Sex Chromosome Status Research Articles

Contribution of maternal mosaicism to false-positive chromosome X loss associated with noninvasive prenatal testing

Objective To report the frequency of maternal mosaicism contributing to false-positive chromosome X loss associated with noninvasive prenatal testing (NIPT) at a single center. Methods Pregnancies undergone NIPT using massively parallel sequencing at Guangzhou Women and Children’s Medical Center between February 2015 and May 2020 were included in this study. Fetal karyotyping, quantitative fluorescence PCR (QF-PCR) or microarray analysis was provided to patients with abnormal sex chromosomal aneuploidy (SCA) results for confirmatory testing, and QF-PCR was also employed to detect maternal sex chromosome status. Results cffDNA testing of 40682 pregnancies revealed 86 cases with NIPT results positive for chromosome X loss (0.21%). Among the 86 high-risk cases, 73 women had undergone confirmatory testing in our center, whereas 13 declined. Of the 73 women verified by invasive prenatal diagnosis, 27.4% (20/73) were true positive cases including six cases of monosomy X, two cases of microdeletion of Xp22.33, one case of deletion Xq27.2q28, one case of 47, XXX and ten cases with fetal sex chromosome mosaicism. Of the remaining 53 patients with fetal normal results, 30 cases had undergone QF-PCR analysis of maternal white blood cells. QF-PCR indicated that 36.7% (11/30) patients had an altered or mosaic maternal sex chromosome status. Statistical analysis indicated that cell-free fetal DNA (cffDNA) concentration estimated by chromosome X in maternal mosaic cases was significantly higher than that in the non-maternal mosaicism group (p < .05) and was related to maternal mosaicism rate (r = 0.88, p < .05). Conclusions Our findings indicated that maternal mosaicism of sex chromosome was not uncommon in false-positive NIPT chromosome X loss cases. We recommend that this information should be disclosed to pregnancies during clinical counseling and maternal sex chromosome status should be confirmed for the cases with NIPT chromosome X loss.

Sex Chromosomes Are Severely Disrupted in Gastric Cancer Cell Lines.

Sex has not received enough attention as an important biological variable in basic research, even though the sex of cells often affects cell proliferation, differentiation, apoptosis, and response to stimulation. Knowing and considering the sex of cells used in basic research is essential as preclinical and clinical studies are planned based on basic research results. Cell lines derived from tumor have been widely used for proof-of-concept experiments. However, cell lines may have limitations in testing the effect of sex on cell level, as chromosomal abnormality is the single most characteristic feature of tumor. To examine the status of sex chromosomes in a cell line, 12 commercially available gastric carcinoma (GC) cell lines were analyzed using several different methods. Loss of Y chromosome (LOY) accompanied with X chromosome duplication was found in three (SNU-484, KATO III, and MKN-1) out of the six male-derived cell lines, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Two (SNU-5 and MKN-28) out of six female-derived cell lines showed a loss of one X chromosome, while SNU-620 gained one extra copy of the X chromosome, resulting in an XXX karyotype. We found that simple polymerase chain reaction (PCR)-based sex determination gives a clue for LOY for male-derived cells, but it does not provide detailed information for the gain or loss of the X chromosome. Our results suggest that carefully examining the sex chromosome status of cell lines is necessary before using them to test the effect of sex on cell level.

PPP2R3B Is a Sex-Linked Melanoma Tumor Suppressor Gene

Abstract PPP2R3B dosage is associated with sex chromosome status and outcome in melanoma.

The electrophoretic separation of spermatozoa: an analysis of genotype, surface carbohydrate composition and potential for capacitation

