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.

Cross-sectional study shows that impaired bone mineral status and metabolism are found in nonmosaic triple X syndrome.

AIM: The effect of a supernumerary X chromosome on bones has not been reported, and this study evaluated bone mineral status and metabolism in nonmosaic triple X syndrome. METHODS: This cross-sectional study comprised 19 girls, with a median age of 10.9 years, with nonmosaic triple X syndrome and a control group matched for age and body size. We studied ionised and total calcium, phosphate, parathyroid hormone (PTH), 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D, osteocalcin, bone alkaline phosphatase levels and urinary deoxypyridinoline concentrations. We also measured the phalangeal amplitude-dependent speed of sound (AD-SoS) and the bone transmission time (BTT) Z-scores. RESULTS: Patients with nonmosaic triple X syndrome showed significantly reduced AD-SoS (p < 0.005) and BTT Z-scores (p < 0.0001) compared to the control group, and these results persisted when we divided the sample into prepubertal and pubertal patients (p < 0.05). These patients also had significantly reduced ionised calcium (p < 0.005) and 25(OH)D levels (p < 0.005) and higher phosphate (p < 0.0001) and PTH (p < 0.0001) levels. CONCLUSION: Subjects with nonmosaic triple X syndrome exhibited a significant impairment in bone mineral status and metabolism similar to other X polisomy, such as Klinefelter's syndrome. This suggests the presence of a primary bone deficit and the need for regular and close monitoring of these subjects.

Hypogonadotropic hypogonadism in a trisomy X carrier: phenotype description and genotype correlation.

We report on a 31-year old female who presented at genetic counseling for a small uterus, secondary amenorrhea and sterility. Gonadotropic hormone levels were low, suggesting a Hypogonadotropic Hypogonadism (HH) condition. Cytogenetic analysis demonstrated the presence of Trisomy X associated to an interstitial deletion of chromosome 4q13.2, resulting in the complete loss of a copy of the GNRHR gene. As GNRHR is known to be responsible for an autosomal recessive form of HH, we checked the status of the undeleted allele and we found the Q106R substitution. In conclusion, the results of our cytogenetic and molecular analyses have allowed us to clarify the etiology of the patient's condition.

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.

The Turner syndrome in patient with 45X/47XXX mosaic karyotype--case report.

BACKGROUND: Turner syndrome (TS) is a gonadal dysgenesis related to partial or total lack of one of the X chromosomes. It this report we describe a young patient presenting some somatic features of TS, who underwent spontaneous puberty and was eumenoorheic up to the age of 23. METHODS: Using fluorescent in situ hybridization (FISH) mosaic karyotype (45X[131]/47XXX[9]) of TS and triple X syndrome was found. RESULTS: She presented uncommon for TS somatic hemihypotrophy and underwent growth hormone and surgical therapy. The patient was diagnosed with premature ovarian failure when she was 23, with absent follicular reserve. Clinical features of this case and a few published cases will be reviewed briefly.

Fold-change correction values for testicular somatic transcripts in gene expression studies of human spermatogenesis.

STUDY QUESTION: What are the reference values for delineating altered somatic cell gene expression from transcript enrichment/dilution in gene expression studies of human spermatogenesis? SUMMARY ANSWER: We have designed a crosstable and rule-of-thumb values for different stages of spermatogenic impairment that define the reference cut-off values for altered gene expression in Sertoli and Leydig cells in the context of impaired spermatogenesis. WHAT IS KNOWN ALREADY: Morphometrical studies have shown that on the cellular level, impaired spermatogenesis results in a relative enrichment of somatic cell types. However, until now it is not known how this affects transcript levels in gene expression studies. STUDY DESIGN, SIZE DURATION: In this study, 31 testis biopsies from men with different stages of spermatogenic impairment (full spermatogenesis, hypospermatogenesis, meiotic arrest, spermatogonial presence, Sertoli cell-only syndrome, complete tubular atrophy) were used to define reference ratios of somatic transcript enrichment/dilution. The reference ratios were validated on an independent test set of 28 samples and on gene expression data from men with Y-chromosomal microdeletions. PARTICIPANTS/MATERIAL, SETTING, METHODS: High-quality microarray data were filtered with respect to Sertoli- and Leydig-cell-specific genes. General reference enrichment/dilution factors for these two cell types for all combinations of spermatogenic impairment were calculated using robust permutation statistics. To validate the specificity of the filtered transcripts, we calculated ratios for an independent test set of spermatogenic impairment and for transcriptional data from men with Y-chromosomal microdeletions, and checked the functional enrichment (gene ontology) and cellular localization of the corresponding proteins in a histological database and assessed their correlation with testicular size. MAIN RESULTS AND THE ROLE OF CHANCE: Filtering of Sertoli- and Leydig-cell-specific genes resulted in a set of 54 and 332 transcripts, respectively. These were used in defining robust reference dilution/enrichment factors of somatic transcripts for all spermatogenic levels and were compiled in a reference crosstable. Validation on an independent test set showed ratios within 0.5 units of our reference crosstable. Analysis of the resulting transcripts with respect to functional enrichment for Sertoli- and Leydig-cell-specific functions and protein expression, as obtained from an immunohistochemical database, indicated filtering of data sets highly enriched for Sertoli and Leydig cell function. The dilution/enrichment ratios differed significantly when transcripts were interrogated in samples with Y-chromosomal microdeletions, pointing to an overall decreased expression of somatic markers in a genetically altered background. LIMITATIONS, REASONS FOR CAUTION: The defined reference ratios might apply with some restrictions in samples that display very heterogeneous histology (e.g. Sertoli cell only with a significant proportion of spermatogenic foci) or when spermatogenic impairment is a consequence of an altered genetic background. WIDER IMPLICATIONS OF THE FINDINGS: The reference dilution/enrichment values for somatic testicular transcripts as defined in this study are to be seen as cut-off values for discriminating between simple transcript dilution/enrichment as a consequence of an altered germ cell composition and actual transcriptional regulation. Future studies dealing with transcriptional changes in testicular somatic cells in a background of altered germ cell quantities should consider these correction factors in order to avoid the description of transcriptional changes that are based simply on shifts in somatic cellular quantities. STUDY FUNDING/COMPETING INTEREST(S): Financial support was from grant Sp721/1-3 of the German Research Foundation. There are no competing interests to be declared.

