Ken McElreavey

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.

The southwestern and Central Asian corridor has played a pivotal role in the history of humankind, witnessing numerous waves of migration of different peoples at different times. To evaluate the effects of these population movements on the current genetic landscape of the Iranian plateau, the Indus Valley, and Central Asia, we have analyzed 910 mitochondrial DNAs (mtDNAs) from 23 populations of the region. This study has allowed a refinement of the phylogenetic relationships of some lineages and the identification of new haplogroups in the southwestern and Central Asian mtDNA tree. Both lineage geographical distribution and spatial analysis of molecular variance showed that populations located west of the Indus Valley mainly harbor mtDNAs of western Eurasian origin, whereas those inhabiting the Indo-Gangetic region and Central Asia present substantial proportions of lineages that can be allocated to three different genetic components of western Eurasian, eastern Eurasian, and south Asian origin. In addition to the overall composite picture of lineage clusters of different origin, we observed a number of deep-rooting lineages, whose relative clustering and coalescent ages suggest an autochthonous origin in the southwestern Asian corridor during the Pleistocene. The comparison with Y-chromosome data revealed a highly complex genetic and demographic history of the region, which includes sexually asymmetrical mating patterns, founder effects, and female-specific traces of the East African slave trade.

The mammalian Y chromosome carries the SRY gene, which determines testis formation. Here we review data on individuals who are XX but exhibit male characteristics: some have SRY; others do not. We have analyzed three families containing more than one such individual and show that these individuals lack SRY. Pedigree analysis leads to the hypothesis that they carry recessive mutations (in a gene termed Z) that allow expression of male characteristics. We propose that wild-type Z product is a negative regulator of male sex determination and is functional in wild-type females. In males, SRY product represses or otherwise negatively regulates Z and thereby allows male sex determination. This hypothesis can also explain other types of sex reversal in mammals, in particular, XY females containing SRY. Some of these individuals may have mutations at the Z locus rendering them insensitive to SRY. Recessive mutations (such as the polled mutation of goats) leading to sex reversal are known in a variety of animals and might be used to map and ultimately clone the human Z gene.

SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated (SRY, SOX9) or over-expressed (DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination.

In many centres, Y chromosome deletion analysis is still not performed routinely and if so, the results are used for genetic counselling but are not considered as having a useful prognostic value. The type of deletion (AZFa, b or c) has been proposed as a potential prognostic factor for sperm retrieval in men undergoing TESE. AZFc deletions and partial AZFb deletions are associated with sperm retrieval in approximately 50% of cases while in the case of a patient with complete AZFb deletion the probability of finding mature spermatozoa is virtually nil. Therefore the extent and position of a Y microdeletion is important (complete or partial). The prognostic value of Y chromosome deletion analysis in cases of oligozoospermia is important when one considers the progressive decrease of sperm number over time in men with AZFc deletions. Cryo-conservation of spermatozoa in these cases could avoid invasive techniques, such as TESE/ICSI, in the future. Male offspring that are conceived by ICSI or IVF techniques from father with oligozoospermia or azoospermia would also benefit from knowledge of their Y status, since the identification of the genetic defect will render future medical or surgical therapies unnecessary. Y microdeletion screening is therefore important, not only to define the aetiology of spermatogenic failure, but also because it gives precious information for a more appropriate clinical management of both the infertile male and his future male child.

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%–50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of <i>NR5A1</i> have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in <i>NR5A1</i> cause male infertility, we sequenced <i>NR5A1</i> in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in <i>NR5A1</i>. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. <i>NR5A1</i> mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY. Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.

Conference Article| February 01 1991 Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord KENNETH D. McELREAVEY; KENNETH D. McELREAVEY *Department of Haematology, The Queen's University of Belfast, Belfast BT7 1NN Northern Ireland, U.K. Search for other works by this author on: This Site PubMed Google Scholar ALEXANDRA I. IRVINE; ALEXANDRA I. IRVINE *Department of Haematology, The Queen's University of Belfast, Belfast BT7 1NN Northern Ireland, U.K. Search for other works by this author on: This Site PubMed Google Scholar KEVIN T. ENNIS; KEVIN T. ENNIS 2Department of Medical Genetics, The Queen's University of Belfast, Belfast BT7 1NN Northern Ireland, U.K. Search for other works by this author on: This Site PubMed Google Scholar W.H. IRWIN McLEAN W.H. IRWIN McLEAN 2Department of Medical Genetics, The Queen's University of Belfast, Belfast BT7 1NN Northern Ireland, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (1): 29S. https://doi.org/10.1042/bst019029s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation KENNETH D. McELREAVEY, ALEXANDRA I. IRVINE, KEVIN T. ENNIS, W.H. IRWIN McLEAN; Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord. Biochem Soc Trans 1 February 1991; 19 (1): 29S. doi: https://doi.org/10.1042/bst019029s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: FCS, foetal calf serum, FITC, fluorescein isothiocyanate This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

<b>Background:</b> Complete deletion of the complete <i>AZFc</i> interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial <i>AZFc</i> deletions (gr/gr and b1/b3) that remove some copies of all <i>AZFc</i> genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. <b>Methods:</b> To determine the incidence of various partial <i>AZFc</i> deletions and their effect on fertility, we combined quantitative and qualitative analyses of the <i>AZFc</i> interval at the <i>DAZ</i> and <i>CDY1</i> loci in 300 infertile men and 399 control men. <b>Results:</b> We detected 34 partial <i>AZFc</i> deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p&gt;0.05). Our data provide evidence for two large <i>AZFc</i> inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate <i>DAZ1/2</i>, <i>DAZ3/4</i>, <i>CDY1a</i> (proximal), and <i>CDY1b</i> (distal) and define four types of <i>DAZ</i>-<i>CDY1</i> gr/gr deletion. <b>Conclusions:</b> The only deletion type to show an association with infertility was <i>DAZ3/4</i>-<i>CDY1a</i> (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the <i>CDY1a</i> SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

The origins and dispersal of farming and pastoral nomadism in southwestern Asia are complex, and there is controversy about whether they were associated with cultural transmission or demic diffusion. In addition, the spread of these technological innovations has been associated with the dispersal of Dravidian and Indo-Iranian languages in southwestern Asia. Here we present genetic evidence for the occurrence of two major population movements, supporting a model of demic diffusion of early farmers from southwestern Iran-and of pastoral nomads from western and central Asia-into India, associated with Dravidian and Indo-European-language dispersals, respectively.

The role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genes (SPINK5, KLK7 and FLG) have been associated with atopic dermatitis. A functional relevance has already been established for filaggrin variants, but not for SPINK5 and KLK7 polymorphisms. The objectives of this study were to confirm the association between SPINK5, KLK7, FLG variants and atopic dermatitis and to assess how variants influence selected phenotypic traits. This cross-sectional study was carried out over 20 months in 99 children and adults with atopic dermatitis (median age 7 years). The following items were analysed: SCORAD, TEWL, ichthyosis vulgaris, presence of asthma, total IgE serum levels. The SPINK5 E420K SNP, the KLK7 4bp insertion polymorphism and the filaggrin mutants (R510X and 2282del4) were analysed as described previously. The control group for genetic analysis was recruited in an ethnically matched, phenotypically anonymous cohort (n=102). The allelic frequencies were 0.525 for SPINK5, 0.26 for KLK7 polymorphisms, 0.101 and 0.075 for 2282del4 and R501X FLG mutants, respectively. The association of atopic dermatitis with filaggrin variants was confirmed, but not that of SPINK5 or KLK7 polymorphisms. SCORAD and TEWL measurements were not influenced by any of the variants. The SPINK5 polymorphism was associated with high IgE serum levels (p=0.011). Abnormal barrier genes do not influence the severity of atopic dermatitis. The SPINK5 gene polymorphism may modulate systemic immune effects favouring the IgE response to atopens. TEWL does not allow the characterization of subsets of patients with or without abnormal barrier genes.

Microdeletions of the long arm of the Y chromosome (Yq) are a common cause of male infertility. Since large structural rearrangements of the Y chromosome are commonly associated with a 45,XO/46,XY chromosomal mosaicism, we studied whether submicroscopic Yq deletions could also be associated with the development of 45,XO cell lines. We studied blood samples from 14 infertile men carrying a Yq microdeletion as revealed by polymerase chain reaction (PCR). Patients were divided into two groups: group 1 (n = 6), in which karyotype analysis demonstrated a 45,X/46,XY mosaicism, and group 2 (n = 8) with apparently a normal 46,XY karyotype. 45,XO cells were identified by fluorescence in-situ hybridization (FISH) using X and Y centromeric probes. Lymphocytes from 11 fertile men were studied as controls. In addition, sperm cells were studied in three oligozoospermic patients in group 2. Our results showed that large and submicroscopic Yq deletions were associated with significantly increased percentages of 45,XO cells in lymphocytes and of sperm cells nullisomic for gonosomes, especially for the Y chromosome. Moreover, two isodicentric Y chromosomes, classified as normal by cytogenetic methods, were detected. Therefore, Yq microdeletions may be associated with Y chromosomal instability leading to the formation of 45,XO cell lines.

Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias.We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein.Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations.

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10−5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.

Significance Sex determination involves antagonistic interactions between the testis-determining (SRY-SOX9-FGF9) and ovary-promoting (RSPO1-WNT/β-catenin-FOXL2) pathways, but the underlying molecular mechanisms remain unclear. We show that ZNRF3, an E3 ubiquitin ligase that inhibits WNT signaling and is a direct target of RSPO1-mediated membrane clearance, is testis-determining in mice. Testis determination defects in the absence of ZNRF3 arise due to ectopic canonical WNT signaling in XY gonads at the sex-determining stage. We identify human ZNRF3 sequence variants in cases of 46,XY disorders of sex development with XY female presentation. In vitro functional assays show that these variants disrupt ZNRF3 function. Our data reveal a sex-determining role for ZNRF3 and indicate that interactions between ZNRF3 and RSPO1 regulate mammalian sex determination.

Disorders of Sex Development (DSD) are a heterogeneous group of disorders affecting gonad and/or genito‐urinary tract development and usually the endocrine‐reproductive system. A genetic diagnosis is made in only around 20% of these cases. The genetic causes of 46,XX‐ SRY negative testicular DSD as well as ovotesticular DSD are poorly defined. Duplications involving a region located ∼600 kb upstream of SOX9 , a key gene in testis development, were reported in several cases of 46,XX DSD. Recent studies have narrowed this region down to a 78 kb interval that is duplicated or deleted respectively in 46,XX or 46,XY DSD. We identified three phenotypically normal patients presenting with azoospermia and 46,XX testicular DSD. Two brothers carried a 83.8 kb duplication located ∼600 kb upstream of SOX9 that overlapped with the previously reported rearrangements. This duplication refines the minimal region associated with 46,XX‐ SRY negative DSD to a 40.7–41.9 kb element located ∼600 kb upstream of SOX9 . Predicted enhancer elements and evolutionary‐conserved binding sites for proteins known to be involved in testis determination are located within this region. © 2015 Wiley Periodicals, Inc.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

In 46,XY men, testis is determined by a genetic network(s) that both promotes testis formation and represses ovarian development. Disruption of this process results in a lack of testis-determination and affected individuals present with 46,XY gonadal dysgenesis (GD), a part of the spectrum of Disorders/Differences of Sex Development/Determination (DSD). A minority of all cases of GD are associated with pathogenic variants in key players of testis-determination, SRY, SOX9, MAP3K1 and NR5A1. However, most of the cases remain unexplained. Recently, unbiased exome sequencing approaches have revealed new genes and loci that may cause 46,XY GD. We critically evaluate the evidence to support causality of these factors and describe how functional studies are continuing to improve our understanding of genotype-phenotype relationships in genes that are established causes of GD. As genomic data continues to be generated from DSD cohorts, we propose several recommendations to help interpret the data and establish causality.

We have isolated the human homologue of the mouse germ cell-specific transcript Tpx2, which we had previously mapped to mouse chromosome 17. Sequence analysis shows that the human gene is part of the DAZ (Deleted in Azoospermia) family, represents the human homologue of the mouse Dazla and Drosophila boule genes, and is termed DAZLA. Like Dazla and boule, DAZLA is single copy and maps to 3p25. This defines a new region of synteny between mouse chromosome 17 and human chromosome 3. Unlike DAZ, which has multiple DAZ repeats, DAZLA encodes a putative RNA-binding protein with a single RNA-binding motif and a single DAZ repeat. DAZLA is more closely related to Dazla in the mouse than to the Y-linked homologue DAZ (88% identity overall with mouse Dazla compared to 76% identity with the human DAZ protein sequence). Southern blot analysis showed that DAZLA is autosomal in all mammals tested and that DAZ has been recently translocated to the Y chromosome, sometime after the divergence of Old World and New World primates. To investigate the evolutionary relatedness of DAZLA and DAZ further, their partial genomic structures were obtained and compared. This revealed that the genomic organization of both genes in the 5' region is highly conserved. DAZLA is a new member of the DAZ family of genes, which is associated with spermatogenesis and male sterility. Familial cases of male infertility in humans show an autosomal recessive mode of inheritance. It is possible that some of these families may carry mutations in the DAZLA gene.

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] [11] and as many as 5% of males are infertile [11] [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.

Several new genes and pathways have been identified in recent years associated with human errors of sex-determination or DSD. SOX family gene mutations, as well as mutations involving GATA4, FOG2 and genes involved in MAP kinase signaling have been associated with virilization in 46,XX individuals or with 46,XY gonadal dysgenesis. Furthermore, mutations involving another key gene in sex-determination, NR5A1, are now known to be an important cause spermatogenic failure in the male and ovarian insufficiency in the female. These new findings offer insights into human sex-determination and highlight important differences between the human and mouse model. This review will critically examine the evidence linking gene mutations, especially MAP3K1, to non-syndromic forms of human 46,XY gonadal dysgenesis or XX testicular/ovotesticular.

XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown. We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS. Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10−10). Five variants are de novo (P value = 1.5 × 10−5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis. DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; In the last 15 years, the care provided for individuals born with differences of sex development (DSD) has evolved, with a strong emphasis on interdisciplinary approaches. However, these developments have not convinced some stakeholders to embrace the current model of care. This care model has also paid insufficient attention to socio-cultural differences and global inequalities. &lt;b&gt;&lt;i&gt;Summary:&lt;/i&gt;&lt;/b&gt; This article is an opinion statement, resulting from in-depth discussions and reflection among clinicians, patients, and family support organizations based in the USA and Europe, where we seek areas of common ground and try to identify opportunities to further develop resources. The product of these conversations is summarized in 10 panels. The corresponding sections provide additional discussion on some of the panel items. &lt;b&gt;&lt;i&gt;Key Messages:&lt;/i&gt;&lt;/b&gt; Participants identified areas of agreement, gained a deeper understanding of the reasons behind disagreements on certain matters, and identified the necessary steps to foster future consensus. We offer preliminary recommendations for guiding clinical management and resource allocation. By promoting a broader consensus, we aim to enhance the quality of care and well-being for individuals of all ages who have a DSD.

Although SRY was first identified 10 years ago, we still know remarkably little about its mode of action or downstream target genes. Recently, potential protein partners have been identified and there has been considerable activity to understand the roles of WT1, SF-1, DAX-1 and SOX9 in gonadogenesis. The emerging picture is one of complex interactions, involving both positive and negative regulatory signals that, depending on the cellular and promoter context, drive the expression of male-specific genes. Despite recent advances, however, we are still unable to explain the genetic cause of most cases of 46,XY gonadal dysgenesis or even a single case of Y-chromosome-negative 46,XX maleness.

The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1-55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in non-idiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eIF-1A, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients.