This study examines the properties of an electrophoretic device designed to effect the rapid isolation of spermatozoa for assisted conception purposes. In light of previous reports suggesting that X- and Y-bearing spermatozoa can be separated in an electric field, the first characteristic examined was the sex chromosome status of electrophoretically isolated spermatozoa. Exploiting sex chromosome-specific differences in the structure of the amelogenin gene, a quantitative PCR protocol was designed that allowed the rapid genotyping of isolated sperm suspensions. Reassuringly, application of this procedure demonstrated that the electrophoretic method did not result in a significant skewing of the ratio of X- and Y-bearing spermatozoa. Analysis of the molecular basis for electrophoretic sperm isolation demonstrated that sperm suspensions prepared in this manner were enriched in surface sialic acid residues that bound the Sambucus nigra agglutinin (SNA) lectin. Western blot analyses demonstrated the presence of four major SNA binding proteins, three of which were identified by MALDI-Tof mass spectrometry as aminopeptidase B, fucosyltransferase and prostatic acid phosphatase. The ability of neuraminidase to significantly suppress the electrophoretic isolation of spermatozoa emphasized the causative nature of this association between cell surface sialation and sperm behaviour in an electric field. Finally, seminal plasma proteins possessing decapacitation properties were shown to co-migrate with spermatozoa during their electrophoresis, necessitating their removal prior to in vitro fertilization. In terms of function, electrophoretically isolated cells were found to capacitate normally, exhibiting high levels of tyrosine phosphorylation and a capacity for extensive binding to homologous zonae pellucidae. We conclude that the electrophoretic procedure rapidly isolates functional spermatozoa via mechanisms that are independent of their genotype but reliant upon a net electronegative charge that is largely conferred by sperm surface glycoproteins.

Diagnostic accuracy: theoretical models for preimplantation genetic testing of a single nucleus using the fluorescence in situ hybridization technique

The aim of this study was to develop and use theoretical models to investigate the accuracy of the fluorescence in situ hybridization (FISH) technique in testing a single nucleus from a preimplantation embryo without the complicating effect of mosaicism. Mathematical models were constructed for three different applications of FISH in preimplantation genetic testing (sex determination for sex-linked diseases, two-way reciprocal translocations and sporadic chromosome aneuploidy). The input values were the degree of aneuploidy (initially set at 3% per chromosome for sporadic aneuploidy) and the accuracy per probe (initially set at 95%), defined as the proportion of normal diploid nuclei with a normal signal pattern. The primary statistic was the predictive value of the test result. Testing two chromosome pairs to determine sex chromosome status or detect unbalanced translocation products had high predictive value: at least 99.5% for a normal test result (95% CI: 99-100%), and 90% for an abnormal test result (95% CI: 88-92%). However, the predictive value of an abnormal test result testing five chromosomes for sporadic chromosome aneuploidy was 41% (95% CI: 36-46%); 90% would be achieved with an aneuploidy rate per chromosome of 20.3% (equivalent to 99.5% prevalence for 23 chromosomes) rather than 3%, or with an accuracy per probe of 99.6% rather than 95%, or when testing 23 chromosome pairs, rather than 5 pairs, with either 8.3% aneuploidy (86.4% prevalence) or 99.5% accuracy. Testing a single cell using the FISH technique has the potential to achieve acceptable analytical performance for sex determination and two-way reciprocal translocations, but is unlikely to achieve adequate performance testing for sporadic chromosome aneuploidy. New techniques for detecting the copy number of every chromosome are emerging, but it remains to be seen if the high accuracy required will be achieved.

A Simpler, Cheaper and Quicker Method to Study Somatic Chromosomes from Goat, Capra hircus (L.)

A modified method for the preparation of somatic chromosomes from goat (Capra hircus) has been introduced. The method is simpler, cheaper and quicker to all the existing protocols. The material is easily available at a low cost and the method produces almost equally effective results within a short period of time. Therefore, this material and the method have been recommended both for classroom demonstration of somatic chromosomes and research. In essence, the model describes the use of HBSS as incubation medium (replacing costly culture media) for bone marrow cells collected from slaughtered goat and incubated in HBSS in presence of a metaphase arrester (colchicine) for varying length of time to standardize the optimum dose. The cells were collected by centrifugation, suspended in hypotonic solution followed by fixation in an appropriate chromatin fixative. The slides were prepared by flame dry technique, stained in Giemsa solution for observation. The diploid number has been conformed as 60 acrocentric chromosomes of varying length. Construction of karyotype revealed a heteromorphic sex chromosome status, in male 2n=60, XY and in female 2n=60, XX. Spontaneous ploidal variation and the occurrence of Robertsonian fusion have been recorded. The advantage of this modified ‘pocket friendly’ technique has been discussed.

