Sex chromosome DSD individuals with mosaic 45,X0 and aberrant Y chromosomes in 46,XY cells: distinct gender phenotypes and germ cell tumour risks§.

"Differences of Sexual Development (DSD)," individuals with rearranged Y chromosome breaks in their 46,XY cells are reported with male and female gender phenotypes and differences in germ cell tumour (GCT) risk. This raised the question of whether male or female gender and GCT risk depends on the site of the break and/or rearrangement of the individual´s Y chromosome. In this paper, we report molecular mapping of the breakpoint on the aberrant Y chromosome of 22 DSD individuals with a 45,X/46,XY karyotype reared with a different gender. Their Y chromosome breaks are found at different sites on the long and short Y arms. Our data indicate that gender rearing is, neither dependent on the site of Y breakage, nor on the amount of 45,X0 cells in the individuals' leukocytes. Most prominent are secondary rearrangements of the Y chromosome breaks forming di-centric Y-structures ("dic-Y"). Duplications of the short Y arm and the proximal part of the long Y arm are the results. A putative GCT risk has been analysed with immunohistochemical experiments on some dysgenetic gonadal tissue sections. With specific antibodies for OCT3/4 expression, we marked the pluripotent germ cell fraction being potential tumour precursor cells. With specific antibodies for DDX3Y, TSPY, and UTY we analyzed their putative Gonadoblastoma Y (GBY) tumour susceptibility function in the same specimen. We conclude GBY expression is only diagnostic for GCT development in the aberrant germ cells of these DSD individuals when strong OCT3/4 expression has marked their pluripotency.

Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99.

Mosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.

Possible misdiagnosis of 46,XX testicular disorders of sex development in infertile males.

Objectives: The 46,XX disorders of sex development (DSD) is a rare genetic cause of male infertility and possible misdiagnosis of this condition has never been reported. We aim to investigate clinical characteristics and laboratory results of infertile males with possibly misdiagnosed 46,XX DSD. Methods: Between January 2008 and December 2017, a retrospective case series study was performed involving sixteen 46,XX DSD males without azoospermia factor (AZF) deletion. Demographics, clinical features, laboratory results and assisted reproductive technology (ART) outcomes of these patients were depicted, and the underlying accurate diagnosis was also discussed. Results: The mean age was 30.06 ± 5.40 years old. Thirteen patients (81.25%) merely obtained secondary school education. Gynaecomastia occurred in one case, and cryptorchidism appeared in two cases. Testicular volumes were equal to 15 mL on two sides in one patient who had severe asthenozoospermia. Thirteen patients (81.25%) had bilateral atrophic testes which were below 5 mL. The majority of patients were observed with elevated levels of gonadotropic hormones and decreased testosterone values. Neither AZF region nor sex-determining region Y gene was absent among all patients. Twelve patients had normal ejaculatory function, whereas four were diagnosed with ejaculatory dysfunction. Eleven patients (68.75%) were diagnosed with azoospermia. Testicular sperm aspiration was performed in six subjects (37.50%). The pathological results showed that Leydig cell hyperplasia with spermatic failure was found in each case, and no sperm was found in testicular tissue. ART with donor sperm was conducted in 15 patients. Live birth was achieved in three cases through artificial insemination by donor and in one case using in-vitro fertilization by donor. Conclusions: Chromosomal analysis rarely yields 46,XX karyotype combined with no deletion of AZF in infertile males. Under this condition, molecular analysis should be conducted to avoid potential misdiagnosis and false interpretation of other findings.

New insights into the potential mechanisms of spermatogenic failure in patients with idiopathic azoospermia.

Idiopathic azoospermia (IA) refers to azoospermia without a clear aetiology. Due to the unclear aetiology and pathological mechanism of IA, there is no effective treatment for IA. The development of assisted reproductive and microsperm extraction technologies has brought hope to patients with IA with fertility problems. However, there are still many patients with IA whose testes lack healthy sperm, causing infertility. Therefore, it is key to identify how testicular spermatogenic failure can be reversed to promote spermatogenesis in patients with IA to resolve fertility problems; these goals are a great challenge in reproductive medicine. The underlying genetic factors seem to be important pathological factors of IA. Understanding the role of genetic factors in the pathological mechanism of spermatogenic failure in patients with IA is of great value for future studies and treatments and is also an important reference for the reproductive health of males and their offspring. A method combining sequencing technology and bioinformatics analysis is an important means to understand the genetic pathological mechanisms. We used bioinformatics analysis to study the public human IA dataset. We found that the pathogenic mechanism of IA may be related to abnormal ciliary structure and function and disrupted RNA metabolism in spermatogenic cells. Disrupted m6A regulation of spermatogenesis may be an important pathological mechanism of IA and warrants attention. Finally, we screened for key genes and potential therapeutic drugs to determine future research directions.

