromosomes in Geometridae (Lepidoptera) Identified by Sex Chromatin.

Sex chromatin is a conspicuous body that occurs in polyploid nuclei of most lepidopteran females and consists of numerous copies of the W sex chromosome. It is also a cytogenetic tool used to rapidly assess the W chromosome presence in Lepidoptera. However, certain chromosomal features could disrupt the formation of sex chromatin and lead to the false conclusion that the W chromosome is absent in the respective species. Here we tested the sex chromatin presence in 50 species of Geometridae. In eight selected species with either missing, atypical, or normal sex chromatin patterns, we performed a detailed karyotype analysis by means of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The results showed a high diversity of W chromosomes and clarified the reasons for atypical sex chromatin, including the absence or poor differentiation of W, rearrangements leading to the neo-W emergence, possible association with the nucleolus, and the existence of multiple W chromosomes. In two species, we detected intraspecific variability in the sex chromatin status and sex chromosome constitution. We show that the sex chromatin is not a sufficient marker of the W chromosome presence, but it may be an excellent tool to pinpoint species with atypical sex chromosomes.

P–519 Investigation of embryo chromosomal constitution and live birth rate after vitrified-warmed euploid single blastocyst transfer across ranges of maternal body-mass-index

Abstract Study question Does maternal body-mass-index (BMI) associate with blastocysts’ chromosomal constitution and clinical outcomes in infertile patients undergoing preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer A higher euploidy rate per biopsied blastocyst was reported among underweight women. Overweight women were instead subject to higher miscarriage (MR) and lower live-birth-rates (LBR). What is known already Different studies in the literature revealed an association between BMI and infertility, suggesting a J-shaped relationship: both underweight and overweight women can suffer from infertility issues. Even if IVF might increase the success rate in both these categories of patients, it seems insufficient per se to overcome the complex and multifactorial fertility impairment derived from unbalanced nutritional intakes. Miscarriage, in particular, is common in both underweight and overweight women. However, most of the literature is based on chromosomally-untested embryos. Study design, size, duration: Retrospective observational study. Only the first IVF cycle with ≥1 biopsied blastocyst from each woman was included. The primary outcome was the association between maternal BMI (underweight, BMI&amp;lt;18.5, n = 160; normal-weight, BMI=18–25, N = 1392; overweight, BMI&amp;gt;25, N = 259) and the mean euploidy rate per cohort of biopsied blastocysts (m-ER). The secondary outcomes were the association between maternal BMI with clinical (mainly MR and LBR), gestational and perinatal outcomes after first vitrified-warmed single euploid blastocyst transfers. Participants/materials, setting, methods We included 1811 women undergoing PGT-A at a private IVF center between April–2013 and March–2020. The secondary outcomes were investigated on 1125 first vitrified-warmed single euploid blastocyst transfers from all patients obtaining ≥1 transferable blastocyst. Only ICSI with ejaculated sperm and continuous culture in standard incubators were performed. Logistic regressions were conducted to identify putative confounders and adjust the results accordingly. Main results and the role of chance Except for a lower maternal age among underweight women (38.3±3.1 versus 38.9±3.4 yr, p &amp;lt; 0.01) and higher among overweight ones (39.3±3.6 yr, p = 0.04), no difference was reported with respect to normal-weight women in terms of duration of infertility, hormonal levels, main cause of infertility, sperm quality, and reproductive history. The mean number of biopsied blastocysts was ∼3 in all groups. The m-ER shows a decreasing trend as the maternal BMI increases between 17 and 22–23, to then plateau. In fact, a significant difference was reported between underweight (50.8%±36.4%) and normal-weight women (41.4%±37.5%, p &amp;lt; 0.01). A linear regression adjusted for maternal age confirmed this moderate association between increasing BMI and m-ER (unstandardized-coefficient-B –0.6%, 95%CI:–1.1% to –0.1%, p = 0.02). Morphological quality and day of full-blastulation among transferred euploid blastocysts was similar in the three groups. Overweight women showed higher MR per pregnancy (N = 20/75, 26.7%, 95%CI:17.4%–38.3% versus N = 67/461, 14.5%, 95%CI:11.5%–18.2%; OR 2.0, 95%CI:1.1–3.6, p = 0.01) and lower LBR per transfer (N = 55/154, 35.7%, 95%CI:28.3%–43.8% versus N = 388/859, 45.2%, 95%CI:41.8%–48.6%; OR adjusted for euploid blastocysts’ features 0.67, 95%CI:0.46–0.96, p = 0.03). Clinical outcomes were instead similar among underweight and normal-weight women. All gestational and perinatal outcomes were comparable in the tree groups. Limitations, reasons for caution Our study is limited by its retrospective nature, and the fact that maternal BMI was measured only before oocyte retrieval and not before embryo transfer. Moreover, the reduced sample size did not allow for further relevant sub-analyses among solely obese women. Wider implications of the findings: When possible nutritional/lifestyle modifications should be encouraged to adjust maternal BMI before IVF. Overweight patients should be especially informed of their higher risk for miscarriage. Yet, BMI is just a gross marker, future studies based on body fat localization and percentage (e.g. by bioelectrical impedance analyses) are desirable. Trial registration number None