‘Your true and proper gender’: the Barr body as a good enough science of sex

In the late 1940s, a microanatomist from London Ontario, Murray Barr, discovered a mark of sex chromosome status in bodily tissues, what came to be known as the ‘Barr body’. This discovery offered an important diagnostic technology to the burgeoning clinical science community engaged with the medical interpretation and management of sexual anomalies. It seemed to offer a way to identify the true, underlying sex in those whose bodies or lives were sexually anomalous (intersexuals, homosexuals and transsexuals). The hypothesis that allowed the Barr body to stand in for ‘chromosomal’ or ‘genetic’ sex was provisional, but it supported the expectation that genetic information established one’s primary identity, and the conviction that the animal world could be neatly divided into two, and only two, sexes. Ultimately, this provisional hypothesis, and its status as an unambiguous arbiter of true sex, was overturned. But during much of the 1950s, Barr’s thesis about the identity of the Barr body was consistent with a coherent set of theories and evidence explaining sexual development and sexual pathology. Though provisional, the scientific status of the sex chromatin within this system of knowledge was good enough to support a flourishing research enterprise in the clinical sciences.

Neonatal mice possessing an Sry transgene show a masculinized pattern of progesterone receptor expression in the brain independent of sex chromosome status.

To assess the relative roles of sex chromosome genes and gonadal steroid hormones in producing sex differences in progesterone receptor (PR) expression in the forebrain of neonatal mice, we used mice in which the Sry gene had been deleted from the Y-chromosome and inserted as a transgene on an autosome in both XX and XY genotypes. Levels of PR immunoreactivity (PRir) in the anteroventral periventricular nucleus, the medial preoptic nucleus, and the ventromedial nucleus were significantly higher in mice that possessed an Sry transgene compared with mice that lacked an Sry transgene, regardless of their complement of sex chromosomes (XX vs. XY). This result suggests that sexual differentiation of PR expression in these regions is likely controlled mainly by gonadal hormones, not by the genetic sex of the brain cells. No differences in PRir were detected between wild-type XY mice with the Sry gene on the Y-chromosome and XY mice with the Sry transgene, suggesting that testicular hormones produced in these two genotypes have comparable effects on neural tissue.

DNA methylation patterns in mouse and human zygotes

Objective: Epigenetic modification during embryogenesis is characterized by selective gene expression achieved through methylation of 5-methylcytosine. In mammals dynamic reprogramming of DNA methylation occurs during gametogenesis and embryogenesis. Changes in environment, e.g., in vivo vs in vitro culture, can alter the process of epigenetic modification. Staining of mouse zygotes with an antibody against 5-methylcytosine (Anti-MeC) has revealed that by 4–6 h post-fertilization the chromatids in the male pronucleus (PN) have been actively demethylated. This study examines whether a similar pattern of DNA methylation takes place in human zygotes. Design: Normally fertilized (2PN) mouse and abnormally fertilized human (3PN) zygotes were examined with Anti-MeC immunofluorescence staining. Sex chromosome status, assessed by fluorescence in situ hybridization (FISH), was used to identify the parental origin of each pronucleus. Materials and Methods: Mouse zygotes were obtained from in vitro fertilization of oocytes harvested from oviducts 16 h post-hCG. Abnormally fertilized zygotes (3PN) were obtained from patients undergoing conventional IVF or ICSI with patient consent (IBRA Protocol H 6092). Each zygote was fixed with methanol:acetic acid (3:1). DNA methylation patterns were detected in mouse zygotes by immunofluorescence staining with an Anti-MeC antibody (Barton et al 2001, Human Molecular Genetics, 26, 2983–2987). Human pronuclei were subjected to FISH to identify the X and Y chromosomes prior to staining with the anti-MeC antibody. Results: Staining patterns in mouse revealed active demethylation between sperm head decondensation and male PN formation. In contrast to the decline in paternal chromatid staining intensity, maternal chromatid staining remained strong throughout PN formation. All 19 conventionally inseminated human zygotes displayed one strongly and two weakly stained PNs. All 21 PNs with a Y chromosome stained poorly as did 17 of 34 PNs with an X chromosome, most likely those of paternal origin. In contrast, 8 zygotes from ICSI were digynic, displaying two strongly and one weakly staining PNs. Interestingly, an ICSI zygote displayed one strongly and two weakly fluorescing PNs, but it was diandric with 2 Y chromosomes. However, 2 ICSI zygotes presented 3 brightly stained PNs with no differences in intensity; these zygotes were X/X/Y and XX/X/O. Conclusion: Selective, active demethylation of the male PN occurs in both mouse and human zygotes. However, abnormal methylation patterns can occur in some zygotes and may indicate a dysfunctional ooplasm. A full investigation of the methylation patterns in donated normal embryos will provide more insights into the epigenetic modifications that occur during in vitro embryogenesis.

