XX Cells Research Articles

Prevalence, diagnostic features, and medical outcomes of females with Turner syndrome with a trisomy X cell line (45,X/47,XXX): Results from the InsighTS Registry.

Turner syndrome (TS) is defined by partial or complete absence of a sex chromosome. Little is known about the phenotype of individuals with TS mosaic with trisomy X (45,X/47,XXX or 45,X/46,XX/47,XXX) (~3% of TS). We compared the diagnostic, perinatal, medical, and neurodevelopmental comorbidities of mosaic 45,X/47,XXX (n = 35, 9.4%) with nonmosaic 45,X (n = 142) and mosaic 45,X/46,XX (n = 66). Females with 45,X/47,XXX had fewer neonatal concerns and lower prevalence of several TS-related diagnoses compared with 45,X; however the prevalence of neurodevelopmental and psychiatric diagnoses were not different. Compared to females with 45,X/46,XX, the 45,X/47,XXX group was significantly more likely to have structural renal anomalies (18% vs. 3%; p = 0.03). They were twice as likely to have congenital heart disease (32% vs. 15%, p = 0.08) and less likely to experience spontaneous menarche (46% vs. 75% of those over age 10, p = 0.06), although not statistically significant. Congenital anomalies, hypertension, and hearing loss were primarily attributable to a higher proportion of 45,X cells, while preserved ovarian function was most associated with a higher proportion of 46,XX cells. In this large TS cohort, 45,X/47,XXX was more common than previously reported, individuals were phenotypically less affected than those with 45,X, but did have trends for several more TS-related diagnoses than individuals with 45,X/46,XX.

A multicenter analysis of individuals with a 47,XXY/46,XX karyotype

PurposeKlinefelter syndrome, a sex chromosome aneuploidy (SCA), is associated with a 47,XXY chromosomal complement and is diagnosed in ∼1:600 live male births. Individuals with a 46,XX cell line, in addition to 47,XXY, are less common with a limited number of published case reports. MethodologyTo better understand the implications of a 47,XXY/46,XX karyotype, we conducted a retrospective, multicenter analysis of the cytogenetic findings and associated clinical records of 34 patients diagnosed with this SCA across 14 institutions. ResultsPresence of the XX cell line ranged from 5% to 98% in patient specimens. Phenotypes also exhibited significant heterogeneity with some reporting a single reason for referral and others presenting with a constellation of symptoms, including ambiguous genitalia and ovotestes. Ovotestes were present in 12% of individuals in this cohort, who had a significantly higher percentage of XX cells. Notably, 2 patients were assigned female sex at birth. ConclusionThese findings highlight the variability of the clinical phenotypes associated with this SCA, as well as the challenges of clinical management for this population. Karyotype or fluorescence in situ hybridization analysis, which offer single-cell resolution, rather than chromosomal microarray or molecular testing, is the ideal test strategy in these instances as mosaicism can occur at low levels.

Post-transcriptional cross- and auto-regulation buffer expression of the human RNA helicases DDX3X and DDX3Y.

The Y-linked gene DDX3Y and its X-linked homolog DDX3X survived the evolution of the human sex chromosomes from ordinary autosomes. DDX3X encodes a multi-functional RNA helicase, with mutations causing developmental disorders and cancers. We find that, among X-linked genes with surviving Y homologs, DDX3X is extraordinarily dosage-sensitive. Studying cells of individuals with sex chromosome aneuploidy, we observe that when the number of Y chromosomes increases, DDX3X transcript levels fall; conversely, when the number of X chromosomes increases, DDX3Y transcript levels fall. In 46,XY cells, CRISPRi knockdown of either DDX3X or DDX3Y causes transcript levels of the homologous gene to rise. In 46,XX cells, chemical inhibition of DDX3X protein activity elicits an increase in DDX3X transcript levels. Thus, perturbation of either DDX3X or DDX3Y expression is buffered - by negative cross-regulation of DDX3X and DDX3Y in 46,XY cells, and by negative auto-regulation of DDX3X in 46,XX cells. DDX3X-DDX3Y cross-regulation is mediated through mRNA destabilization - as shown by metabolic labeling of newly transcribed RNA - and buffers total levels of DDX3X and DDX3Y protein in human cells. We infer that post-transcriptional auto-regulation of the ancestral (autosomal) DDX3 gene transmuted into auto- and cross-regulation of DDX3X and DDX3Y as these sex-linked genes evolved from ordinary alleles of their autosomal precursor.

Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Sexual development disorder in a dog

A 1-year intact, phenotypic female Doberman Pinscher dog, was evaluated for suspected sexual development disorder. Patient had a history of white mucoid vaginal discharge with no estrous signs or behavior. Clitoral hypertrophy with a palpable os clitoris were noted. Transabodminal ultrasonography revealed a right gonad and tubular structures (possibly uterus). Exploratory laparotomy revealed 2 underdeveloped gonads in caudodorsal abdomen and a markedly underdeveloped right uterine horn. Histopathology confirmed bilateral ovotestes. Karyotyping (number and morphology of chromosomes) and fluorescence in situ hybridization (XX and XY cells) results were normal. Samples were PCR positive for sex-determining region Y (SRY) and X-linked androgen receptor gene. We concluded that the patient was either a mosaic or chimera with normal female 78,XX and normal male 78,XY cells and genetically a mix of male and female. Final diagnosis was sex chromosome (78,XX/XY), SRY-positive, ovotesticular, disorder of sexual development with female phenotype.

Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development

BackgroundDisorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry.MethodsWe employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches.ResultsSLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells.ConclusionsThe trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

Reproductive Outcomes of Women with Turner Syndrome Undergoing Oocyte Vitrification: A Retrospective Multicenter Cohort Study.

Turner syndrome (TS) is accompanied with premature ovarian insufficiency. Oocyte vitrification is an established method to preserve fertility. However, data on the oocyte yield in women with TS who vitrify their oocytes and the return rate to utilize the oocytes are scarce. Retrospective multicenter cohort study. Data was collected from medical records of women with TS who started oocyte vitrification between 2010 and 2021. Thirty-three women were included. The median cumulative number of vitrified oocytes was 20 per woman. Complications occurred in 4% of the cycles. Significant correlations were found between the cumulative number of vitrified oocytes and AMH (r = 0.54 and p < 0.01), AFC (r = 0.49 and p < 0.01), percentage of 46,XX cells (r = 0.49 and p < 0.01), and FSH (r = -0.65 and p < 0.01). Spontaneous (n = 8) and IVF (n = 2) pregnancies occurred in 10 women ± three years after vitrification. So far, none of the women have returned to utilize their vitrified oocytes. Oocyte vitrification is a feasible fertility preservation option for women with TS, particularly in those with 46,XX cell lines or sufficient ovarian reserve. Multiple stimulation cycles are recommended to reach an adequate number of vitrified oocytes for pregnancy. It is too early to draw conclusions about the utilization of vitrified oocytes in women with TS.

Modeling unveils sex differences of signaling networks in mouse embryonic stem cells.

For a short period during early development of mammalian embryos, both X chromosomes in females are active, before dosage compensation is ensured through X-chromosome inactivation. In female mouse embryonic stem cells (mESCs), which carry two active X chromosomes, increased X-dosage affects cell signaling and impairs differentiation. The underlying mechanisms, however, remain poorly understood. To dissect X-dosage effects on the signaling network in mESCs, we combine systematic perturbation experiments with mathematical modeling. We quantify the response to a variety of inhibitors and growth factors for cells with one (XO) or two X chromosomes (XX). We then build models of the signaling networks in XX and XO cells through a semi-quantitative modeling approach based on modular response analysis. We identify a novel negative feedback in the PI3K/AKT pathway through GSK3. Moreover, the presence of a single active X makes mESCs more sensitive to the differentiation-promoting Activin A signal and leads to a stronger RAF1-mediated negative feedback in the FGF-triggered MAPK pathway. The differential response to these differentiation-promoting pathways can explain the impaired differentiation propensity of female mESCs.

Quantification of Female Chimeric Cells in the Tonsils of Male Children and Their Determinants.

The factors influencing mother-to-child cell trafficking and persistence over children's lives have yet to be established. The quantification of maternal microchimerism was previously reported through HLA-based approaches, which introduced bias regarding the tolerogenic environment. We aimed to identify cells of maternal origin irrespective of the HLA repertoire and to ascertain the determinants of microchimeric cells. This case-control study enrolled 40 male infants attending pediatric surgery from January 2022 to October 2022. Female cells were quantified in infants' tonsil tissue by using cytogenetic fluorescent in situ hybridization (FISH) coupled with optimized automated microscopy. Out of the 40 infants, half (47.4%) had been breastfed for more than one month, a quarter for less a month, and 10 children (26.3%) were never breastfed. XX cells were observed in male tonsils in two-thirds of participants at a median density of 5 cells per 100,000 cells. In univariate analyses, child age was negatively associated with a high female cell density. In exploratory multivariate analyses, previous breastfeeding is a likely determinant of the persistence of these cells in the host, as well as the rank among siblings. Part of the benefit of breastmilk for child health may therefore be driven by breastfeeding-related microchimerism.