Extreme downregulation of chromosome Y and Alzheimer's disease in men.

Research has revealed scarcely any biological factors of Alzheimer's disease (AD) that are specific to men. Here, we found that the extreme downregulation of chromosome Y (EDY) increases the age-related risk of AD in men. We considered that EDY was a possible male-specific pathway toward AD because EDY is the most likely consequence of the mosaic loss of chromosome Y, which has been recently associated with AD. We studied EDY in the undiseased brain of 371 individuals and observed that it co-occurred across multiple brain regions (p < 10-4) and associated with rs114241159 (p = 1.53 × 10-7) within ACSS3/PPFIA2, previously linked to amyloid beta concentrations. We also analyzed the 5 largest transcriptomic case-control studies, publicly available to date on AD (cases/controls = 556/462) and found a significant interaction with age (OREDY × age = 1.22, p = 0.0038). Our analyses suggest that aging men who live longer by avoiding EDY are more resilient to AD than those who do not.

Mosaic loss of human Y chromosome: what, how and why.

Y chromosome (ChrY), the male-specific sex chromosome, has been considered as a genetic wasteland. Aging-related mosaic loss of ChrY (LOY) has been known for more than half a century, but it was constantly considered as a neutral karyotype related to normal aging. These views have been challenged with genome-wide association studies identifying mosaic LOY in human somatic cells is the most commonly acquired mutation in male's genome and is associated with a wide spectrum of human diseases including cancer, Alzheimer's disease, and cardiovascular disease. These previously undescribed clinical significances deeply modify our perception on ChrY and open up a range of new questions. Here, we review the latest advances in our knowledge of the biological origins and clinical consequences of mosaic LOY. We highlight the association of mosaic LOY to pathogenic conditions and evaluate the cause-and-consequence relationships between mosaic LOY and pathogenesis. The known risk factors of mosaic LOY including age, genetic variants, ChrY structural aberrations and environmental stressors are discussed. In light of evidence from pioneering and more recent studies, we propose the micronucleation hypothesis and centromere-dysfunction and telomere-attrition models to explain how mosaic LOY occurs and why ChrY is prone to lose. We believe it is importantly and timely to extend mosaic LOY research from epidemiological associations to mechanistic studies. In this regard, we outline important gaps and assess several future directions from a biological and clinical perspective. An improved understanding of mosaic LOY will open new pathways to modify and increase healthy aging in males.

Age-dependent DNA methylation patterns on the Y chromosome in elderly males.

The Y chromosome, a sex chromosome that only exists in males, has been ignored in traditional epigenetic association studies for multiple reasons. However, sex differences in aging-related phenotypes and mortality could suggest a critical role of the sex chromosomes in the aging process. We obtained blood-based DNA methylation data on the Y chromosome for 624 men from four cohorts and performed a chromosome-wide epigenetic association analysis to detect Y-linked CpGs differentially methylated over age and cross-validated the significant CpGs in the four cohorts. We identified 40-219 significant CpG sites (false discovery rate <0.05) with >82% of them hypermethylated with increasing age, which is in strong contrast to the patterns reported on the autosomal chromosomes. Comparing the rate of change in the Y-linked DNA methylation across cohorts that represent different age intervals revealed a trend of acceleration in DNA methylation with increasing age. The age-dependent DNA methylation patterns on the Y chromosome were further examined for their association with all-cause mortality with results suggesting that the predominant pattern of age-related hypermethylation on the Y chromosome is associated with reduced risk of death.

Usefulness of imprint cytology of gonadoblastoma with dysgerminoma in a patient with Turner syndrome and a Y chromosome: A case report and literature review.

Ovarian gonadoblastoma coexisting with a dysgerminoma is extremely rare in patients with Turner syndrome (TS) and a Y chromosome. The cytological findings, including imprint cytology, of these unusual ovarian tumors have rarely been reported. We report a rare patient with a gonadoblastoma with dysgerminoma, 3.0 × 2.0 cm in size; she was a 19-year-old woman with TS and a Y chromosome. She underwent laparoscopic bilateral gonadectomy, and the tumor was classified as stage IA (pT1aNxM0) according to the International Federation of Gynecology and Obstetrics classification system. Intraoperative imprint cytology revealed two types of neoplastic cells: small tumor cells surrounding light green-stained or eosinophilic hyaline globules with marked calcification, suspicious for gonadoblastoma; and large, round, atypical cells with abundant cytoplasm, macronucleoli, and marked lymphocytic infiltration (two-cell pattern), suspicious for dysgerminoma. The cytology results in our patient may represent the second reported results of imprint cytology describing a gonadoblastoma with dysgerminoma. They are the first reported results in a patient with TS and a Y chromosome.