Although it has been established since the 1970s that deletions of the long arm of the Y chromosome are associated with spermatogenic failure, only in the last few years have these regions been described at the molecular level. In parallel, Y-linked genes and gene families that may explain the phenotypes of men carrying these deletions have been identified. The first association between spermatogenic failure and an underlying genetic cause was demonstrated by Tiepolo and Zuffardi (Tiepolo and Zuffardi, 1976Tiepolo L Zuffardi O Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm.Hum Genet. 1976; 34: 119-124Crossref PubMed Scopus (800) Google Scholar) in a report of six azoospermic patients carrying microscopically detectable deletions of the distal portion of Yq. In four patients, the deletion was de novo—that is, their fathers were tested and were found to carry intact Y chromosomes. On this basis, Tiepolo and Zuffardi (Tiepolo and Zuffardi, 1976Tiepolo L Zuffardi O Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm.Hum Genet. 1976; 34: 119-124Crossref PubMed Scopus (800) Google Scholar) proposed the existence of a spermatogenesis factor, called the “azoospermia factor” (AZF), encoded by a gene on distal Yq. However, the assumption that AZF represented a single Y-linked gene was overturned when Vogt et al. (Vogt et al., 1996Vogt PH Edelmann A Kirsch S Henegariu O Hirschmann P Kiesewetter F Kohn FM et al.Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11.Hum Mol Genet. 1996; 5: 933-943Crossref PubMed Scopus (1058) Google Scholar) observed that Y chromosome microdeletions follow a certain deletion pattern, with three recurrently deleted nonoverlapping subregions in proximal, middle, and distal Yq11, designated “AZFa,” “AZFb,” and “AZFc,” respectively. In addition, it became clear that these deletions were not exclusively associated with azoospermia (Reijo et al. Reijo et al., 1996aReijo R Alagappan RK Patrizio P Page D Severe oligospermia resulting from deletions of azoospermia factor gene on Y chromosome.Lancet. 1996a; 347: 1290-1293Abstract PubMed Scopus (397) Google Scholar). Deletions are associated with a wide range of histological profiles, from Sertoli cell–only syndrome (SCOS) to spermatogenic arrest (SGA) and severe hypospermatogenesis. The physical size of these regions has been estimated to be 1–3 Mb for AZFa and AZFb and ∼1.4 Mb for AZFc. Recent studies have shown that ∼10%–15% of azoospermic and ∼5%-10% of severely oligozoospermic men have Yq microdeletions. However, despite these advances, there are still many unanswered questions, which are the subject of this review. Several combined clinical and molecular studies have sought (1) to define recurrently deleted regions of Yq, (2) to determine the incidence of microdeletions among azoospermic and oligozoospermic men, and (3) to correlate the size and position of the deletions that cause the infertile phenotype. The reported incidence of microdeletions in infertile men varies enormously between studies, within the range 1%–55% (Reijo et al. Reijo et al., 1995Reijo R Lee TY Salo P Alagappan R Brown LG Rosenberg M Rozen S et al.Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene.Nat Genet. 1995; 10: 383-393Crossref PubMed Scopus (1070) Google Scholar; Quereshi et al. Quereshi et al., 1996Quereshi SJ Ross AR Ma K Cooke HJ Intyre MAM Chandley AC Hargreave TB PCR screening for Y chromosome microdeletions: a first step towards the diagnosis of genetically determined spermatogenic failure in men.Mol Hum Reprod. 1996; 2: 775-779Crossref PubMed Scopus (155) Google Scholar; Stuppia et al. Stuppia et al., 1996Stuppia L Mastroprimiano G Calabrese G Peila R Tenaglia R Palka G Microdeletions in interval 6 of the Y chromosome detected by STS-PCR in 6 of 33 patients with idiopathic oligo- and azoospermia.Cytogenet Cell Genet. 1996; 72: 155-158Crossref PubMed Scopus (106) Google Scholar, Stuppia et al., 1997Stuppia L Gatta V Mastroprimiano G Pompetti F Calabrese G Guanciali Franchi P Morizio E et al.Clustering of Y chromosome deletions in subinterval E of interval 6 supports the existence of an oligozoospermia critical region outside the DAZ gene.J Med Genet. 1997; 34: 881-883Crossref PubMed Scopus (31) Google Scholar; Foresta et al. Foresta et al., 1997Foresta C Ferlin A Garolla A Rossato M Barbaux S Bortoli A Y-chromosome deletions in idiopathic severe testiculopathies.J Clin Endocrinol Metab. 1997; 82: 1075-1080Crossref PubMed Scopus (145) Google Scholar, Foresta et al., 1998Foresta C Ferlin A Garolla A Moro E Pistorello M Barbaux S Rossato M High frequency of well-defined Y-chromosome deletions in idiopathic Sertoli cell-only syndrome.Hum Reprod. 1998; 13: 302-307Crossref PubMed Scopus (195) Google Scholar; Pryor et al. Pryor et al., 1997Pryor JL Kent-First M Muallem A Van Bergen AH Nolten WE Meisner L Roberts KP Microdeletions in the Y chromosome of infertile men.N Engl J Med. 1997; 336: 534-539Crossref PubMed Scopus (452) Google Scholar; Simoni et al. Simoni et al., 1997Simoni M Gromoll J Dworniczak B Rolf C Abshagen K Kamischke A Carani C et al.Screening for deletions of the Y chromosome involving the DAZ (deleted in azoospermia) gene in azoospermia and severe oligozoospermia.Fertil Steril. 1997; 67: 542-547Abstract Full Text PDF PubMed Scopus (196) Google Scholar; Van der Vent et al. Van der Vent et al., 1997Van der Vent K Montag M Peshka B Leygraaf J Schwanitz G Haidl G Krebs D et al.Combined cytogenetic and Y chromosome microdeletion screening in males undergoing intracytoplasmic sperm injection.Mol Hum Reprod. 1997; 3: 699-704Crossref PubMed Scopus (137) Google Scholar), but study design probably accounts for much of this variation. Study populations have included azoospermic patients, azoospermic and oligozoospermic patients, or azoospermic/oligozoospermic and infertile normospermic patients. Most clinical studies “select” individuals with idiopathic azoospermia or oligozoospermia, although others include “unselected” infertile men with known or unknown causes of infertility. Unfortunately, however, there is no general agreement on what constitutes idiopathic infertility. Varicocele and history of cryptorchidism are considered idiopathic in some studies and nonidiopathic in others. Variation in reported deletion frequency also seems to be affected by the number of patients in the study; in general, studies with low patient numbers report a higher deletion frequency, perhaps because these studies select patients more stringently. Another variable that may also affect Yq deletion frequency is marker density or the position of markers. Despite these caveats, it is possible that differences in deletion frequency and/or localization, between studies, may reflect genuine geographic or ethnic differences, perhaps related to a particular Y chromosome haplogroup, the genetic background, or environmental influences. Vogt et al. (Vogt et al., 1996Vogt PH Edelmann A Kirsch S Henegariu O Hirschmann P Kiesewetter F Kohn FM et al.Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11.Hum Mol Genet. 1996; 5: 933-943Crossref PubMed Scopus (1058) Google Scholar) originally proposed that AZFa deletions result in type I SCOS, in which no spermatogonia develop, whereas deletions in AZFb cause SGA, usually at the spermatocyte stage, and deletions in AZFc are associated with a more variable phenotype, ranging from type II SCOS (absence of germ cells in most testis tubules) to hypospermatogenesis (presence of all germ-cell types, albeit in reduced numbers). In general, subsequent studies have supported these findings, but there have been exceptions: both AZFa and AZFb deletions have been reported in oligozoospermic men, and we have found oligozoospermia associated with AZFb deletions (authors' unpublished data). Another problem in the definition of genotype/phenotype correlations is the variability of the phenotype in the same man, over time. In one study, an individual with an AZFc deletion showed a progressive decrease in sperm concentration, from severe oligozoospermia to azoospermia, over 30 mo (Girardi et al. Girardi et al., 1997Girardi SK Mielnik A Schlegel PN Submicroscopic deletions in the Y chromosome of infertile men.Hum Reprod. 1997; 12: 1635-1641Crossref PubMed Scopus (171) Google Scholar). Nevertheless, some patterns have emerged from a survey of the clinical literature on these deletions. First, microdeletions are found almost exclusively in males affected by azoospermia or severe oligozoospermia or, occasionally, in patients with other abnormal andrological findings. Second, a higher frequency of Yq deletions is found in azoospermic men, compared with oligozoospermic men, and in men with well-defined idiopathic infertility, compared with men for whom the etiology of the infertility is known. Third, large deletions generally are associated with more-severe spermatogenic defects. Finally, AZFa deletions, which are relatively uncommon (frequency of 1%–5%), generally are associated with SCOS type I, whereas AZFc and AZFc+AZFb deletions, the most frequent form of these lesions, may be associated with a variety of spermatogenesis defects, including oligozoospermia. The relatively high frequency of de novo Y deletions indicates that the Y chromosome is susceptible to the spontaneous loss of genetic material. The instability of the Y chromosome may be related to the high frequency of repetitive elements clustered along the length of the chromosome, and deletions may occur through aberrant recombination events (between areas of homologous or similar sequence repeats, between the X and Y chromosomes, or by Y chromosome unbalanced sister-chromatid exchange) or by slippage during DNA replication. There also may be particular Y chromosome sequences that promote deletion of the AZF regions; consequently, some individuals may be more susceptible to de novo deletions than are others. Indeed, Jobling et al. (Jobling et al., 1998Jobling MA Williams G Schiebel K Pandya A McElreavey K Salas L Rappold GA et al.A selective difference between human Y-chromosomal DNA haplotypes.Curr Biol. 1998; 8: 1391-1394Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) defined one Y chromosome haplotype that is susceptible to aberrant X/Y exchange during male meiosis, leading to Y-positive 46,XX maleness and infertility. Advanced paternal age also might promote the loss of Y sequences, although this hypothesis needs to be examined by correlation of deletion incidence with the age of the father at conception. Paternal age effects have been described in Marfan syndrome, neurofibromatosis, and Apert syndrome. However, in most of these cases, the mutations are 1-bp substitutions, rather than deletions. Several genes and gene families have been identified on the long arm of the Y chromosome (Lahn and Page Lahn and Page, 1997Lahn BT Page D Functional coherence of the human Y chromosome.Science. 1997; 278: 675-680Crossref PubMed Scopus (686) Google Scholar; also see Lau Lau, 1999Lau Y-FC Gonadoblastoma, testicular and prostate cancers, and the TSPY gene.Am J Hum Genet. 1999; 64 (in this issue): 921-927Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar [in this issue]). Some of these genes fall within AZF deletion intervals and therefore may underlie the observed deletion phenotypes (tables 1 and 2). These genes can be divided into those that may be involved in cellular “housekeeping” activities and those that are expressed solely in the testis. The former group (table 1) includes Drosophila fats facets related Y (DFFRY), dead box Y (DBY), ubiquitous tetratricopeptide repeat (TPR) motif Y (UTY), the eukaryotic translation-initiation–factor 1A Y isoform (eIF-1AY), selected mouse cDNA on the Y (SMCY), and the thymosin β4 Y isoform (Tβ4Y). These ubiquitously expressed genes each exist in a single copy on the Y chromosome, and each possesses a closely related X-linked homologue that escapes X inactivation. The testis-specific group (table 2) includes the RNA-binding–motif Y chromosome gene (RBMY) and its relatives, deleted in azoospermia (DAZ), chromodomain Y (CDY), XK-related Y (XKRY), protein-tyrosine phosphatase BAS-like (PTP-BL)–related Y (PRY), and the genes for basic proteins Y1 and Y2 (BPY1 and BPY2). These genes are present in multiple copies on the Y and do not appear to have X homologues.Table 1Ubiquitously Expressed Housekeeping Genes That Map to AZF-Deleted Regions and That Have Been Implicated in Male InfertilityGene SymbolGene NameComment(s)X-Linked HomologueAmino Acid Identity (%)DFFRYDrosophila fats facets related YHomologous to Drosophila deubiquinating enzyme (Brown et al. Brown et al., 1998Brown GM Furlong RA Sargent CA Erickson RP Longepied G Mitchell M Jones MH et al.Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene.Hum Mol Genet. 1998; 7: 97-107Crossref PubMed Scopus (182) Google Scholar)DFFRX91DBYDead box YContains a DEAD (amino acid sequence Asp-Glu-Ala-Asp) box motif; may function as an RNA helicase (Linder et al. Linder et al., 1989Linder P Lasko PF Ashburner M Leroy P Nielsen PJ Nishi K Schnier J et al.Birth of the D-E-A-D box.Nature. 1989; 337: 121-122Crossref PubMed Scopus (598) Google Scholar)DBX91Tβ4YThymosin β4 YMay be involved in actin sequestration (Gondo et al. Gondo et al., 1987Gondo H Kudo J White JW Barr C Selvanayagam P Saunders GF Differential expression of the human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes.J Immunol. 1987; 139: 3840-3848PubMed Google Scholar)Tβ4X93UTYUbiquitous TPR motif YContains 10 tandem TPR motifs that may be involved in protein-protein interactions (Greenfield et al. Greenfield et al., 1996Greenfield A Scott D Pennisi D An H-YDb epitope is encoded by a novel mouse Y chromosome gene.Nat Genet. 1996; 14: 474-478Crossref PubMed Scopus (158) Google Scholar)UTX85SMCYSelected mouse cDNA on the YEncodes an H-Y antigen epitope (Agulnik et al. Agulnik et al., 1994aAgulnik AI Mitchell MJ Lerner JL Woods DR Bishop CE A mouse Y chromosome gene encoded by a region essential for spermatogenesis and expression of male-specific histocompatibility antigens.Hum Mol Genet. 1994a; 3: 873-878Crossref PubMed Scopus (115) Google Scholar,Agulnik et al., 1994bAgulnik AI Mitchell MJ Mattei MG Borsani G Avner PA Lerner JL Bishop CE A novel X gene with a widely transcribed Y homologue escapes X inactivation in mouse and human.Hum Mol Genet. 1994b; 3: 879-884Crossref PubMed Scopus (152) Google Scholar)SMCX84eIF-1AYEukaryotic translation-initiation–factor 1AEukaryotic translation-intiation factor (Pestova et al. Pestova et al., 1998Pestova TV Borukhov SI Hellen CTU Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons.Nature. 1998; 394: 854-859Crossref PubMed Scopus (304) Google Scholar).eIF1-1AX98Note.