TurnerFertility trial: fertility preservation in young girls with Turner syndrome by freezing ovarian cortex tissue—a prospective intervention study

To evaluate which girls with Turner syndrome (TS) could benefit from fertility preservation by ovarian tissue cryopreservation on the basis of karyotype, puberty status, and hormonal data. Prospective intervention study; participants were included between 2018 and2020. Tertiary hospital in the Netherlands. In total, 106 girls with TS aged between 2 and 18 years were included. Girls with minor X chromosome deletions, Ychromosomal content, active infections, or contraindications for surgery were excluded. A laparoscopic unilateral ovariectomy was performed to obtain ovarian cortical tissue for cryopreservation. One tissue fragment per participant was used to determine the number of follicles per ovary by serial sectioning and staining. Chromosome analysis was performed on lymphocytes and buccal cells. A blood sample was taken before the ovariectomy for hormonal analysis. The presence of follicles in ovarian cortex tissue from girls with TS in relation to karyotype, puberty status, and hormonal data. A unilateral ovariectomy was performed on 93 girls with TS. Complications after surgery occurred in 5 girls, including luxation of psychological symptoms in 2 girls. In 13 (14%) girls, a 46,XX cell line was found in buccal cells that was absent in lymphocytes. Follicles were observed in 30 (32%) of the 93 girls and were found mainly in girls with a 46,XX cell line in lymphocytes or buccal cells (Phi coefficient = 0.55). Spontaneous onset of puberty (Phi coefficient = 0.59), antimüllerian hormone (AMH; point-biserial correlation [r] = 0.82), inhibin B (r = 0.67), and follicle-stimulating hormone (r = -0.46) levels were also correlated strongly with the presence of follicles. Furthermore, AMH levels had a significant correlation with the number of follicles per ovary (r = 0.66). Favorable predictive markers for the presence of follicles included either a 46,XX cell line, spontaneous onset of puberty, or a combination of measurable AMH and normal follicle-stimulating hormone levels. Karyotyping of two peripheral cell lines in girls with TS is recommended to reveal hidden mosaicisms. Ovarian tissue cryopreservation should be offered with caution in a research setting to those with a sufficient ovarian reserve, considering the significant loss of follicles after ovarian tissue cryopreservation and autotransplantation. Physicians should pay attention to the mental health of the girls during the whole process. Trial registration number: NCT03381300- Preservation of Ovarian Cortex Tissue in Girls With Turner Syndrome - Full Text View - ClinicalTrials.gov. Registered on: December 21, 2017. First patient recruited on January 1, 2018.

Concerns over the declining human Y-chromosome and social functioning

In humans, like other mammals, females have two X chromosomes and males have one X and one Y chromosome. The Y chromosome contains a sex-determining gene SRY that kick-starts male development in the embryo and activates other genes that regulate the testicular development. Researchers have found that the Y chromosome seems to be disappearing, as seen in many other mammals, including rodents. The male eastern Europe vole and the spiny rats of Japan have completely lost their Y chromosomes and the Sry gene. However, the SOX9 protein plays a key role there in determining their sex. The new finding so support the possibility that humans too, may evolve a new sex determining mechanism. The sex chromosome genes act within cells to cause differences in phenotypes of XX and XY cells throughout the body. In the gonad, they determine the type of gonad, leading to differences in secretion of testicular vs. ovarian hormones, which cause further sex differences in tissue function. The human brains, as shown by neuroimaging, demonstrate variation in different areas of the brain that are associated with both differences in gonadal hormones, and in the number of X and Y chromosomes. In humans, males and females are different because of innate biological differences, and also they have different social and physical environments. Biologists tend to emphasize the importance of biological variables, and sociologists tend to emphasize the role of gendered environments. The Sry and the other Y-linked genes not only determine the differences in gonad development, they are also linked to maintaining gender balance (biologically) and various functions in the brain, including appropriate social responses by supporting behavioral flexibility, attention and emotion. In this article, we have reviewed the function of the Y chromosome and the Sry gene and their possible alternatives in case of their future extinction.