Multibatch TMT Reveals False Positives, Batch Effects and Missing Values.

Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, tandem mass tags (TMT) in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We identified a significant inflation rate of protein missing values as multiple batches are integrated and show that this pattern is aggravated at the peptide level. We also show that without normalization strategies to address the batch effects, the high precision of quantitation within a single multiplexed TMT batch is not reproduced when data from multiple TMT batches are integrated.Further, the incidence of false positives was studied by using Y chromosome peptides as an internal control. The iPSC lines quantified in this data set were derived from both male and female donors, hence the peptides mapped to the Y chromosome should be absent from female lines. Nonetheless, these Y chromosome-specific peptides were consistently detected in the female channels of all TMT batches. We then used the same Y chromosome specific peptides to quantify the level of ion coisolation as well as the effect of primary and secondary reporter ion interference. These results were used to propose solutions to mitigate the limitations of multi-batch TMT analyses. We confirm that including a common reference line in every batch increases precision by facilitating normalization across the batches and we propose experimental designs that minimize the effect of cross population reporter ion interference.

Gonadoblastoma Y locus genes expressed in germ cells of individuals with dysgenetic gonads and a Y chromosome in their karyotypes include DDX3Y and TSPY.

STUDY QUESTION: Which Y genes mapped to the 'Gonadoblastoma Y (GBY)' locus on human Y chromosome are expressed in germ cells of individuals with some Differences of Sexual Development (DSD) and a Y chromosome in their karyotype (DSD-XY groups)? SUMMARY ANSWER: The GBY candidate genes DDX3Y and TSPY are expressed in the germ cells of DSD-XY patients from distinct etiologies: patients with mixed gonadal dysgenesis (MGD) and sex chromosome mosaics (45,X0/46,XY; 46,XX/46,XY); patients with complete androgen insensitivity (CAIS), patients with complete gonadal dysgenesis (CGD; e.g. Swyer syndrome). WHAT IS KNOWN ALREADY: A GBY locus was proposed to be present on the human Y chromosome because only DSD patients with a Y chromosome in their karyotype have a high-although variable-risk (up to 55%) for germ cell tumour development. GBY was mapped to the proximal part of the short and long Y arm. TSPY located in the proximal part of the short Y arm (Yp11.1) was found to be a strong GBY candidate gene. It is expressed in the germ cells of DSD-XY patients with distinct etiologies but also in foetal and pre-meiotic male spermatogonia. However, the GBY region extends to proximal Yq11 and therefore includes probably more than one candidate gene. STUDY DESIGN, SIZE, DURATION: Protein expression of the putative GBY candidate gene in proximal Yq11, DDX3Y, is compared with that of TSPY in serial gonadal tissue sections of 40 DSD-XY individuals from the three DSD patient groups (MGD, Complete Androgen Insensitivity Syndrome [CAIS], CGD) with and without displaying malignancy. Expression of OCT3/4 in the same tissue samples marks the rate of pluripotent germ cells. PARTICIPANTS/MATERIALS, SETTING, METHOD: A total of 145 DSD individuals were analysed for the Y chromosome to select the DSD-XY subgroup. PCR multiplex assays with Y gene specific marker set score for putative microdeletions in GBY Locus. Immunohistochemical experiments with specific antisera mark expression of the GBY candidate proteins, DDX3Y, TSPY, in serial sections of the gonadal tissue samples; OCT3/4 expression analyses in parallel reveal the pluripotent germ cell fraction. MAIN RESULTS AND THE ROLE OF CHANCE: Similar DDX3Y and TSPY protein expression patterns were found in the germ cells of DSD-XY patients from each subgroup, independent of age. In CAIS patients OCT3/4 expression was often found only in a fraction of these germ cells. This suggest that GBY candidate proteins are also expressed in the non-malignant germ cells of DSD-XY individuals like in male spermatogonia. LIMITATIONS, REASONS FOR CAUTION: Variation of the expression profiles of GBY candidate genes in the germ cells of some DSD-XY individuals suggests distinct transcriptional and translational control mechanisms which are functioning during expression of these Y genes in the DSD-XY germ cells. Their proposed GBY tumour susceptibility function to transform these germ cells to pre-malignant GB/Germ Cell Neoplasia in Situ (GB/GCNIS) cells seems therefore to be limited and depending on their state of pluripotency. WIDER IMPLICATIONS OF THE FINDINGS: These experimental findings are of general importance for each individual identified in the clinic with DSD and a Y chromosome in the karyotype. To judge their risk of germ cell tumour development, OCT3/4 expression analyses on their gonadal tissue section is mandatory to reveal the fraction of germ cells still being pluripotent. Comparative expression analysis of the GBY candidate genes can be helpful to reveal the fraction of germ cells with genetically still activated Y chromosomes contributing to further development of malignancy if at high expression level. STUDY FUNDING/COMPETING INTEREST(S): This research project was supported by a grant (01GM0627) from the BMBF (Bundesministerium für Bildung und Forschung), Germany to P.H.V. and B.B. The authors have no competing interests.