—These genes are present in a single copy on the Y, but they all have X homologues that escape X inactivation. In all cases, the degree of sequence identity between the X and Y homologues is ≥84%. Open table in a new tab Table 2Genes and Gene Families with Expression Restricted to the Testis and That Map to the AZF-Deleted Regions of the Y chromosomeGene SymbolGene NameComment(s)X-Linked or Autosomal HomologueRBMYRNA-binding–motif YSubfamilies include RBMY1 and RBMY2; RBMY1 may be functional and is predicted to bind RNARBMY may be an ancestral hnRNPG geneDAZDeleted in azoospermiaPredicted to bind RNA, as Xenopus Dazl does in vitroDAZL1 chromosome 3p25XKRYXK-related YSimilar to XK, a putative membrane-transport protein (Ho et al. Ho et al., 1994Ho M Chelly J Carter N Danek A Crocker P Monaco AP Isolation of the gene for McLeod syndrome that encodes a novel membrane transport protein.Cell. 1994; 77: 869-880Abstract Full Text PDF PubMed Scopus (220) Google Scholar)None knownCDYChromodomain YContains chromodomain (James and Elgin James and Elgin, 1986James TC Elgin SC Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.Mol Cell Biol. 1986; 6: 3862-3872Crossref PubMed Scopus (462) Google Scholar); may be involved in chromatid modification during spermatogenesisNone knownPRYPTP-BL–related YSimilar to PTP-BL, a putative membrane-transport protein (Hendriks et al. Hendriks et al., 1995Hendriks W Schepens J Bachner D Rijss J Zeeuwen P Zechner U Hameister H et al.Molecular cloning of a mouse epithelial protein-tyrosin phosphatase with similarities to submembranous proteins.J Cell Biochem. 1995; 59: 418-430Crossref PubMed Scopus (47) Google Scholar)None knownBPY1Basic protein Y1Basic protein of unknown functionNone knownBPY2Basic protein Y2Basic protein of unknown functionNone knownNote.—Table modified from the report by Lahn and Page (Lahn and Page, 1997Lahn BT Page D Functional coherence of the human Y chromosome.Science. 1997; 278: 675-680Crossref PubMed Scopus (686) Google Scholar). Open table in a new tab Note.— These genes are present in a single copy on the Y, but they all have X homologues that escape X inactivation. In all cases, the degree of sequence identity between the X and Y homologues is ≥84%. Note.— Table modified from the report by Lahn and Page (Lahn and Page, 1997Lahn BT Page D Functional coherence of the human Y chromosome.Science. 1997; 278: 675-680Crossref PubMed Scopus (686) Google Scholar). DFFRY, DBY, and UTY all fall within the AZFa deletion interval; therefore, one or more of these genes may be implicated in SCOS or other male-fertility disorders. AZFb includes copies of CDY and XKRY, as well as of SMCY, eIF-1AY, and RBMY. Although sequences related to RBMY are found throughout the Y chromosome, functional copies appear to be restricted to AZFb, since deletions of distal AZFb lead to the absence of RBMY epitopes in testicular sections (Elliott et al. Elliott et al., 1997Elliott DJ Millar MR Oghene K Ross A Kiesewetter F Pryor J McIntyre M et al.Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm.Proc Natl Acad Sci USA. 1997; 94: 3848-3853Crossref PubMed Scopus (228) Google Scholar). A number of transcripts are found within AZFc. At least six copies of DAZ are found in this region (Saxena et al. Saxena et al., 1996Saxena R Brown LG Hawkins T Alagappan RK Skaletsky H Reeve MP Reijo R et al.The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned.Nat Genet. 1996; 14: 292-299Crossref PubMed Scopus (373) Google Scholar; Yen et al. Yen et al., 1997Yen PH Chai NN Salido EC The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat regions.Mamm Genome. 1997; 8: 756-759Crossref PubMed Scopus (73) Google Scholar), as are multiple copies of PRY, BPY2, CDY, and XKRY. Any of these genes may contribute to the AZFc-deletion phenotype, and most AZFc deletions probably remove all these genes. Individuals with AZFc deletions can present with oligozoospermia, and some even father children; hence, it is clear that these genes are not essential for spermatogenesis. At present, we know very little about the biochemistry or biology of Y-encoded proteins. Only RBMY and DAZ have been studied extensively. More than 30 RBMY genes and pseudogenes occur over both arms of the Y chromosome (Ma et al. Ma et al., 1993Ma K Inglis JD Sharkey A Bickmore WA Hill RE Prosser EJ Speed RM et al.A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling spermatogenesis.Cell. 1993; 75: 1287-1295Abstract Full Text PDF PubMed Scopus (478) Google Scholar; Prosser et al. Prosser et al., 1996Prosser J Inglis JD Condie A Ma K Kerr S Thakrar R Taylor K et al.Degeneracy in human multi-copy RBM (YRRM), a candidate spermatogenesis gene.Mamm Genome. 1996; 7: 835-842Crossref PubMed Scopus (50) Google Scholar; Chai et al. Chai et al., 1997Chai NN Salido EC Yen PH Multiple functional copies of the RBM gene family, a spermatogenesis candidate on the human Y chromosome.Genomics. 1997; 45: 355-361Crossref PubMed Scopus (78) Google Scholar), and these sequences can be divided into several subfamilies. The RBMY1 subfamily has at least seven members, all of which appear to be clustered in the AZFb region (Prosser et al. Prosser et al., 1996Prosser J Inglis JD Condie A Ma K Kerr S Thakrar R Taylor K et al.Degeneracy in human multi-copy RBM (YRRM), a candidate spermatogenesis gene.Mamm Genome. 1996; 7: 835-842Crossref PubMed Scopus (50) Google Scholar; Chai et al. Chai et al., 1997Chai NN Salido EC Yen PH Multiple functional copies of the RBM gene family, a spermatogenesis candidate on the human Y chromosome.Genomics. 1997; 45: 355-361Crossref PubMed Scopus (78) Google Scholar, Chai et al., 1998Chai NN Zhou H Hernandez J Najmabadi H Bhasin S Yen PH Structure and organization of the RBMY genes on the human Y chromosome: transposition and amplification of an ancestral autosomal hnRNPG gene.Genomics. 1998; 49: 283-289Crossref PubMed Scopus (48) Google Scholar). These genes encode germ-cell specific nuclear proteins that contain an RNA-binding motif (RBM), as well as four copies of an SRGY (Serine-Arginine-Glycine-Tryosine motif) repeat. RBMY2 genes share 88% homology with RBMY1 genes and encode an RBM and a single SRGY repeat. The RBMY1 sequence is 67% similar to the autosomally expressed hnRNPG (ribonucleoprotein G) protein, a nuclear glycoprotein with RNA-binding activities but with no known biological function (Soulard et al. Soulard et al., 1993Soulard M Valle VD Siomi M Pinol-Roma S Codogno P Bauvy C Bellini M et al.hnRNPG: sequence and characterization of a glycosylated RNA-binding protein.Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar). RBMY1 genes may derive from an hnRNPG gene that translocated to the Y chromosome and subsequently was amplified (Delbridge et al. Delbridge et al., 1997Delbridge ML Harry JL Toder R O'Neill RJ Ma K Chandley AC Graves JA A human candidate spermatogenesis gene, RBM1, is conserved and amplified on the marsupial Y chromosome.Nat Genet. 1997; 15: 131-136Crossref PubMed Scopus (97) Google Scholar). In humans, RBMY1 can be detected by immunostaining of pachytene spermatocytes, an interesting observation in view of the SGA often seen in association with AZFb deletions (Elliott et al. Elliott et al., 1997Elliott DJ Millar MR Oghene K Ross A Kiesewetter F Pryor J McIntyre M et al.Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm.Proc Natl Acad Sci USA. 1997; 94: 3848-3853Crossref PubMed Scopus (228) Google Scholar, Elliott et al., 1998Elliott DJ Oghene K Makarov G Makarova Hargreave TB Chandley AC Eperon IC et al.Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during germ cell development.J Cell Sci. 1998; 111: 1255-1265PubMed Google Scholar). In spermatocytes, RBMY1 colocalizes with pre–mRNA-splicing components in a discrete area of the nucleus, but, by late meiosis, it is found diffusely throughout the nucleoplasm of round spermatids. Hence, RBMY1 may play distinct roles during different phases of spermatogenesis. Like RBMY, DAZ encodes a testis-specific protein that has a single RBM and a series of 8–24 copies of a 24–amino-acid unit termed the “DAZ repeat” (Reijo et al. Reijo et al., 1995Reijo R Lee TY Salo P Alagappan R Brown LG Rosenberg M Rozen S et al.Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene.Nat Genet. 1995; 10: 383-393Crossref PubMed Scopus (1070) Google Scholar; Yen et al. Yen et al., 1997Yen PH Chai NN Salido EC The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat regions.Mamm Genome. 1997; 8: 756-759Crossref PubMed Scopus (73) Google Scholar). The biological function of this motif is unknown, and DAZ genes differ substantially in the sequence and organization of these repeats (Yen et al. Yen et al., 1997Yen PH Chai NN Salido EC The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat regions.Mamm Genome. 1997; 8: 756-759Crossref PubMed Scopus (73) Google Scholar). DAZ is homologous to an autosomal gene with a single DAZ repeat, named “DAZL1” (DAZ-like autosomal 1; Saxena et al. Saxena et al., 1996Saxena R Brown LG Hawkins T Alagappan RK Skaletsky H Reeve MP Reijo R et al.The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned.Nat Genet. 1996; 14: 292-299Crossref PubMed Scopus (373) Google Scholar; Yen et al. Yen et al., 1996Yen PH Chai NN Salido EC The human autosomal gene DAZLA: testis specificity and a candidate for male infertility.Hum Mol Genet. 1996; 5: 2011-2013Crossref Scopus (136) Google Scholar), and the Y-linked DAZ probably originated from the translocation and amplification of this ancestral autosomal gene. Mice lack the Y-located DAZ gene, but they do carry a single autosomal Dazl1 gene (Cooke et al. Cooke et al., 1996Cooke HJ Lee M Kerr S Ruggiu M A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads.Hum Mol Genet. 1996; 5: 513-516Crossref PubMed Scopus (192) Google Scholar; Reijo et al. Reijo et al., 1996bReijo R Seligman J Dinulos MB Jaffe T Brown LG Disteche CM Page DC Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans is expressed in male germ cells before and after puberty.Genomics. 1996b; 35: 346-352Crossref PubMed Scopus (125) Google Scholar). Immunostaining has revealed human DAZ in the innermost layer of male germ-cell epithelium and in the tails of spermatozoa (Habermann et al. Habermann et al., 1998Habermann B Mi HF Edelmann A Bohring C Backert IT Kiesewetter F Aumuller G et al.DAZ (deleted in azoospermia) genes encode proteins located in human late spermatids and in sperm tails.Hum Reprod. 1998; 13: 363-369Crossref PubMed Scopus (108) Google Scholar). This observation is consistent with the expression of DAZ transcripts just inside the perimeter of seminiferous tubules in spermatogonia (Menke et al. Menke et al., 1997Menke DB Mutter GL Page DC Expression of DAZ, an azoospermia factor candidate, in human spermatogonia.Am J Hum Genet. 1997; 60: 237-241PubMed Google Scholar). However, some caution must be used when these results are interpreted, since cross-hybridization with DAZL mRNA or protein cannot be excluded. Insights into human DAZ function may come from the analysis of its autosomal homologues in other species. Targeted disruption of Dazl1 in mice leads to a complete absence of gamete production in both testis and ovary, demonstrating that Dazl1 is essential for the development or survival of germ cells (Ruggiu et al. Ruggiu et al., 1997Ruggiu M Speed R Taggart M McKay SJ Kilanowski F Saunders P Dorin J et al.The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis.Nature. 1997; 389: 73-77Crossref PubMed Scopus (499) Google Scholar). In Drosophila, mutation of the boule gene, another homologue of DAZL, results in spermatocyte arrest at the G2/M transition and complete azoospermia (Castrillon et al. Castrillon et al., 1993Castrillon DH Gönczy P Alexander S Rawson R Eberhart CG Viswanathan S Dinardo S et al.Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis.Genetics. 1993; 135: 489-505Crossref PubMed Google Scholar; Eberhart et al. Eberhart et al., 1996Eberhart CG Maines JZ Wasserman SA Meiotic cell requirement for a fly homologue of human deleted in azoospermia.Nature. 1996; 381: 783-785Crossref PubMed Scopus (335) Google Scholar). The boule protein occurs in the nucleus of primary spermatocytes until the end of the meiotic prophase, after which it is found in the cytoplasm. In Xenopus, Xdazl is expressed in premeiotic germ cells in adult testis (Houston et al. Houston et al., 1998Houston DW Zhang J Maines JZ Wasserman SA King ML A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule.Development. 1998; 125: 171-180PubMed Google Scholar). Interestingly, the Xenopus Xdazl gene can rescue meiotic entry of spermatocytes in Drosophila boule mutants, suggesting functional conservation of the DAZ family over evolutionary time (Houston et al. Houston et al., 1998Houston DW Zhang J Maines JZ Wasserman SA King ML A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule.Development. 1998; 125: 171-180PubMed Google Scholar). Xdazl protein has RNA-binding properties in vitro, and perhaps other members of the DAZ family play a role in RNA metabolism during gamete development (Houston et al. Houston et al., 1998Houston DW Zhang J Maines JZ Wasserman SA King ML A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule.Development. 1998; 125: 171-180PubMed Google Scholar). Other genes on the long arm of the Y also may be involved in RNA metabolism. DBY is predicted to act as an RNA helicase (Lahn and Page Lahn and Page, 1997Lahn BT Page D Functional coherence of the human Y chromosome.Science. 1997; 278: 675-680Crossref PubMed Scopus (686) Google Scholar), and eIF-1AY encodes an essential translation-initiation factor (Pestova et al. Pestova et al., 1998Pestova TV Borukhov SI Hellen CTU Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons.Nature. 1998; 394: 854-859Crossref PubMed Scopus (304) Google Scholar). During the latter stages of spermatogenesis, transcription terminates and posttranscriptional regulation plays a primary role (see reviews by Braun [Braun, 1998Braun RE Post-transcriptional control of gene expression during spermatogenesis.Semin Cell Dev Biol. 1998; 9: 483-489Crossref PubMed Scopus (126) Google Scholar] and Hecht [Hecht, 1998Hecht NB Molecular mechanism of male germ cell differentiation.Bioessays. 1998; 20: 555-561Crossref PubMed Scopus (369) Google Scholar]). RNA synthesis peaks during the spermatocyte stage, is gradually reduced in subsequent stages, and ceases as round spermatids differentiate into elongated spermatids. Numerous mRNA that are under posttranslational control during spermatogenesis have been identified. It is tempting to speculate that many of the factors encoded by Y-linked genes play key roles in this process. This work was supported by the Institut National de la Santé et de la Recherche Médicale and by the Association pour la Recherche sur la Cancer.