Spermatid development in XO male mice with varying Y chromosome short-arm gene content: evidence for a Y gene controlling the initiation of sperm morphogenesis.

We recently used three XO male mouse models with varying Y short-arm (Yp) gene complements, analysed at 30 days post partum, to demonstrate a Yp gene requirement for the apoptotic elimination of spermatocytes with a univalent X chromosome at the first meiotic metaphase. The three mouse models were i) XSxr(a)O in which the Yp-derived Tp(Y)1Ct(Sxr-a) sex reversal factor provides an almost complete Yp gene complement, ii) XSxr(b)O,Eif2s3y males in which Tp(Y)1Ct(Sxr-b) has a deletion completely or partially removing eight Yp genes - the Yp gene Eif2s3y has been added as a transgene to support spermatogonial proliferation, and iii) XOSry,Eif2s3y males in which the Sry transgene directs gonad development along the male pathway. In this study, we have used the same mouse models analysed at 6 weeks of age to investigate potential Yp gene involvement in spermiogenesis. We found that all three mouse models produce haploid and diploid spermatids and that the diploid spermatids showed frequent duplication of the developing acrosomal cap during the early stages. However, only in XSxr(a)O males did spermiogenesis continue to completion. Most strikingly, in XOSry,Eif2s3y males, spermatid development arrested at round spermatid step 7 so that no sperm head restructuring or tail development was observed. In contrast, in XSxr(b)O,Eif2s3y males, spermatids with substantial sperm head and tail morphogenesis could be easily found, although this was delayed compared with XSxr(a)O. We conclude that Sxr(a) (and therefore Yp) includes genetic information essential for sperm morphogenesis and that this is partially retained in Sxr(b).

Cytogenetic, Y chromosome microdeletion, sperm chromatin and oxidative stress analysis in male partners of couples experiencing recurrent spontaneous abortions.

PURPOSE: Etiology in majority of couples experiencing recurrent spontaneous abortions (RSA) is still unknown. The aim of the study was to find the role of cytogenetic abnormalities, Y chromosome microdeletion, oxidative stress (OS) and sperm DNA fragmentation in male partners of couples experiencing RSA. METHODS: Forty-eight couples with history of RSA and 20 fertile controls were included in the study. The study subjects were divided into male partners of RSA couples with abnormal sperm parameters (SA) (N = 16), male partners of RSA couples with normal sperm parameters (NS) (N = 32) and age-matched fertile controls with normal sperm parameters (FC) (N = 20). RESULTS: One of 48 men (2%) showed 46, XY (1qh-) chromosomal complement. None of the cases including FC showed deletion in any of the 3 AZF loci on Y chromosome long arm. Sperm count was found be significantly lower in SA cases as compared to group NS cases (P < 0.0001) and FC (P < 0.005). Sperm forward motility was found to be significantly (P < 0.05) lower in SA cases as compared to NS and FC. Male partners of RSA couples with abnormal sperm parameters had higher reactive oxygen species (ROS) levels (P < 0.005) and sperm DNA damage (P < 0.0001), however, in male partners of RSA couples with normal sperm parameters had only increased (P < 0.0001) sperm DNA damage. CONCLUSION: Other than chromosomal anomalies, sperm DNA fragmentation and seminal OS may be the underlying pathology in RSA, thus screening for seminal ROS levels and DNA fragmentation has diagnostic and prognostic capabilities.