X CHROMOSOME-DERIVED MECHANISMS OF SEX DIFFERENCES IN LIFESPAN AND BRAIN AGING

Abstract Women live longer than men worldwide – and also show cognitive resilience in many aging populations. One major source of biologic difference between the sexes is that females have two X chromosomes and males have one. This difference in sex chromosome complement causes unique X-derived mechanisms that are sex-specific. In mammalian development, one X randomly inactivates in XX cells. One X-derived sex difference is that females are mosaics with the active X chromosome in each cell being either maternally-derived (Xm) or paternally-derived (Xp), whereas males harbor only a maternally-derived X (Xm) in all cells. Interestingly, some females show considerable or complete skew toward Xm or Xp. We utilized several genetic models of sex biology to understand mechanisms of sex difference in aging. We found that the X chromosome contributes to longevity and better cognition in male and female mice. In aging, a genetic manipulation in females to express only the maternally-derived X (Xm), like males, accelerated cognitive decline and epigenetic brain aging. This suggests that Xm is harmful and that female mosaicism (Xm+Xp) provides a buffer to deleterious processes in aging. To assess if Xm alters transcription, we used mice with nuclear localized genetic reporters and sorted Xm from Xp neurons from young and aging XX hippocampi. We found that Xm imprinted several genes within aging hippocampal neurons, suggesting silenced cognitive loci. Our data suggests that Xm – the maternal X – accelerates brain aging and causes cognitive deficits. Understanding how Xm impairs brain function could increase understanding of female heterogeneity and of sex differences in cognitive heath – and unlock new X-derived pathways against cognitive deficits and brain aging of males, females, or both.

XX/XY Chimerism in Internal Genitalia of a Virilized Heifer.

Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.

P-485 The TurnerFertility study: data on fertility preservation (FP) by ovarian tissue cryopreservation (OTC) in young girls with Turner syndrome (TS)

Abstract Study question Which girls with TS could benefit from FP by OTC, based on the presence of follicles in relation to karyotype, clinical and hormonal data? Summary answer Girls with TS who have favourable predictive parameters (e.g 46,XX cell line, a measurable AMH or spontaneous puberty) could benefit from FP by OTC. What is known already Infertility due to premature ovarian insufficiency is a major concern for girls with TS and their parents. Physicians are often asked about possible options to preserve their fertility. However, evidence for successful FP by OTC in these girls is lacking. Without evidence on the effectiveness of OTC in TS girls, it should only be offered in a research setting. Study design, size, duration A national prospective exploratory intervention study. Ovarian cortex will be obtained after unilateral ovariectomy from 106 girls with TS aged 2-18 years. Patients will be included between 2017 and 2022. Participants/materials, setting, methods All girls with TS who have completed the diagnostic work up of TS were included. After unilateral ovariectomy, one fragment of the ovarian cortex was used to determine the number of follicles by serial sectioning and staining. Karyotyping of ovarian cells, lymphocytes, buccal cells and urine cells was performed by Fluorescence in situ hybridization (FISH). Blood samples obtained before oophorectomy and during the yearly clinical visit after oophorectomy will provide information on hormonal parameters. Main results and the role of chance Currently, we have received 106 informed consent forms and 86 TS patients (age 3-19) had a unilateral ovariectomy. Oocytes were found in 32,6% (n = 28; age 5-19) of which 11 were prepubertal, 16 had a spontaneous puberty, 22 had numerical chromosome X aberrations, of whom one 45,X monosomy and 6 had structural chromosome X aberrations. In 6 patients with structural aberrations we found a low follicle density. In 24/28 patients AMH was measurable (0.1 - 4.79µg/L) and 25/28 patients had a FSH below 15 E/L. FISH was used to karyotype the ovarian cortex cells of 12 patients with a numerical aberration and revealed that 112 of the 119 oocytes (94,1%) had a normal X-chromosomal content. Granulosa cells were largely 45,X, but showed different levels of X chromosome mosaicism, not only between patients but also between individual follicles of the same patient. Despite the level of aneuploidy (0–80%) of ovarian stromal cells, no obvious morphological/histological abnormalities were observed in the ovarian cortex tissue. Hormone values and the chromosome pattern were found to be predictive parameters for the presence of follicles. The chance of finding follicles in girls with a 46,XX cell line was three times higher than in girls without a 46,XX cell line. Limitations, reasons for caution The final analysis will be performed when the dataset of 106 TS girls is completed. Further research is necessary to determine the effects of ovarian mosaicism on folliculogenesis, i.e. are follicles capable to grow to antral stages, and to elucidate if OTC is an effective method for FP in TS. Wider implications of the findings A combination of clinical, hormonal and karyotypic data could provide predictive parameters to define which girls with TS might benefit from fertility preservation. These parameters could help physicians during FP counselling to determine if OTC is an option for a certain girl with TS. Trial registration number NCT03381300