The geographic location of Egypt, at the interface between North Africa, the Middle East, and southern Europe, prompted us to investigate the genetic diversity of this population and its relationship with neighboring populations. To assess the extent to which the modern Egyptian population reflects this intermediate geographic position, ten Unique Event Polymorphisms (UEPs), mapping to the nonrecombining portion of the Y chromosome, have been typed in 164 Y chromosomes from three North African populations. The analysis of these binary markers, which define 11 Y-chromosome lineages, were used to determine the haplogroup frequencies in Egyptians, Moroccan Arabs, and Moroccan Berbers and thereby define the Y-chromosome background in these regions. Pairwise comparisons with a set of 15 different populations from neighboring European, North African, and Middle Eastern populations and geographic analysis showed the absence of any significant genetic barrier in the eastern part of the Mediterranean area, suggesting that genetic variation and gene flow in this area follow the "isolation-by-distance" model. These results are in sharp contrast with the observation of a strong north-south genetic barrier in the western Mediterranean basin, defined by the Gibraltar Strait. Thus, the Y-chromosome gene pool in the modern Egyptian population reflects a mixture of European, Middle Eastern, and African characteristics, highlighting the importance of ancient and recent migration waves, followed by gene flow, in the region.

In industrialized countries, many couples are choosing to delay childbearing, and the proportion of couples having children after age 30–35 years has increased. This has highlighted the effect of age on reproductive failure (van Balen et al, 1997). The effects of maternal age have been thoroughly investigated in the past few decades, and a major effect of maternal age over 35 years has been demonstrated on infertility, ectopic pregnancy, and miscarriage (van Noord-Zaadstra et al, 1991; van Balen et al, 1997; Nybo Andersen et al, 2000). In contrast, little attention has been paid to the possible effects of paternal age. Most studies that have dealt with this factor have focused on changes in sperm characteristics with age, and physicians have tried to set an upper age limit for sperm donors. The American Society for Reproductive Medicine (1998) and the British Andrology Society (1999) have fixed the upper age limit for sperm donation at 40 years old on the basis of the increased risk of genetic abnormalities in children of older fathers (Bordson and Leonardo, 1991). In discussions of the effects of paternal age with a view toward setting age limits for sperm donors, the possibility that paternal age affects the likelihood of reproductive failure was not considered. In other respects, some demographic studies have analyzed the effects of paternal age on effective fecundity, which is the probability of initiating a pregnancy leading to a live birth (Anderson, 1975; Mineau and Trussell, 1982; Goldman and Montgomery, 1989; Strassmann and Warner, 1998). These studies, based on large data sets from populations not using birth control methods, showed a decrease in effective fecundity with increasing paternal age. We reviewed the existing literature, analyzing the effect of paternal age on 2 major reproductive failures: infertility and miscarriage. We searched MEDLINE, using the PubMed Searching system developed by the National Center for Biotechnology Information at the U.S. National Library of Medicine. We considered only references that dealt with studies of humans published in English. We selected articles analyzing reproductive failures on the basis of their having at least one of the following MEDLINE major keywords: infertility; fertility; fertilization; abortion, spontaneous; pregnancy outcome; fetal death; embryo loss; and pregnancy, ectopic. We selected references in which the term ‘paternal age’ was a key word. We also included all references in which the term “paternal age” was present in the title or in the abstract, to reduce any search review bias toward the exclusion of nonsignificant results. Our MEDLINE query1 identified 35 references. We excluded 28 of these references because they were general reviews on the male reproductive tract (n = 4) or comments on another article (n = 1), they concerned infertile couples (n = 3), or they were irrelevant because they concerned diseases and malformations in the embryo and child (n = 5), paternal germ cell characteristics (n = 4), chromosomal analysis of dead fetuses (n = 4), professional and environmental exposure of the father (n = 3), teenage pregnancies (n = 2), the consequences of treatment for cryptorchidism (n = 1), or the consequences of consanguinity (n = 1). We therefore obtained a total of 7 relevant references from this MEDLINE search. We checked the exhaustiveness of our list of references by cross-checking it with 1) references cited in these 7 articles, 2) the 7 sets of PubMed “related articles,” and 3) our reference database. We extended our search to all papers published since 1965 and identified 7 other references. Five of these 7 references were listed on MEDLINE. However, they were not found in our initial MEDLINE query because the term “paternal age” was absent. Unfortunately, we could not extend our MEDLINE search using synonyms such as “male age,” “father age,” or “husband age” because these terms are not phrases recognized by MEDLINE (PubMed does not actually perform adjacency searching but uses a list of recognized phrases against which search terms are matched). Thus, in total, we identified 14 references that specifically analyzed the effects of paternal age on infertility and miscarriage (Table). Most of these papers (n = 11) concerned couples in the general population who were trying to conceive or who had conceived. In these papers, the authors adjusted for female factors (especially maternal age) by means of multivariate models. A few papers (n = 3) analyzed data from in vitro fertilization (IVF) programs involving ovum donation, to ensure efficient control for maternal factors as in these studies female donor factors were independent from paternal age. By considering such different data, we were able to compare the results of analyses with very different limitations: difficulties adjusting for confounders (especially for sexual confounders such as decreased frequency of intercourse with age) in natural reproduction and problems associated with gamete manipulation and selection bias in data from IVF with ovum donation. Infertility is defined as a failure to conceive in a couple trying to reproduce for “some time.” The World Health Organization has defined infertility as a period of 2 years without conception, but many couples actually seek medical advice after 1 year of infertility. Infertility is usually investigated by calculating time to pregnancy, which is the number of months required to achieve a recognized pregnancy for a couple having regular sexual intercourse in the absence of birth control methods. An increase in time to pregnancy may indicate changes in male and female gametogenesis, the transport of gametes in the male and female reproductive tracts, fertilization, migration of the zygote to the uterus, implantation, and the early survival of the conceptus until detection of the pregnancy (Baird et al, 1986). Thus, time to pregnancy is considered an accurate indicator of human fertility—not only for women but also for men (Joffe, 1997). Joffe and Li (1994) carried out an analysis of a sample of men born in Britain in 1958, followed in a longitudinal study. They analyzed male and female factors affecting the time required to achieve a pregnancy in couples where the men (n = 2576) had fathered at least 1 child by the time of data collection in 1991, when all these men were aged 33 years. They used a multivariate Cox regression model to compare men who started attempting to father a pregnancy when they were 30 to 33 years old with those who attempted to father a pregnancy when they were less than 30 years old. They could not control for maternal age in their model because of incomplete data on female partner age. They found no difference in time to pregnancy between these 2 groups of men differing in paternal age, both of which consisted of men under the age of 33 years. Olsen (1990) investigated the effects of maternal age and paternal age on the risk of taking more than 1 year to conceive for all pregnant women (n = 10 886) in two Danish cities from April 1984 to April 1987. A very weak effect of paternal age was observed in logistic regression analysis after controlling for maternal age. This author considered only pregnant women. This is a major limitation, which may have resulted in an underestimation of the effects of age, because of the exclusion or underrepresentation of sterile and less fecund couples (Juul et al, 2000). The Australian Pregnancy and Lifestyle Study investigated sociodemographic, occupational, and environmental risk factors for infertility and miscarriage by interviewing couples (n = 585) who had planned a pregnancy (Ford et al, 1994). After 9 months of trying, 17.3% of couples had not yet achieved a pregnancy. The authors analyzed the effect of age on the risk of 9 months of infertility in a multivariate regression model in which partner's age effect was controlled. Taking less than 35 years as the reference age class, the authors showed that the risk of infertility was significantly higher in couples in which the man (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.44–3.71) or the woman (OR, 2.19; 95% CI, 1.23–3.99) was more than 35 years old. In the United Kingdom, the Avon Longitudinal Study of Pregnancy and Childhood was carried out between April 1991 and December 1992 on all couples expecting a baby in the Avon Health District. On the basis of planned pregnancies (n = 8515), Ford et al (2000) analyzed the probability of having conceived in a 6-month period and that of having conceived in a 12-month period. Multivariate logistic-regression analysis showed that the probability of conception decreased steadily with paternal age after controlling for maternal age. For example, for the probability of conceiving within a period of 12 months, the OR was 0.51 (95% CI, 0.31–0,86) for men aged 40 years and over when compared with fathers aged 24 years and younger. In this study, the authors analyzed paternal age at the time of conception rather than at the time when the couple started trying to conceive a child, leading to a potential overestimation of the effects of paternal age in cases in which the time to pregnancy was long (resulting in older fathers) (Sallmen and Luukkonen, 2001). A European multicenter study was recently conducted on a large cohort of couples (n = 782) using natural family planning methods to avoid pregnancy (Dunson et al, 2002). The authors estimated the probability of conception on various days in the menstrual cycle. This fertility indicator allowed the analysis of risk factors (such as maternal and paternal age) by taking into account sexual activity. The authors investigated paternal age by controlling for the maternal age effect. For couples in which the woman was aged 35 to 39 years, Dunson et al (2002) observed a decrease in the probability of conception for men in their late thirties or older. For a woman aged 35 years having intercourse on the most fertile day of the menstrual cycle, the probability of conception decreases from 0.29 if the man is aged 35 years to 0.18 if the man is aged 40 years. A few studies have analyzed paternal age using data from IVF programs involving ovum donation. On the one hand, these data allowed analysis of paternal age without possible confusion with sexual activity. On the other hand, maternal factors were controlled more efficiently, because there was no relationship between paternal age and oocyte factors (such as age of the female donor or the number of oocytes retrieved). Watanabe et al (2000) analyzed 288 cycles performed at a French IVF center and concluded that the rate of clinical pregnancy decreased with increasing paternal age when 5 or fewer oocytes were retrieved. An analysis of 316 cycles from an American IVF center and 558 cycles from a Spanish IVF center showed no effect of male age on the rate of clinical pregnancy (Gallardo et al, 1996; Paulson et al, 2001). In these studies, the authors did not control for the number of oocytes retrieved, which is a key predictive factor. Finally, Nieschlag et al (1982) investigated paternal age by comparing 23 grandfathers aged 60 to 88 years with 20 fathers aged 24 to 37 years. The men were recruited by newspaper advertisements and were asked to supply semen samples by masturbation after sexual abstinence for 2 to 7 days. The fertilizing capacity of the sperm was assessed by the Heterologous Ovum (HOP) test for 16 grandfathers and 20 fathers. The authors found no difference between the grandfathers and fathers in these 2 groups, each of which contained only a small number of subjects. As was stated by Nybo Andersen et al (2000), more than 13% of clinically recognized pregnancies end in fetal death. Most of these deaths occur during the first trimester of gestation and are defined as miscarriages. The term “late fetal death” is generally used to refer to deaths occurring after 20 weeks of gestation, and the term “stillbirth” is used for deaths occurring after 28 weeks of gestation. In a case-control study carried out in a University Hospital in Saudi Arabia (n = 226 cases and 226 controls), Al-Ansary and Babay (1994) analyzed the risk factors for miscarriage before 24 weeks of gestation. After controlling for maternal age effect, they showed that the risk of miscarriage increased with paternal age, especially when fathers were aged more than 50 years. Surprisingly, these authors found no evidence of the well-documented effect of maternal age, possibly because of the small number (n = 38) of enrolled women who were aged 35 years or over. Ford et al (1994) used a logistic regression model to analyze risk factors for first trimester miscarriage in couples (n = 484) who achieved a recognized pregnancy in the Pregnancy and Lifestyle Study (Ford et al, 1994). They considered only 2 age classes (<35 and ≥35 years), and, after controlling for maternal age effect, they found that the risk of miscarriage was higher in couples in which the man was aged 35 years or older, with an OR of 2.33 (95% CI, 1.41–3.84), than in men younger than age 35 years. de La Rochebrochard and Thonneau (2002) analyzed data from a large study on subfecundity and infertility carried out in 4 European countries (n = 3174); they used a multivariate logistic regression model to analyze the risk of miscarriage. By controlling for maternal age, they concluded that the risk of miscarriage increased steadily with paternal age for men aged 40 years and older, especially if the mother was aged 35 years or older. Thus, in this study, compared with couples in which both partners were aged 20 to 29 years, the OR for women aged 35 years or older increased with paternal age from 3.38 (95% CI, 1.76–6.47) if the man was aged 35–39 years to 6.73 (95% CI, 3.50–12.95) if the man was aged 40 years or older. In a paper published in 1976, Resseguie (1976) analyzed live births and fetal death certificates from Wisconsin for the years 1968 to 1971. That author compared the risk of late fetal death (after 20 weeks of gestation) for various paternal age classes by chi-square tests in sub-populations defined by birth order, maternal age class, and number of years of maternal education completed. No increase was observed in the risk of late fetal death with paternal age. On the basis of more than 1.5 million live births and fetal death certificates from New York State for the years 1959–1967, Selvin and Garfinkel (1976) analyzed the risk of late fetal death (after 20 weeks of gestation) by multivariate logistic-regression analysis. After controlling for partner's age, they obtained an OR of 1.027 for a 1-year increase in paternal age and of 1.032 for a 1-year increase in maternal age. These authors concluded that paternal age and maternal age have independent and approximately equal effects on the risk of late fetal death. Nevertheless, this model is based on the assumption that the effect of age (both maternal and paternal) is linear. This assumption is controversial—the effect of age on the risk of reproductive failure is usually considered to follow a J-shaped curve (Nybo Andersen et al, 2000). Wunsch and Gourbin (1998) recently studied the effect of paternal age on stillbirth (death after 28 weeks of gestation), neonatal mortality (death during the first 28 days of life), and postneonatal mortality (death in the first 12 months of life) by analyzing birth and death certificates from Belgium (1986–1990) and Hungary (1984–1988). After adjustment for confounding factors (especially maternal age), these authors concluded that paternal age 35 years or older increased the risks of stillbirth and of neonatal mortality. Our analysis provides some evidence that increasing paternal age increases the risk of reproductive failure. Almost all of the published studies on the effects of paternal age on miscarriage have concluded that the risk of miscarriage and late fetal death were higher for couples in which the man was 35 to 40 years or older than in couples in which the man was younger than 35 years. We found that some of the results of studies on paternal age and the risk of infertility were discordant. However, it appeared to us that this discordance resulted principally from the major limitations of inconclusive studies. These were mainly the limitation of observations to a particular male age interval (eg, under 33 years), lack of adjustment for major confounders (such as the number of oocytes retrieved in IVF with ovum donation studies), or consideration only of limited measures of the reproductive process (such as heterologous ovum tests). After detailed discussions of each of these studies, we concluded that, overall, the published studies provided evidence increased risk of infertility with paternal age. Furthermore, the effect of paternal age on infertility was investigated in very heterogeneous populations: couples from the general population who had tried to conceive or had conceived a child versus data for IVF with ovum donation. In both populations, an effect of paternal age was identified in men in their late thirties. Confirmation is required for the results obtained for IVF with ovum donation, but the overall concordance of results obtained for such different populations provides further evidence for the existence of a paternal age effect. This process of comparing results from very different populations has already been used to demonstrate the well-known effect of maternal age. Thus, conclusions concerning the effects of maternal age are based not only on studies of couples trying to conceive or who have conceived (van Balen et al, 1997) but also on data from studies of intrauterine insemination with donor spermatozoa (Schwartz and Mayaux, 1982). In both types of reproductive failure investigated in these reviews, we concluded that the risk may be greater when the man is aged 40 years or older. The age of 40 has also been established as the upper age limit for sperm donors because of an increased risk of genetic abnormalities in children of these fathers (American Society for Reproductive Medicine, 1998; British Andrology Society, 1999). Paternal age over 40 years could thus be a cutoff in the reproductive life of men. In the present review, we considered only the effect of paternal age in the general population and in cases of IVF with ovum donation (used to analyze the effects of paternal age because it concerned an infertile female population). Other unanswered questions remain concerning paternal age, such as its effect on success rates in assisted reproductive techniques (ART). On the basis of 821 intracytoplasmic sperm injections carried out in a New York infertility center, Spandorfer et al (1998) found no effect of paternal age on success rates after adjusting for the effects of maternal age by including only women under age 35 years. In contrast, by analyzing data from intrauterine artificial insemination with the husband's spermatozoa, Mathieu et al (1995) concluded that paternal age over 35 years was an important predictive factor of success, after controlling for maternal age. So, in ART, conclusions concerning the effects of paternal age may differ considerably according to the technique examined. Various hypotheses that might account for the effect of paternal age on the risk of reproductive failure have been considered. For example, the possible contribution of paternal age to the occurrence of fetal trisomies has been disputed and remains controversial (Griffin et al, 1995; Sartorelli et al, 2001). Here, we present only the 2 major hypotheses: changes in sperm production and increased risk of mutation in male germ cells (Vermeulen and Kaufman, 1995; Tserotas and Merino, 1998). Changes in semen with age were demonstrated in a recent exhaustive review, which concluded that semen volume, sperm motility, and sperm morphology deteriorated with age, following an analysis that compared men aged 50 years with men aged 30 years (Kidd et al, 2001). In line with the results obtained by Bonde et al (1998) in a cohort of 430 Danish couples, such age-related changes in sperm concentration and morphology may lead to an increase in time to pregnancy. However, a number of unresolved questions remain concerning changes in sperm characteristics with male age. In particular, Kidd et al (2001) were unable to draw firm conclusions concerning the possible existence of an age threshold or the shape of the relationship (eg, linearity) between age and changes in sperm characteristics. Moreover, studies on changes in sperm characteristics with age must be analyzed with care because of possible confounders (especially duration of abstinence) and selection biases (especially in clinic-based studies). Several authors have also concluded that the effects of paternal age may be mediated by a genetic mechanism, with an increase in the risk of autosomal dominant diseases (American College of Obstetricians and Gynecologists Committee, 1997; Tarin et al, 1998). Indeed diseases such as Apert syndrome, Marfan syndrome, and Waardenburg syndrome all show a strong paternal age effect (see review by Crow, 2000). The vast majority of underlying mutations associated with paternal age are single base-pair substitutions that may be a consequence of the greater ratio of germ cell divisions between males and females. Increased incidence of mutations with age could be the result of reduced fidelity of DNA replication and repair mechanisms. Other types of mutations (small intragenic deletions and chromosome rearrangements) do not show a paternal bias, with the exception of large deletions such as loss of chromosome 18q, 4p, and 5p. However, it remains to be determined whether a paternal age effect is associated with the paternal bias observed for these forms of chromosomal deletion and also with the paternal bias observed in the expansion of trinucleotide repeats in diseases such as Huntington disease and myotonic dystrophy (Crow, 2000). All of these factors could be expected to result in reduced fertility and an increased incidence of miscarriage. In conclusion, our analysis of the existing literature on the effects of paternal age on the risk of reproductive failure suggests that 40 years could be considered to be the “amber light” in the reproductive life of men, just as 35 years is considered to be the “amber light” in the reproductive life of women (Gosden and Rutherford, 1995). Nevertheless, because of the relatively small number of published large-scale studies analyzing paternal age, our hypothesis of a cutoff age in male fertility must be confirmed by analyzing reproductive issues according to male and female ages in other large databases.

Deletions of distal chromosome 9p24 are often associated with 46,XY gonadal dysgenesis and, depending on the extent of the deletion, the monosomy 9p syndrome. We have previously noted that some cases of 46,XY gonadal dysgenesis carry a 9p deletion and exhibit behavioural problems consistent with autistic spectrum disorder. These cases had a small terminal deletion of 9p with limited or no somatic anomalies that are characteristic of the monosomy 9p syndrome. Here, we present a new case of 46,XY partial gonadal dysgenesis and autistic spectrum disorder associated with a de novo deletion of 9p24 that was detected by ultra-high resolution oligo microarray comparative genomic hybridization. The deletion included the candidate sex-determining genes in the region DMRT1 and DMRT3. These data suggest that a gene responsible for autistic spectrum disorder is located within 9p24. It remains to be determined if the gonadal dysgenesis and autistic spectrum disorder are caused by a single gene or if they are caused by distinct genetic entities at 9p24.

The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility.We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations.We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

The protamine locus consists of a 28.5 kb region with a linear array of the protamine (PRM)1, PRM2, PRM3 and transition nuclear protein (TNP)2 genes. Several studies indicate an abnormal expression pattern of protamine genes associated with male infertility, although the molecular mechanism underlying this observation is unclear. Here, we determined the spectrum of DNA variants present in all four genes in men with unexplained infertility compared with an ancestry-matched fertile/normospermic population. A total of 160 control individuals and at least 125 infertile men with either idiopathic azoospermia or oligozoospermia were sequenced for the open reading frame of PRM1, PRM2, PRM3 and TNP2 genes. All individuals carried an apparently intact Y chromosome. Of the 28 variants identified, 21 were previously described in the literature. The novel variants that were observed only in the infertile cohort included the SNP c.65G>A mutation which resulted in an amino acid change at the codon 22 (p.Ser22Asn) in the PRM1 gene, a mutation in the promoter region of PRM2 (−67C>T) and a nonsense mutation in the PRM3 gene. These data are consistent with that of previous studies which have indicated that mutations in the protamine locus may be an infrequent cause of male infertility.

Infertility affects around 1 in 10 men and in most cases the cause is unknown. The Y chromosome plays an important role in spermatogenesis and specific deletions of this chromosome, the AZF deletions, are associated with spermatogenic failure. Recently partial AZF deletions have been described but their association with spermatogenic failure is unclear. Here we screened a total of 339 men with idiopathic spermatogenic failure, and 256 normozoospermic ancestry-matched men for chromosome microdeletions including AZFa, AZFb, AZFc, and the AZFc partial deletions (gr/gr, b1/b3 and b2/b3).AZFa and AZFc deletions were identified in men with severe spermatogenic failure at similar frequencies to those reported elsewhere. Gr/gr deletions were identified in case and control populations at 5.83% and 6.25% respectively suggesting that these deletions are not associated with spermatogenic failure. However, b2/b3 deletions were detected only in men with spermatogenic failure and not in the normospermic individuals. Combined with our previous data this shows an association of the b2/b3 deletion (p = 0.0318) with spermatogenic failure in some populations. We recommend screening for this deletion in men with unexplained spermatogenic failure.

Differences of sex development are conditions with discrepancies between chromosomal, gonadal and phenotypic sex. In congenital hypogonadotropic hypogonadism, a lack of gonadotropin activity results primarily in the absence of pubertal development with prenatal sex development being (almost) unaffected in most patients. To expedite progress in the care of people affected by differences of sex development and congenital hypogonadotropic hypogonadism, the European Union has funded a number of scientific networks. Two Actions of the Cooperation of Science and Technology (COST) programmes — DSDnet (BM1303) and GnRH Network (BM1105) — provided the framework for ground-breaking research and allowed the development of position papers on diagnostic procedures and special laboratory analyses as well as clinical management. Both Actions developed educational programmes to increase expertise and promote interest in this area of science and medicine. In this Perspective article, we discuss the success of the COST Actions DSDnet and GnRH Network and the European Reference Network for Rare Endocrine Conditions (Endo–ERN), and provide recommendations for future research. The COST programme funded two European Actions for the systematic elucidation of differences of sex development and congenital hypogonadotropic hypogonadism. In this Perspective article, the authors describe the achievements of these two related COST Actions and highlight the gaps in research.