Sex-chromosome dosage effects on gene expression in humans.

A fundamental question in the biology of sex differences has eluded direct study in humans: How does sex-chromosome dosage (SCD) shape genome function? To address this, we developed a systematic map of SCD effects on gene function by analyzing genome-wide expression data in humans with diverse sex-chromosome aneuploidies (XO, XXX, XXY, XYY, and XXYY). For sex chromosomes, we demonstrate a pattern of obligate dosage sensitivity among evolutionarily preserved X-Y homologs and update prevailing theoretical models for SCD compensation by detecting X-linked genes that increase expression with decreasing X- and/or Y-chromosome dosage. We further show that SCD-sensitive sex-chromosome genes regulate specific coexpression networks of SCD-sensitive autosomal genes with critical cellular functions and a demonstrable potential to mediate previously documented SCD effects on disease. These gene coexpression results converge with analysis of transcription factor binding site enrichment and measures of gene expression in murine knockout models to spotlight the dosage-sensitive X-linked transcription factor ZFX as a key mediator of SCD effects on wider genome expression. Our findings characterize the effects of SCD broadly across the genome, with potential implications for human phenotypic variation.

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system.

Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility.

Sex chromosome-dependent differential viability of human spermatozoa during prolonged incubation.

STUDY QUESTION: Are there significant differences in the ability of X chromosome-bearing (X) spermatozoa and Y chromosome-bearing (Y) spermatozoa to survive incubation under stressful conditions? SUMMARY ANSWER: Y spermatozoa are more vulnerable to stress than their X counterparts depending on culture period and temperature, and show higher expression of apoptotic proteins. WHAT IS KNOWN ALREADY: The primary sex ratio is determined by there being an equal number of spermatozoa carrying X and Y chromosomes. This balance can be skewed by exposure to stressful environmental conditions such as changes in pH, pollutants or endocrine disruptors. However, less is known about the ability of sperm carrying either sex chromosome to withstand environmental stress. STUDY DESIGN, SIZE, DURATION: The difference in survival between X and Y spermatozoa was evaluated by measuring motility, viability and Y:X chromosome ratio during incubation for 5 days, at three temperatures (4, 22 and 37°C), and three pH conditions (6.5, 7.5 and 8.5). To identify the critical factors that determine the survival of X and Y bearing spermatozoa, we analysed the expression levels of apoptosis-related proteins (Bcl, Bax and Caspase-3), as well as the extent of DNA damage under a subset of conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples were obtained by masturbation from normozoospermic donors after 3 days of sexual abstinence. Four samples with >60% motility from different donors were mixed to obtain sufficient semen and eliminate sampling-related bias. Data are presented as mean ± SD of three independent experiments. Mean age of donors was 28.7 ± 3.2 years. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 58 489 spermatozoa were scored. The viability of Y spermatozoa was lower after exposure to different temperatures and culture periods than that of X spermatozoa (P < 0.05). Increased expression of apoptotic proteins in live Y spermatozoa was observed, despite the addition of tocopherol to the culture medium (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Spermatozoa were cultured in vitro during the treatment period. It is difficult to extrapolate the observed lifespan differences to spermatozoa survival in vivo. The experiments were replicated only three times. WIDER IMPLICATIONS OF THE FINDINGS: The prolonged survival of X spermatozoa under stressful conditions might lead to shifts in the ratio of male-to-female births. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. NRF-2014R1A2A2A01002706). The authors declare no competing financial interests.

Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining.