Integrating Sex Chromosome and Endocrine Theories to Improve Teaching of Sexual Differentiation.

Major sex differences in mammalian tissues are functionally tied to reproduction and evolved as adaptations to meet different reproductive needs of females and males. They were thus directly controlled by gonadal hormones. Factors encoded on the sex chromosomes also cause many sex differences in diverse tissues because they are present in different doses in XX and XY cells. The sex chromosome effects likely evolved not because of demands of reproduction, but as side effects of genomic forces that adaptively reduced sexual inequality. Sex-specific effects of particular factors, including gonadal hormones, therefore, are not necessarily explained as adaptations for reproduction, but also as potential factors offsetting, rather than producing, sex differences. The incorporation of these concepts would improve future teaching about sexual differentiation.

46,XX/46,XY Chimerism &amp; Human Sexual Development

The term chimera refers to an organism with cell lines from two or more distinct zygotes. Human chimerism may occur naturally or artificially. Although rare, advancements in genetics and genomics have resulted in the identification of additional natural human chimeras. Three forms of naturally occurring chimerism have been documented in humans: blood group chimerism, microchimerism, and fusion chimerism. Fusion chimerism may occur through several means. Sex-chromosome discordant chimerism refers to individuals with both XX and XY cell lines. There is a large amount of phenotypic variability among 46,XX/46,XY chimeric individuals. The care of people with intersex traits or DSD (Disorders of Sexual Development) is controversial due in part to a history of unnecessary surgical intervention and power-imbalances between the intersex and medical communities. As more 46,XX/46,XY chimeric individuals with intersex traits or DSD are identified, the implications for their care will need to be clarified.

EP170: When cfDNA screening deceives: A rare case of mosaicism for 46,XX/47,XXY with uniparental isodisomy and genital atypia