Abstract Although true hermaphroditism (TH) accounts for less than 10% of intersex patients, it stands as a diagnostic challenge and has allowed a better understanding of the mechanisms involved in sexual differentiation. In this paper we review the clinical and laboratory data as well as molecular biology findings on 16 TH patients followed up at the Pediatric Endocrine Unit, Instituto da Criança, Hospital das Clínicas, São Paulo University Medical School. They were of a mean age of 3 years 8 months and nine of them were black. All the patients had ambiguous external genitalia as the main complaint. The 46,XX karyotype accounted for 50% of the cases and the ovotestis was the most frequent gonad found (59%). In the eight TH patients with a 46,XX karyotype, the sex-determining region of the Y chromosome (SRY) was negative, posing an intriguing question about the testicular differentiation mechanisms involved in these cases. In 7/19 ovotestes, the ovarian portion of the gonad has been preserved, keeping open the possibility of fertility. The female sex option was made in 10/16 cases (62·5%) and three patients exhibited spontaneous puberty. The mechanism through which testicular tissue develops without SRY has not yet been completely clarified, suggesting the involvement of the X chromosome as well as autosomal genes in the process. European Journal of Endocrinology 136 201–204

The condition termed 46,XY complete gonadal dysgenesis is characterized by a completely female phenotype and streak gonads. In contrast, subjects with 46,XY partial gonadal dysgenesis and those with embryonic testicular regression sequence usually present ambiguous genitalia and a mix of Müllerian and Wolffian structures. In 46,XY partial gonadal dysgenesis gonadal histology shows evidence of incomplete testis determination. In 46,XY embryonic testicular regression sequence there is lack of gonadal tissue on both sides. Various lines of evidence suggest that embryonic testicular regression sequence is a variant form of 46,XY gonadal dysgenesis. The sex-determining region Y chromosome gene (SRY) encodes sequences for the testis-determining factor. To date germ-line mutations in SRY have been reported in approximately 20% of subjects with 46,XY complete gonadal dysgenesis. However, no germ-line mutations of SRY have been reported in subjects with the partial forms. We studied 20 subjects who presented either 46,XY partial gonadal dysgenesis or 46,XY embryonic testicular regression sequence. We examined the SRY gene and the minimum region of Y-specific DNA known to confer a male phenotype. The SRY-open reading frame (ORF) was normal in all subjects. However a de novo interstitial deletion 3' to the SRY-ORF was found in one subject. Although it is possible that the deletion was unrelated to the subject's phenotype, we propose that the deletion was responsible for the abnormal gonadal development by diminishing expression of SRY. We suggest that the deletion resulted either in the loss of sequences necessary for normal SRY expression or in a position effect that altered SRY expression. This case provides further evidence that deletions of the Y chromosome outside the SRY-ORF can result in either complete or incomplete sex reversal.

To the Editor: Sex determination in humans depends on the function of the SRY (sex-determining region, Y) gene. This gene consists of a single exon located on the short arm of the Y chromosome and encodes a protein that has a conserved domain shared by the high-mobility–group nuclear proteins and various transcription factors (Sinclair et al. Sinclair et al., 1990Sinclair AH Berta P Palmer MS Hawkins R Griffiths BL Smith MJ Foster JW et al.A conserved gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif.Nature. 1990; 346: 240-244Crossref PubMed Scopus (2423) Google Scholar). The SRY protein has DNA-binding and DNA-bending activities, suggesting that it may be a transcription factor that controls the expression of downstream genes involved in sex determination and/or differentiation (Pontiggia et al. Pontiggia et al., 1994Pontiggia A Rimini R Harley VR Goodfellow PN Lovell-Badge R Bianchi ME Sex reversing mutations affect the architecture of SRY-DNA complexes.EMBO J. 1994; 13: 6115-6124Crossref PubMed Scopus (249) Google Scholar). Swyer syndrome is characterized by a female phenotype and gonadal dysgenesis leading to a streak gonad, in an individual with a 46,XY chromosome complement (MIM 306100). Affected individuals have normal stature and do not have Turner stigmata (German German, 1970German JL Abnormalities of human sex chromosomes: a unifying concept in relation to gonadal dysgenesis.Clin Genet. 1970; 1: 27-57Google Scholar). Approximately 15% of these cases have mutations in the SRY gene. Duplications of the locus DSS (dosage-sensitive sex reversal) at Xp21.3 also are associated with 46,XY gonadal dysgenesis; however, duplications of Xp are uncommon in 46,XY females (Veitia et al. Veitia et al., 1997Veitia R Ion A Barbaux S Jobling MA Soleyreau N Ennis K Ostrer H et al.aMutations and sequence variants in the testis determining region of the Y chromosome in individuals with a 46,XY female phenotype.Hum Genet. 1997; 99: 648-652Crossref PubMed Scopus (88) Google Scholar). The etiology of the majority of cases is unknown. Partial monosomy of 9p (MIM 158170) is associated with a syndrome characterized by mental retardation; trigonocephaly; upward-slanting palpebral fissures; short, broad, and webbed neck; flat nasal bridge; anteverted nostrils; and long philtrum (Alfi et al. Alfi et al., 1973Alfi O Donnell GN Crandall BF Derencsenyi A Menon R Deletion of the short arm of chromosome 9 (46, 9p−): a new deletion syndrome.Ann Genet. 1973; 16: 17-22PubMed Google Scholar; Huret et al. Huret et al., 1988Huret JL Leonard C Forestier B Rethoré MO Lejeune J Eleven new cases of del (9p) and features from 80 cases.J Med Genet. 1988; 25: 741-749Crossref PubMed Scopus (126) Google Scholar). The presence of ambiguous genitalia has been reported in 70% of XY individuals monosomic for 9p (De Grouchy and Turleau De Grouchy and Turleau, 1982De Grouchy J Turleau C Monosomie 9p2.in: Atlas des maladies chromosomiques. Expansion Scientifique Française, Paris1982: 162-167Google Scholar, pp. 162–167; Schinzel Schinzel, 1984Schinzel A Catalogue of unbalanced chromosome aberrations in man. De Gruyter, New York1984Google Scholar). In addition, an increasing number of reports describe 46,XY partial or complete gonadal-dysgenesis cases associated with deletions of 9p and presenting with dysmorphic features due to either the deletion syndrome or the presence of a trisomic segment (Bennett et al. Bennett et al., 1993Bennett CP Docherty Z Robb SA Ramani P Hawkins JR Grant D Deletion 9p and sex reversal.J Med Genet. 1993; 30: 518-520Crossref PubMed Scopus (138) Google Scholar; Ogata et al. Ogata et al., 1997Ogata T Muroya K Matsuo N Hata J Fukushima Y Suzuki Y Impaired male sex development in an infant with molecularly defined 9p partial monosomy: implication for a testis forming gene (s) on 9p.J Med Genet. 1997; 34: 331-334Crossref PubMed Scopus (31) Google Scholar; Veitia et al. Veitia et al., 1997Veitia R Nunes M Brauner R Doco-Fenzy M Joanny-Flinois O Jaubert F Lortat-Jacob S et al.bDistal deletions of 9p associated with male to female sex reversal: definition of the breakpoints at 9p23.3-p24.1.Genomics. 1997; 41: 271-274Crossref PubMed Scopus (79) Google Scholar; MIM 273350). In three of these cases, the breakpoints have been defined at a molecular level, with the smallest deleted region distal to D9S144 (Veitia et al. Veitia et al., 1997Veitia R Nunes M Brauner R Doco-Fenzy M Joanny-Flinois O Jaubert F Lortat-Jacob S et al.bDistal deletions of 9p associated with male to female sex reversal: definition of the breakpoints at 9p23.3-p24.1.Genomics. 1997; 41: 271-274Crossref PubMed Scopus (79) Google Scholar) and D9S168 (Ogata et al. Ogata et al., 1997Ogata T Muroya K Matsuo N Hata J Fukushima Y Suzuki Y Impaired male sex development in an infant with molecularly defined 9p partial monosomy: implication for a testis forming gene (s) on 9p.J Med Genet. 1997; 34: 331-334Crossref PubMed Scopus (31) Google Scholar), which map ∼21 cM from the telomere. Here, we describe the molecular analysis of two cases of 46,XY gonadal dysgenesis associated with deletions of 9p. One of them had a cytogenetically detectable 9p deletion, and the other had an apparently normal 9p. Patient 1, of Portuguese origin, was born to unrelated parents and presented at birth with a normal somatic phenotype and clitoridomegalia, mild hypotonia, and a left clubfoot. Birthweight was 2,900 g, and height was 49 cm. At age 7 years the patient had affective disorders but a normal IQ. At age 10 years weight was 40 kg, and height was 145 cm. At this time, internal genitalia consisted of a normal vaginal cavity, uterus, and fallopian tubes and of bilateral streak gonads. Karyotype analysis indicated a 46,XYdel(9)(p23) chromosome complement. Hormonal evaluation at age 9 years was consistent with partial gonadal failure: plasma follicle-stimulating hormone 33 mU/ml; luteinizing hormone 21 mU/ml; testosterone 46.9 ng/dl (normal 0–30 ng/dl) and, after hCG stimulation, 68.7 ng/dl; and dihydrotestosterone 12 ng/dl (normal 3–10 ng/dl) and, after hCG stimulation, 20 ng/dl. The gonads consisted of a fibrous stroma without primary follicles (fig. 1). Small islands of Leydig-like cells were observed. On the basis of the combined endocrinology, histology, and phenotype of the patient, the diagnosis of Swyer syndrome was made. The analysis, by SSCP, of 4 kb around the SRY gene failed to show the presence of mutations. A group of 10 completely or partially 46,XY sex-reversed patients (for whom the DNA of the parents was available) also were analyzed, for the presence of a loss of heterozygosity (LOH) at 9p. These cases previously had been screened for mutations in the SRY and DSS regions (Veitia et al. Veitia et al., 1997Veitia R Ion A Barbaux S Jobling MA Soleyreau N Ennis K Ostrer H et al.aMutations and sequence variants in the testis determining region of the Y chromosome in individuals with a 46,XY female phenotype.Hum Genet. 1997; 99: 648-652Crossref PubMed Scopus (88) Google Scholar). In one case, a small deletion was found (for details of molecular analysis, see below). This individual, patient 2, of French origin, presented at birth (birthweight 2,000 g, height 49 cm) with ambiguous external genitalia (Prader 3) and one gonad palpable in the right genital fold. At laparatomy, normal uterus, vagina, fallopian tubes, and bilateral rudimentary gonads were observed. Wolffian structures such as the epididymis were also present. Gonad histology showed the presence of syncytial Sertolian cords and some spermatogonia. The patient had no dysmorphic features, and there was no evidence of psychomotor retardation, except for learning difficulties and affective disorders. High-resolution karyotype analysis indicated an apparently normal 46,XY chromosome complement. Final height, at age 19 years, was 163 cm. The patient was diagnosed as having partial gonadal dysgenesis. The breakpoints were mapped, in both cases, by analysis of the segregation of polymorphic microsatellite alleles. The markers used in this study were, from telomere to centromere, D9S1779, G10023, D9S1858, VLDLR trinucleotide repeat, D9S1813, D9S1810, D9S937, G10183, D9S286, D9S144, D9S168, D9S256, D9S267, and D9S254. Primer sequences for all markers were obtained from Genome Database and Whitehead Institute Database. The PCR reactions were performed under standard conditions for 30 cycles, in the presence of a fluorescently labeled dUTP derivative (R6G; Perkin Elmer, Applied Biosystems). Analysis was performed with an ABI370A automatic sequencer provided with GeneScan software (ABI), according to the manufacturer's instructions. Patient 1 carried a deletion of one chromosome 9, involving 9p23 (distal-middle) to pter. The rearranged chromosome 9 was found to be of paternal origin, and its breakpoint was located between D9S144 and D9S267 (fig. 2). In spite of carrying a large deletion, the patient did not present with the specific stigmata of the monosomy-9p syndrome. The hypotonia in this and other 9p-deleted patients could be caused by an alteration of the high-affinity glutamate-transporter (excitatory amino acid carrier) gene (SLC1A1), which maps to the monosomic region (Smith et al. Smith et al., 1994Smith CP Weremowics S Kanai Y Stelzner M Morton CC Hediger MA Assignment of the gene coding for the high affinity glutamate transporter EAAC1 to 9p24: potential role in dicarboxylic aciduria and neurodegenerative disorders.Genomics. 1994; 20: 335-336Crossref PubMed Scopus (40) Google Scholar). Monosomy-9p syndrome is due to the heterozygous deletion of a critical region mapping between D9S286 and D9S267 (Schwartz et al. Schwartz et al., 1997Schwartz S Crowe CA Conroy JM Haren JM Micale MA Becker LA Chromosome breakage in 9p and delineation of the critical region for the 9p deletion syndrome.Ann Hum Genet. 1997; 61: 222Google Scholar). However, Ogata et al. (Ogata et al., 1997Ogata T Muroya K Matsuo N Hata J Fukushima Y Suzuki Y Impaired male sex development in an infant with molecularly defined 9p partial monosomy: implication for a testis forming gene (s) on 9p.J Med Genet. 1997; 34: 331-334Crossref PubMed Scopus (31) Google Scholar) have described a sex-reversed patient carrying a pure 9p deletion who presented with a phenotype compatible with the 9p-deletion syndrome. The breakpoint was located distal to D9S168 (20 cM from the telomere), suggesting that the monosomy-9p critical region must lie distal to this marker. Our results suggest that this critical region is proximal to D9S144, because the absence of the 9p distal portion including this marker in patient 1 is not associated with the characteristic dysmorphology. Taken together, these data indicate that the minimum region responsible for the syndrome may be located between D9S144 and D9S168. Notably, both markers have been unambiguously mapped to YAC 784-B-4 (Whitehead Institute Database), which may contain genes involved in brain, skeletal, and craniofacial development. These data also indicate that the 9p deletion–syndrome locus and the sex-reversal locus are distinct entities and that some 46,XY females (or even normal individuals) without somatic anomalies may harbor 9p deletions. Consistent with this hypothesis, we identified an individual (patient 2) with an apparently normal 9p who was hemizygous for markers D9S1858 and VLDLR (fig. 2). All alleles present were of paternal origin. The most distal heterozygous marker was D9S1813, which maps to a point 8 cM from the telomere. Since VLDLR resides in the contig WC1422, ∼2 cM telomeric with respect to D9S1813 (the distal end of contig WC844), the breakpoint in this patient may lie 6–8 cM from the telomere. The presence of a deletion was confirmed by PCR amplification of somatic-cell hybrids containing the rearranged chromosome 9. This individual did not present with hypotonia, and, consistent with the hypothesis described earlier, the marker WI-7925 (SLC1A1), mapping proximal to D9S1813 (Whitehead Institute Database), could be amplified from the hybrids containing the deleted chromosome 9. The molecular analysis of this patient confirmed that a deletion of 9p may be associated with 46,XY sex reversal in the absence of monosomy-9p syndrome and that the critical region for the sex-reversing locus is distal to D9S1813. Although, given the small number of samples studied, no statistical conclusions can be drawn from this screening it is remarkable that 1 of 10 patients carried a deletion. These deletions may be undetectable by ordinary cytogenetic techniques, because of their very-terminal character (Huret et al. Huret et al., 1988Huret JL Leonard C Forestier B Rethoré MO Lejeune J Eleven new cases of del (9p) and features from 80 cases.J Med Genet. 1988; 25: 741-749Crossref PubMed Scopus (126) Google Scholar). Recently, a case of sex reversal associated with a familial translocation with a breakpoint at 9p24 has been described; however, the position of the breakpoint has not been defined at a molecular level (McDonald et al. McDonald et al., 1997McDonald MT Flejter W Sheldon S Putzi MJ Gorski JL XY sex reversal and gonadal dysgenesis due to 9p24 monosomy.Am J Med Genet. 1997; 73: 321-326Crossref PubMed Scopus (51) Google Scholar). Elsewhere, we also have described a patient with a complex 9p rearrangement associated with 46,XY partial gonadal dysgenesis (Ion et al. Ion et al., 1998Ion R Telvi L Chaussain JL Barbet JP Nunes M Safar A Rethore MO et al.Failure of testicular development associated with a rearrangement of 9p24.1 proximal to the SNF2 gene.Hum Genet. 1998; 102: 151-156Crossref PubMed Scopus (20) Google Scholar). This patient had an inverted duplication of 9p, with a distal breakpoint proximal to the SNF2 gene, at 9p24.1. In this case, we failed to identify any rearrangement distal to this gene, suggesting that the phenotype may be caused by a position effect. . The authors wish to thank Nicole Souleyreau for excellent technical assistance and Drs. Marguerite Prieur, Catherine Turleau, Géraldine Viot, and Michel Vekemans for helpful discussions. R.A.V. has been supported by the Fondation pour La Recherche Médicale.