Chromosome missegregation into a micronucleus can cause complex and localized genomic rearrangements known as chromothripsis, but the underlying mechanisms remain unresolved. Here we developed an inducible Y centromere-selective inactivation strategy by exploiting a CENP-A/histone H3 chimaera to directly examine the fate of missegregated chromosomes in otherwise diploid human cells. Using this approach, we identified a temporal cascade of events that are initiated following centromere inactivation involving chromosome missegregation, fragmentation, and re-ligation that span three consecutive cell cycles. Following centromere inactivation, a micronucleus harbouring the Y chromosome is formed in the first cell cycle. Chromosome shattering, producing up to 53 dispersed fragments from a single chromosome, is triggered by premature micronuclear condensation prior to or during mitotic entry of the second cycle. Lastly, canonical non-homologous end joining (NHEJ), but not homology-dependent repair, is shown to facilitate re-ligation of chromosomal fragments in the third cycle. Thus, initial errors in cell division can provoke further genomic instability through fragmentation of micronuclear DNAs coupled to NHEJ-mediated reassembly in the subsequent interphase.

Y chromothripsis?

Micronucleation of missegregated chromatin can lead to substantial chromosome rearrangements via chromothripsis. However, the molecular details of micronucleus-based chromothripsis are still unclear. Now, an elegant system that specifically induces missegregation of the Y chromosome provides insight into this process, including a role for non-homologous end joining.

How Many Non-coding RNAs Does It Take to Compensate Male/Female Genetic Imbalance?

Genetic sex determination in mammals relies on dimorphic sex chromosomes that confer phenotypic/physiologic differences between males and females. In this heterogametic system, X and Y chromosomes diverged from an ancestral pair of autosomes, creating a genetic disequilibrium between XX females and XY males. Dosage compensation mechanisms alleviate intrinsic gene dosage imbalance, leading to equal expression levels of most X-linked genes in the two sexes. In therian mammals, this is achieved through inactivation of one of the two X chromosomes in females. Failure to undergo X-chromosome inactivation (XCI) results in developmental arrest and death. Although fundamental for survival, a surprising loose conservation in the mechanisms to achieve XCI during development in therian lineage has been, and continues, to be uncovered. XCI involves the concerted action of non-coding RNAs (ncRNAs), including the well-known Xist RNA, and has thus become a classical paradigm to study the mode of action of this particular class of transcripts. In this chapter, we will describe the processes coping with sex chromosome genetic imbalance and how ncRNAs underlie dosage compensation mechanisms and influence male-female differences in mammals. Moreover, we will discuss how ncRNAs have been tinkered with during therian evolution to adapt XCI mechanistic to species-specific constraints.

DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs.

Deletions of the AZFa region (AZoospermia Factor-a) region of the human Y chromosome cause irreversible spermatogenic failure that presents clinically in men as Sertoli-cell only (SCO) pathology of the testis. Deletions of the AZFa region typically encompass two genes: DDX3Y and USP9Y. However, human genetic evidence indicates that SCO is most tightly linked to deletion of DDX3Y and that deletions/mutations of USP9Y can be transmitted from one generation to the next. Here, we generated stable iPSC lines with AZFa deletions, tested complementation via introduction of DDX3Y, and assessed ability to form germ cells in vivo in a xenotransplantation model. We observed a quantifiable improvement in formation of germ cell like cells (GCLCs) from complemented donor iPSCs. Moreover, expression of UTF1, a prospermatogonial protein, was restored in cells complemented by introduction of DDX3Y on the AZFa background. Whole-genome RNA sequencing of purified GCLCs revealed an enrichment of genes involved in translational suppression and transcriptional control in DDX3Y-rescued GCLCs over mutant GCLCs, which maintained a molecular phenotype more similar to undifferentiated iPSCs. This study demonstrates the ability to probe fundamental genetics of human germ cell formation by complementation and indicates that DDX3Y functions in the earliest stages of human germ cell development.

Rapid and simultaneous screening of 47,XXY and AZF microdeletions by quadruplex real-time polymerase chain reaction.

We developed a quadruplex real-time PCR assay that allows rapid and simultaneous detection of 47,XXY and azoospermia factor (AZF) microdeletions on Y chromosome. The quadruplex assay consisted of four hydrolysis probes and primer sets. Three probes and the corresponding primers were used to qualitatively detect AZFa, AZFb, and AZFc deletions. For the detection of 47,XXY, the hydrolysis probe-mediated melting analysis was conducted to analyze the relative amounts of X and Y chromosomes. The quadruplex assay for detecting 47,XXY was characterized by very high analytical specificity (100%) in a wide template DNA range (2-100 ng). The detection limit of the assay was 2 ng of genomic DNA, and the optimal template DNA amount for the detection of 47,XXY was 25 ng. The quadruplex assay for detecting 47,XXY and AZF microdeletions has also demonstrated very high diagnostic sensitivity and specificity (100%). The assay was found to be rapid, sensitive, reliable, and inexpensive. This method is suggested to be applied as a first-step tool in genetic screening of patients with non-obstructive azoospermia and severe oligospermia.