Sex chromosome aneuploidy (SCA) identified on cell free DNA screening (cfDNA) presents a challenge for both prenatal counseling, and care planning for the infant after delivery. It is well understood that post-natal evaluation must include chromosomal analysis to confirm SCAs and evaluate for mosaicism. This is traditionally achieved using karyotype, with or without FISH for Y-component. The findings are important for diagnosis care planning, including for individuals with Intersex/Differences of Sex Development (I/DSD) traits. In the case of I/DSD traits, recommendations of gender of rearing are complex and require consideration of multiple pieces of evidence that include, but are not limited to, chromosomes. Despite being a screening rather than a diagnostic test, the advent of cfDNA analysis has generated added prenatal information that can often color the complex medical and psychosocial thought process of assignment of gender of rearing in cases of ambiguity at birth. I/DSD traits are defined as atypical/non-binary development of chromosomal, gonadal and/or anatomic sex. SCAs are a frequent cause of DSD, most commonly mosaic monosomy X (“Turner cells”) with Y chromosome material. Mosaic SCAs involving 47,XXY (“Klinefelter cells”) is a rare cause of DSDs. Mosaicism involving 47,XXY is typically 46,XY/47,XXY, and is not associated with genital atypia. We present a case of a patient who was initially erroneously reported as 46, XX on cfDNA screening. Post-natal confirmation using genome-wide SNP array showed instead 46,XX/47,XXY mosaicism. Furthermore, both cell lines contained uniparental isodisomy of the entire chromosome X. The infant was evaluated at 6-months-old for non-binary genital (“ambiguity”) first noted at birth. Patient was naturally conceived, born to a G1P0-P1 mother, and with normal prenatal screening and ultrasounds by report. CfDNA screen report was low risk with 46,XX, and the infant was therefore assigned female at birth. Patient was SGA with non-dysmorphic facies, and external genitalia exam notable for: Prader 3 clitorophallus, with one identifiable opening on the ventral aspect, and fused, ruggated scrotolabial folds without palpable gonads. Labs at the time of mini-puberty of infancy (8 weeks of life) provided evidence of gonadal function, despite absence of gonads on imaging: testosterone (188 ng/dL, normal range for 46,XX <10 ng/dL; normal range for 46,XY 60-400 ng/dL); AMH (27.49 ng/mL, normal range for 46,XX 0.53-7.78 ng/mL); estradiol (1.9 pg/mL, normal range for 46,XX 5-50 pg/mL); and pubertal LH and FSH concentrations. This suggested the presence of functional testicular tissue. Karyotype and SNP microarray were completed. SNP initially reported this patient as mosaic for chromosomal complement 45,X/46,XY (arr[hg19] Yp11.32q12(10,814-59,339,753)x0 [75%]). However, karyotype results were then noted to be mosaic for 46, XX [25]/47, XXY [5], with further analysis showing the patient had total uniparental isodisomy of the entire X chromosome in 100% of her cells, with deletion of the entire Y as above in 75% of cells. Uniparental isodisomy of the entire X chromosome is exceedingly rare and is due to monosomy rescue in the case of a 45,X complement, or maternal nondisjunction and lack of paternal sex chromosome complement. It is most commonly reported in the literature in 46,XX female patients with X-linked conditions such as Duchene muscular dystrophy or Fragile X. Our patient showed no true 45,X complement on her microarray, which would be expected in the setting of monosomy rescue. Such a monosomy rescue mechanism is also difficult to reconcile with the presence of Y chromosome. Our hypothesis is that the initial zygote was 47,XXY with the UPD X due to maternal non-disjunction at meiosis II. The Y was later lost in one cell line. However, it is also atypical that her X shows no evidence of crossing over if this was in fact a meiosis II non-disjunction event. Her Y also must have been lost early on in development, as her placenta likely had predominantly 46, XX cells for her cfDNA screen results to show no Y component. Less than ten patients have been previously reported with mosaicism for 46,XX/47,XXY. Of note, the majority of these patients did not have genital differences at birth, and all were assigned male. This is despite well-documented cases in the literature that mosaicism of 45,X/46,XY is commonly associated with genital atypia. Patients with mosaicism for Y containing cells are often picked up as having a Y component on their cfDNA results, where sensitivity to determine presence of a Y chromosome is 99.4%. It is important to understand cfDNA analysis as a screening test, and not a diagnostic test like a karyotype or SNP microarray. Under some circumstances, chromosomal aneuploidies, including SCAs, can be missed. Therefore, regardless of cfDNA screen results, karyotype and SNP microarray are important tests to consider in the setting of a DSD where mosaicism for a Y-containing line is a common diagnosis.

Ontogenes and Chromosome Nondisjunction in the &amp;lt;i&amp;gt;D. melanogaster&amp;lt;/i&amp;gt; Meiosis

A mutation in an ontogene acts as a conditional dominant lethal: it is lethal in a certain genotype but not lethal in another. In total, 30 mutations of this type residing in the Drosophila melanogaster X chromosome have been assayed for their ability to cause meiotic nondisjunction. The level of X nondisjunction in the females heterozygous for the mutation in ontogene appears to be very high. The share of matroclinous daughters reaches 24.7% of the overall offspring and of patroclinous males, 24.9%. Neither inversion in the opposite X chromosome nor additional Y chromosome has any effect on the X nondisjunction. The balance of the XX and X0 egg cells is disturbed: exceptional daughters are prevalent in the offspring of the females with a normal opposite X chromosome and exceptional sons, in the offspring of the females with an inverted X chromosome. In addition, 12% of the matroclinous daughters of the females with a normal opposite X chromosome are homozygous for the marker of one of the maternal X chromosomes (“equational” nondisjunction). A “fading” parental effect of the mutation in ontogene on the X chromosome nondisjunction is also observed. Under experimental conditions, the mutant ontogenes reside in meiotic densely compacted X chromosomes. We infer that the ontogenes are DNA regions with controlled compaction. It is postulated that the genetic activity of ontogenes is determined by this compaction and has a biophysical (electromagnetic) nature. In a meiotic cell, ontogenes induce physical fields providing the operation of meiotic proteins. The structure of these fields is distorted in the mutants for ontogenes, thereby decreasing the efficiency of proteins and, as a consequence, causing meiotic defects.