Abstract A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y‐chromosome instability. We studied 17 clinically well‐characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y‐chromosome specific‐sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y‐chromosome loci hosting genes other than SRY , and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y‐chromosome instability. © 2005 Wiley‐Liss, Inc.

Recent data emphasized the implication of polymerase gamma (POLG) CAG repeats in infertility, making it a very attractive gene for study. A comparison of POLG CAG repeats in infertile and fertile men showed a clear association between the absence of the usual 10-CAG allele and male infertility, excluding azoospermia. It has also been suggested that the POLG gene polymorphism should be considered as a possible contributing factor in unexplained couple infertility where semen parameters are normal. In this study, we investigated the POLG CAG repeats, in a well-defined population of patients with severe male factor infertility.We conducted a large study of POLG CAG repeats in 433 infertile and 91 fertile, normozoospermic and healthy males. In all subjects, phenotypic data, including semen parameters, hormonal status and clinical profiles, were available.Thirteen 'homozygous mutants' (3%) were found among the 433 idiopathic infertile patients. The follow-up of the 13 'homozygous mutant' resulted in pregnancy for more than half of the couples, through assisted reproductive techniques or even spontaneously. In addition, one 'homozygous mutant' was identified in 91 fertile men (1.1%)Under our conditions, our study does not confirm any relationship between the polymorphic CAG repeat in the POLG gene and male infertility.

No statistically significant difference in deletion frequency was found between infertile patients and a control group. It is suggested that in some populations the gr/gr deletion might be an inconsequential polymorphism.

The aim of this study is to determine if Müllerian agenesis has a genetic basis linked to the WNT genes. Genomic DNA analyses for mutations in the coding sequences of four members of this family in a series of 11 women with Mayer-Rokitansky-Kuster-Hauser syndrome found four variants in the coding sequence of these genes, but causal mutations were not observed. This supports the hypothesis that mutations in the coding sequence of WNT4, WNT5A, WNT7A, and WNT9B are not responsible for the Mayer-Rokitansky-Kuster-Hauser syndrome.

To evaluate for the first time the frequency of Y chromosome microdeletions and the occurrence of the partial deletions of AZFc region in Moroccan men, and to discuss the clinical significance of AZF deletions.We screened Y chromosome microdeletions and partial deletions of the AZFc region of a consecutive group of infertile men (n = 149) and controls (100 fertile men, 76 normospermic men). AZFa, AZFb, AZFc and partial deletions of the AZFc region were analyzed by polymerase chain reaction (PCR) according to established protocols.Among the 127 infertile men screened for microdeletion, four subjects were found to have microdeletions: two AZFc deletions and two AZFb+AZFc deletions. All the deletions were found only in azoospermic subjects (4/48, 8.33%). The overall AZFc deletion frequency was low (4/127, 3.15%). AZF microdeletions were not observed in either oligoasthenoteratozoospermia (OATS) or the control. Partial deletions of AZFc (gr/gr) were observed in a total of 7 of the 149 infertile men (4.70%) and 7 partial AZFc deletions (gr/gr) were found in the control group (7/176, 3.98%). In addition, two b2/b3 deletions were identified in two azoospermic subjects (2/149, 1.34%) but not in the control group.Our results suggest that the frequency of Y chromosome AZF microdeletions is elevated in individuals with severe spermatogenic failure and that gr/gr deletions are not associated with spermatogenic failure.

Deletions in the azoospermia factor (AZF) region of the Y chromosome are frequent in infertile men. The clinical consequences and the mode of inheritance of these deletions are not yet clear.Y chromosome deletion mapping and quantitative PCR analysis of the DAZ-gene copy number, supplemented with haplogroup typing in deleted patients, were performed, in combination with clinical assessments in 264 fathers and their sons conceived by assisted reproduction techniques (ART), and in 168 fertile men with normal sperm concentration.In the ART fathers group, a complete AZFc deletion was detected in 0.4% (1/264). AZFc rearrangements/polymorphisms were found in 6.8% (18/264; 95% CI: 4.4-10.5), which was significantly more frequent (P = 0.021) than in the controls (3/168; 1.8%, 95% CI: 0.6-5.1). All deletions were transmitted to the sons, without any clinical symptoms in early childhood. In the fathers, there was no significant correlation between the DAZ copy number and the severity of spermatogenic failure.AZFc rearrangements/polymorphisms are transmitted to sons and may represent a risk factor for decreased testis function and male subfertility, which needs confirmation in further studies in larger cohorts. However, deletions of two DAZ gene copies are compatible with normal spermatogenesis and fertility.

In a majority of individuals with disorders/differences of sex development (DSD) a genetic etiology is often elusive. However, new genes causing DSD are routinely reported and using the unbiased genomic approaches, such as whole exome sequencing (WES) should result in an increased diagnostic yield. Here, we performed WES on a large cohort of 125 individuals all of Algerian origin, who presented with a wide range of DSD phenotypes. The study excluded individuals with congenital adrenal hypoplasia (CAH) or chromosomal DSD. Parental consanguinity was reported in 36% of individuals. The genetic etiology was established in 49.6% (62/125) individuals of the total cohort, which includes 42.2% (35/83) of 46, XY non-syndromic DSD and 69.2% (27/39) of 46, XY syndromic DSD. No pathogenic variants were identified in the 46, XX DSD cases (0/3). Variants in the AR, HSD17B3, NR5A1 and SRD5A2 genes were the most common causes of DSD. Other variants were identified in genes associated with congenital hypogonadotropic hypogonadism (CHH), including the CHD7 and PROKR2. Previously unreported pathogenic/likely pathogenic variants (n = 30) involving 25 different genes were identified in 22.4% of the cohort. Remarkably 11.5% of the 46, XY DSD group carried variants classified as pathogenic/likely pathogenic variant in more than one gene known to cause DSD. The data indicates that variants in PLXNA3, a candidate CHH gene, is unlikely to be involved in CHH. The data also suggest that NR2F2 variants may cause 46, XY DSD.

The human Y chromosome is strictly paternally inherited and, in most of its length, does not recombine during male meiosis. These features make the Y a very useful genetic marker for different purposes. In the last decade, the Y has been increasingly used to investigate the evolution, migrations and range expansions of modern humans. The possibility to construct highly informative Y chromosome haplotypes has also had a significant impact in forensic studies and paternity testing. All these studies assume that the Y chromosome markers used are selectively neutral. However, recent experimental and statistical analyses suggest that both positive and negative selection are acting on the Y chromosome and, consequently, may influence Y chromosome haplotype distribution in the general population. Current data suggest that the effects of selection on patterns of Y chromosome distribution are minimal, however as interest focuses on biological functions of the Y chromosome which have a major impact on male fitness such as fertility, these assumptions may be challenged. This review briefly describes the genes and biological functions of the human Y chromosome and its use in disentangling the origin and history of human populations. An overview of the role of selection acting on the Y chromosome from the perspective of human population histories and disease is given.

The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provided a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences were observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gave a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. We therefore hypothesize that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55 000-200 000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations showed that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race.

Although most of the human Y does not normally recombine with the X chromosome, there are two limited regions of sequence identity with the X that permit pairing and recombination during male meiosis (see Rappold 1993). These are the pseudoautosomal regions located at the distal portions of the short and long arms of the Y chromosome. The Yp pseudoautosomal region consists of 2.6 Mb. Absence of this region is associated with short stature, and male infertility. During meiotic prophase, germ cells require the Y chromosome as a pairing partner for the X. In the absence of pairing, caused by deletions of the pseudoautosomal region, germ cells undergo meiotic arrest resulting in azoospermia. The Yq pseudoautosomal region is 0.4 Mb in size and contains the genes for the interleukin 9 receptor (ILR9) and synaptobevine (SYBL1). Both genes have X homologues. The gene for ILR9 escapes X-inactivation and is expressed from the Y whereas SYBL1 is subject to X-inactivation and is not expressed from the Y chromosome (Vermeesch et al. 1997). The boundary between the pseudoautosomal region and the non-recombining region is defined by an Alu element of 303 bp followed by 220 bp with 78% identity, after which the sequences diverge completely (Ellis et al. 1989). This Alu repeat element inserted at a pre-existing boundary sometime after the Old World monkey and great ape lineages diverged. During male meiosis there is an obligatory crossing-over event between Xp and Yp pseudoautosomal regions which maintains X-Y identity. There is a gradient of recombination which decreases as one approaches the pseudoautosomal boundary (Rouyer et al. 1986).

Y chromosome microdeletions have been reported as a possible genetic factor of male infertility. Despite a large number of studies in this subject, there is still considerable debate and confusion surrounding the role of Y chromosome microdeletions in male infertility. This has been further compounded by observations of Y microdeletions in fertile males. The aim of the present study was to evaluate: 1) the incidence of Y microdeletions in control male population and infertile males, where complete semen and hormonal analysis was available to define whether Y microdeletions are specific for spermatogenic failure or if they can be found also in normospermic men; and 2) whether the suboptimal semen quality reported in Denmark is associated with a higher incidence of Y microdeletions in respect to other populations. Double-blind molecular study of deletions was performed in 138 consecutive patients seeking intracytoplasmic sperm injection treatment, 100 men of known fertility, and 107 young military conscripts from the general Danish population. Microdeletions or gene-specific deletions were not detected in normospermic subjects or in subfertile men with a sperm count of more than 1 x 10(6)/mL. Deletions of the Azoospermia factor (AZF)c region were detected in 17% of individuals with idiopathic azoo/cryptozoospermia and in 7% of individuals with nonidiopathic azoo/cryptozoospermia. The data indicate that: 1) the composition of the study population is the major factor in determining deletion frequency; 2) Y chromosome microdeletions are specifically associated with severe spermatogenic failure; therefore, the protocol described here is reliable for the routine clinical workup of severe male factor infertility; and 3) the frequency of Yq microdeletions in the Danish population is similar to that from other countries and argues against the involvement of microdeletions in the relatively low sperm count of the Danish population.