Development and targeted application of a rapid QF-PCR test for sex chromosome imbalance.

A QF-PCR test has been developed to diagnose sex chromosome imbalances in prenatal samples and has been applied to a diagnostic service. The test uses a PCR multiplex with eight primer pairs: six X-chromosome polymorphic markers, including two markers from Xp (a region not included in previously published sex chromosome aneuploidy tests), one polymorphic marker for a locus common to the long arms of the X and Y chromosomes, and the non-polymorphic amelogenin marker. Homozygosity for all X-chromosome markers and the absence of the Y-chromosome amelogenin marker is highly likely (907 : 1) to represent monosomy X (Turner syndrome), but interphase FISH is always used to confirm such a result. Blind studies were carried out to validate the test and the first year of clinical use has been reported. Results are usually issued within one working day, and the test is more efficient than interphase FISH. The sex chromosome imbalance test has been targeted to prenatal samples displaying a clear ultrasound indication consistent with Turner syndrome, and has also been used to identify fetal sex in pregnancies at risk of inheriting a sex-linked molecular disorder. No misdiagnoses were made. It is concluded that QF-PCR can rapidly and accurately diagnose sex chromosome status and imbalances, reducing maternal anxiety and aiding in efficient pregnancy management.

Derivation of nephrogenic adenomas from renal tubular cells in kidney-transplant recipients.

Nephrogenic adenomas are benign, tumor-like lesions within the urothelial mucosa of the urinary tract that are not uncommon in renal-transplant recipients. We investigated the origin of nephrogenic adenomas in renal-transplant recipients. Tissue sections were analyzed by fluorescence in situ hybridization with the use of probes for the X and Y chromosomes, by immunohistochemical methods with the use of antibodies to renal tubular antigens, and by lectin histochemical methods. Forty-six nephrogenic adenomas from 29 patients were analyzed. All nephrogenic adenomas in 14 female recipients of transplants from male donors and 10 male recipients of transplants from female donors showed the same sex-chromosome status as the donor kidney, but not the same sex-chromosome status as the recipient's surrounding bladder tissue. The nephrogenic adenomas from all 6 female recipients of transplants from female donors showed female chromosomes, and those from the 16 male recipients of transplants from male donors showed male chromosomes. The presence of aquaporin 1, PAX2, and lectin-binding capacity for peanut agglutinin, Lotus tetragonolobus agglutinin, and Sophora japonica agglutinin in nephrogenic adenomas indicated an origin from renal tubular cells. Nephrogenic adenomas in renal-transplant recipients are derived from tubular cells of the renal transplants and are not metaplastic proliferations of the recipient's bladder urothelium.

Analysis of meiosis in intratesticular germ cells from subjects affected by classic Klinefelter's syndrome.

Azoospermic subjects affected by Klinefelter's syndrome may occasionally show the presence of intratesticular residual foci of spermatogenesis, and the retrieval of mature spermatozoa from the testis may permit fertility and paternity by means of intracytoplasmic sperm injection. Previous studies have demonstrated that these subjects show the presence of an increased incidence of hyperaploid spermatozoa. Here we analyzed, by fluorescence in situ hybridization using specific probes for chromosomes 8, X, and Y, the spermatogenic process and the meiotic progression of 47,XXY germ cells retrieved by fine needle aspiration of the testis in ten azoospermic patients affected by classic Klinefelter's syndrome. All patients had lower testicular volume, higher gonadotropins, and lower testosterone plasma levels compared with control subjects. Cytological analysis of the testicular cells retrieved by fine needle aspiration showed the presence of Sertoli cells only in eight subjects, while germ cells were observed in two patients. In each patient Sertoli cells showed a 47,XXY karyotype, and the same chromosome pattern was observed in spermatogonia and primary spermatocytes of patients presenting a residual spermatogenesis. Secondary spermatocytes, spermatids, and mature spermatozoa showed different sex chromosome patterns, reflecting their origin from 47,XXY spermatogonia. In conclusion, this study demonstrated that, in subjects affected by Klinefelter's syndrome, residual germ cells may be present in the testis and that 47,XXY spermatogonia are able to undergo and complete the spermatogenic process leading to mature spermatozoa. These data further suggest the need to evaluate the sex chromosome status of sperm from patients affected by Klinefelter's syndrome undergoing assisted reproductive techniques.