P–154 The role of the X Chromosome in early human embryo metabolism

Abstract Study question What role does the X chromosome play in early embryo metabolism? Does X chromosome copy number contribute to sex differences in early embryonic metabolism? Summary answer Chromosome X contains several metabolism-related genes that are expressed prior to X-inactivation, suggesting that their dosage plays a role in sex-biased regulation of embryo metabolism. What is known already Published reports indicate that sex differences in preimplantation embryo metabolism exist across mammalian species, including humans. Two observations supporting this are that male embryos reach blastocyst stage earlier than their female counterparts, and that glucose uptake and processing is thought to be higher in female compared to male embryos. It has been hypothesized that these differences reflect the location of the metabolism gene G6PD, the rate limiting enzyme in the Pentose Phosphate Pathway, on Chromosome X. Study design, size, duration This study is a reanalysis of publicly available RNA-seq data, including 1176 single cells from 59 blastocysts (24 E5, 18 E6, 17 E7) published in one study (Petropoulos et al 2016). Participants/materials, setting, methods Cells were subjected to a digital karyotype inference algorithm and aneuploid samples were removed from the dataset. Sex differential gene expression analyses (DE) were then performed in euploid trophectoderm cells (TE; 233 XY from 16 embryos and 180 XX cells from 12 embryos). Cell numbers from ICM were too sparse to compare. Main results and the role of chance Analysis of XX and XY TE revealed 618 significantly differentially expressed genes (DEGs; 507 upregulated in XX cells, and 111 upregulated in XY cells). These genes are spread across autosomes and sex chromosomes. Interestingly, G6PD is not significantly more highly expressed in XX cells. Gene Ontology (GO) analysis of the XX-biased DEGs revealed a transcriptional sex bias in metabolism-related GO categories, including “mitochondrial ATP synthesis coupled electron transport”, and “respiratory chain complex I”. Gene-level assessment revealed that the drivers of these enrichments are spread across the genome, but 28/64 reside on Chromosome X (hypergeometric p-value = 5.984473e–27), including NDUFA1, NDUFB11, and COX7B (components of the electron transport chain), and SLC25A5 (an ATP/ADP transporter involved in maintaining mitochondrial membrane potential). This indicates a direct role for multiple X-linked genes in sex-biased regulation of embryo metabolism. Metabolic genes that are not sex-biased are distributed across the genome, with no significant enrichment on Chromosome X (76/266, hypergeometric p-value=0.607). Together, these data indicate that GO metabolic term X enrichment is a feature of sex-biased expression and not due to an accumulation of metabolism-related genes on the X. Limitations, reasons for caution This analysis draws on publicly available data, and thus we are unable to perform orthogonal validation of karyotype calls. Additionally, while the initial dataset is large, the quality-filtered dataset (euploid XX and XY TE) is small, and single cell data is infamously variable. Further data collection is required. Wider implications of the findings: Our analysis of sex-biased gene expression in early human embryos suggests a more important role for the X chromosome in modulating sex biases in early embryo metabolism than previously recognized. This study provides insight into the mechanisms underlying the development of metabolic sex differences throughout the lifespan. Trial registration number NA

Functional and regulatory aspects of oxidative stress response in X monosomy

The partial/complete loss of one X chromosome in a human female leads to Turner syndrome (TS). TS individuals display a range of phenotypes including short stature, osteoporosis, ovarian malfunction, diabetes, and thyroid dysfunction. Epigenetic factors and regulatory networks are distinctly different in X monosomy (45, X). In a lifetime, an individual is exposed to a variety of stress conditions. To study whether X monosomy cells display a differential response upon exposure to mild stress as compared to normal 46,XX cells and whether this may contribute to various co-morbidities in aneuploid individuals, we have carried out a transcriptomic analysis of human fibroblasts 45, X and 46, XX after exposure to mild oxidative stress. Under these conditions, over 350 transcripts were seen to be differentially expressed in 45, X and 46,XX cells. Pathways associated with oxidative stress were differentially expressed highlighting the differential regulation of genes and associated phenotypes. It could be seen that X monosomy cells are more susceptible to oxidative stress as compared to normal cells and have altered molecular pathways both in normal conditions and also upon exposure to mild oxidative stress. To explore this aspect in detail, we have mapped the expressions of transcription factors (TFs) in 45,X and 46,XX cells. The network of transcription activating factors is differentially regulated in 45,X and 46,XX cells under stress exposure. It is tempting to speculate that the altered ability of 45,X (Turner) cells to respond to stress may play a significant role in the physiological function and altered phenotypes in Turner syndrome.