Abstract Context: We report herein a remarkable family in which the mother of a woman with 46,XY complete gonadal dysgenesis was found to have a 46,XY karyotype in peripheral lymphocytes, mosaicism in cultured skin fibroblasts (80% 46,XY and 20% 45,X) and a predominantly 46,XY karyotype in the ovary (93% 46,XY and 6% 45,X). Patients: A 46,XY mother who developed as a normal woman underwent spontaneous puberty, reached menarche, menstruated regularly, experienced two unassisted pregnancies, and gave birth to a 46,XY daughter with complete gonadal dysgenesis. Results: Evaluation of the Y chromosome in the daughter and both parents revealed that the daughter inherited her Y chromosome from her father. Molecular analysis of the genes SOX9, SF1, DMRT1, DMRT3, TSPYL, BPESC1, DHH, WNT4, SRY, and DAX1 revealed normal male coding sequences in both the mother and daughter. An extensive family pedigree across four generations revealed multiple other family members with ambiguous genitalia and infertility in both phenotypic males and females, and the mode of inheritance of the phenotype was strongly suggestive of X-linkage. Conclusions: The range of phenotypes observed in this unique family suggests that there may be transmission of a mutation in a novel sex-determining gene or in a gene that predisposes to chromosomal mosaicism.

Distal chromosome 9p contains a locus that, when deleted, is a cause of 46,XY gonadal dysgenesis in the absence of extragenital anomalies. This locus might account for the frequently observed cases of 46,XY pure gonadal dysgenesis who do not harbor mutations in SRY, the sex master regulator gene found in mammalian species. The genomic organization of 9p positional candidate genes is currently being studied and mutational screens are ongoing. Among other positional candidates, including two additional doublesex-related genes, the evidence to support a role for the gene DMRT1 in vertebrate male sexual development is accumulating. Although formal proof of the requirement of DMRT1 in gonadal sex fate choice has not been obtained so far, the particular interest in this gene and perhaps other doublesex-related genes identified in vertebrates lies in that they may provide an entry point to a conserved mechanism of sex determination across animal phyla. We discuss recent results and emerging views on the genetics of sex determination, while stressing that the majority of cases of 46,XY gonadal dysgenesis remain unexplained. The latter is likely to be efficiently addressed by positional cloning efforts, particularly by considering the wealth of sequence data provided by the Human Genome Project.

Intense efforts are currently being pursued to identify autosomal genes associated with 46,XY male-to-female sex reversal. The genes DMRT1 and 2 are located on distal 9p, a region deleted in 46,XY sex-reversed patients. They are considered excellent candidates because of their homology to regulators of sex development in invertebrates. We present the genomic structure of DMRT2, showing that it generates several transcripts with distinct coding potential. In addition to the previously reported 226-amino-acid protein-encoding transcript, we describe other mRNA isoforms that are potentially bicistronic and are predicted to encode an additional 328-amino-acid polypeptide. Finally, a stop codon-containing exon (exon 4) can be skipped by alternative splicing and can generate a transcript that is predicted to encode a fusion protein. The latter shares 58% amino acid identity with a gene recently described in fish, termed terra. Differences in expression pattern exist for DMRT2 mRNA isoforms among the human adult tissues tested, between adult tissues and human embryos, and between DMRT2 and DMRT1 during embryonic development. We failed to detect mutations by sequencing of DMRT2 in a sample of 46,XY female patients. The interesting structure of DMRT2 coupled to preliminary functional studies in fish showing that terra is involved in somitogenesis suggests that validation or exclusion of this gene as a cause of sex reversal will require more in-depth investigations.

Translocations involving the X and Y chromosomes are often associated with anomalies of gonadal development. Transfer of Yp sequences, including the testis-determining SRY gene, to the terminal portion of the short arm of the X chromosome is associated with 46,XX maleness and in rare cases 46,XX true hermaphroditism. Three classes of XX males have been defined on the basis of the extent of Y material transferred to the X chromosome. In one class, the transfer of material involves aberrant recombination between two highly homologous genes, PKRX and PKRY, and there is evidence to suggest that this interchange is influenced by the Y chromosome background. Other types of X-Y translocations associated with anomalies of sex differentiation include Xp-Yq translocations, which result in a functional disomy of Xp sequences including the DSS locus and are associated with 46,XY complete or partial gonadal dysgenesis. In rare cases Yp-Xq translocations have been described in association with 46,XX maleness.

Testicular production of inhibin B is believed to be dependent on the presence of germ cells within the seminiferous tubules. However, this association has recently been questioned in patients with deletions of azoospermia factor (AZF) on the Y chromosome. We have addressed this problem in 442 unselected infertile/subfertile patients (excluding obstructive and iatrogenic forms) who were analyzed for Yq microdeletions. AZFc microdeletions were found in 16 patients (3.8% of the total infertile group, but 9% of the subgroup with azoospermia or severe oligozoospermia with sperm concentration <1 x 10(6)/ml). The reproductive hormone profiles in patients with AZFc microdeletions were analyzed and compared with those in infertile patients without microdeletions and those in fertile control individuals. The mean serum inhibin B concentration in the patients with AZFc microdeletions (39.5 +/- 36.0 pg/ml) was significantly lower than that in the group of infertile patients without microdeletions (134.6 +/- 88.5 pg/ml). However, no significant difference was found compared with that in a matched group of infertile patients with comparably low sperm counts (72.6 +/- 75.5 pg/ml). Bilateral testicular biopsies in the AZFc-deleted patients revealed a variable histological pattern suggestive of a progressive depletion of seminiferous epithelium. An association between testicular pathology and the reproductive hormone profile was found; the more severe forms had lower inhibin B and higher FSH levels. Importantly, if Sertoli cell-only tubules were prevalent in the biopsy, inhibin B was invariably undetectable. In patients with bilateral spermatocytic arrest, inhibin B remained within the normal range, which is consistent with a role of spermatocytes in the maintenance of inhibin B secretion. Our data support the view that, in contrast to recently published data, in patients with AZF microdeletions the serum concentration of inhibin B is dependent upon the functional interaction between Sertoli cells and spermatocytes and/or spermatids.

Determination of mammalian sex depends on the presence or absence of a functional testis. Testes are determined by the activity of the testis determining factor encoded by the sex determining gene, Y (SRY) located on the Y chromosome. Considerable evidence suggests that the SRY gene is the only gene on the Y chromosome that is both necessary and sufficient to initiate testis determination. Other steps in the mammalian sex determining pathway are unknown, although recent advances have shown that mutations in X chromosome and autosomal loci are also associated with sex reversal, suggesting the presence of at least one other sex determining gene. Duplications of sequences on the short arm of the human X chromosome, including the DAX-1 (DSS-AHC critical region on the X chromosome, gene 1) gene, are occasionally associated with XY male-to-female sex reversal. In addition, mutations in the SRY-related gene SOX9 (SRY-related box) are associated with a failure of human testicular determination. Furthermore, the occurrence of inherited sex reversed conditions in both mice and men indicate the presence of at least one other sex determining gene. Breeding the Y chromosome from certain Mus musculus domesticus strains into the laboratory mouse strain C57BL/6J results in XY male-to-female sex reversal. This suggests both allelic variation of the Sry gene and the presence of autosomal sex determining genes. In humans, familial cases of SRY-negative XX males occur. Analysis of the transmission of the trait indicates the segregation of an autosomal or X-linked recessive mutation. The mutation may be in a gene whose wild-type function is to inhibit male sex determination. SRY may trigger male sex determination by repressing or functionally antagonizing the product of this gene.

Deletions of the Y chromosome are a significant cause of spermatogenic failure.Three major deletion intervals have been defined and termed AZFa, AZFb and AZFc.Here, we report an unusual case of a proximal AZFb deletion that includes the Y chromosome palindromic sequence P4 and a novel heat shock factor (HSFY).This deletion neither include the genes EIF1AY, RPS4Y2 nor copies of the RBMY1 genes.The individual presented with idiopathic azoospermia.We propose that deletions of the testis-specific HSFY gene family may be a cause of unexplained cases of idiopathic male infertility.This deletion would not have been detected using current protocols for Y chromosome microdeletion screens, therefore we recommend that current screening protocols be extended to include this region and other palindrome sequences that contain genes expressed specifically in the testis.

Recently, mutations in the X-linked ubiquitin protease 26 (USP26) gene have been proposed to be associated with male infertility. In particular a 371insACA, 494T>C and 1423C>T haplotype, which results in a T123-124ins, L165S and H475Y amino acid change respectively, has been reported to be associated with Sertoli cell-only syndrome (SCOS) and an absence of sperm in the ejaculate. Here, we demonstrate that two of these changes actually correspond to the ancestral sequence of the gene and that the USP26 haplotype is present in significant frequencies in sub-Saharan African and South and East Asian populations, including in individuals with known fertility. This indicates that the allele is not associated with infertility. The pattern of frequency distribution of the derived haplotype (371delACA, 494T), which is present at high frequencies in most non-African populations could be interpreted as either a result of migration followed by simple genetic drift or alternatively as positive selection acting on the derived alleles. The latter hypothesis seems likely, because there is evidence of strong positive selection acting on the USP26 gene.

Summary The detailed analysis of the Y chromosome in men with azoospermia or severe oligozoospermia has resulted in the identification of three regions of the long arm of the human Y chromosome, termed AZFa, AZFb and AZFc, (AZF: AZoospermia Factor) that are currently deleted in men with otherwise unexplained spermatogenic failure. Screening for these deletions is now common in many infertility centres and in some instances they have a prognostic relevance. Recently, attention has turned to partial deletions of the AZFc region. At first sight some of these deletions appear to have little effect on fertility, whilst others appear to be associated with significant risk for developing spermatogenic failure. However, the relationship between these partial AZFc deletions, reduced sperm counts and infertility is the subject of a continuing intense debate. There is a pressing need to clarify the impact of partial AZFc deletions on human spermatogenesis. This requires large‐scale studies on well‐characterized normospermic and oligo/azoospermic individuals of different ethnic origins with multiple informative AZFc markers if the correlation between these deletions and the phenotype is finally to be resolved. The definition of Y chromosome variants (haplotypes) in cases of partial AZFc deletions is likely to play an essential role in understanding the contribution of the deletion to reduced sperm counts.

ObjectiveTo determine if there is a relationship between various forms of partial AZFc deletions and spermatogenic failure.DesignCase-control study.SettingInfertility clinic (Tenon Hospital, Paris).Patient(s)557 men, comprising 364 infertile men from mixed ethnic backgrounds, and 193 men with known fertility (n = 84) and/or normospermic (n = 109).Intervention(s)Characterization of 32 partial AZFc deletions.Main Outcome Measure(s)DAZ gene cluster divided into two families (DAZ1/2 and DAZ3/4), CDY1 gene, and Y-chromosome haplogroups.Result(s)We observed 18 partial AZFc deletions in 364 (4.95%) infertile men compared with 14 out of 193 (7.25%) in the control normospermic/fertile group.Conclusion(s)The analysis of informative Y-chromosome single nucleotide variants combined with Y-chromosome haplogroup definition enabled us to infer seven deletion classes that occur on a minimum of six Y-chromosome parental architectures. We found no relationship between either the presence or the absence of DAZ1/2, DAZ3/4, CDY1a, or CDY1b with spermatogenic failure at least on one Y-chromosome lineage. The DAZ dosage and Southern blot analyses indicated that the majority of individuals tested carried two copies of the DAZ gene, indicating a partial AZFc deletion. Our data are consistent with the hypothesis that, at least in our study populations, partial AZFc deletions may have a limited impact on fertility. To determine if there is a relationship between various forms of partial AZFc deletions and spermatogenic failure. Case-control study. Infertility clinic (Tenon Hospital, Paris). 557 men, comprising 364 infertile men from mixed ethnic backgrounds, and 193 men with known fertility (n = 84) and/or normospermic (n = 109). Characterization of 32 partial AZFc deletions. DAZ gene cluster divided into two families (DAZ1/2 and DAZ3/4), CDY1 gene, and Y-chromosome haplogroups. We observed 18 partial AZFc deletions in 364 (4.95%) infertile men compared with 14 out of 193 (7.25%) in the control normospermic/fertile group. The analysis of informative Y-chromosome single nucleotide variants combined with Y-chromosome haplogroup definition enabled us to infer seven deletion classes that occur on a minimum of six Y-chromosome parental architectures. We found no relationship between either the presence or the absence of DAZ1/2, DAZ3/4, CDY1a, or CDY1b with spermatogenic failure at least on one Y-chromosome lineage. The DAZ dosage and Southern blot analyses indicated that the majority of individuals tested carried two copies of the DAZ gene, indicating a partial AZFc deletion. Our data are consistent with the hypothesis that, at least in our study populations, partial AZFc deletions may have a limited impact on fertility.

ObjectiveTo evaluate the carrier frequency of the pathogenic c.144delC mutation in AURKC gene and the contribution of this mutation in male infertility in a Moroccan population.DesignSanger sequencing of exon 3 in AURKC gene in infertile and control patients in Morocco.SettingResearch institute.Patient(s)A total of 326 idiopathic infertile patients, and 450 age-related men.Intervention(s)The incidence of AURKC c.144delC mutation was determined in men with unexplained spermatogenic failure and a control cohort of normospermic fertile men.Main Outcome Measure(s)Genomic DNA was extracted from peripheral blood lymphocytes and the screening of the c.144delC mutation in AURKC gene performed by polymerase chain reaction and sequencing.Result(s)The c.144delC mutation in AURKC gene was found in patients at homozygous and heterozygous states, with an allelic frequency of 2.14%, whereas in controls this mutation was found only in the heterozygous state, with lower frequency (1%). Homozygous patients were characterized by macrocephalic and multiflagellar spermatozoa.Conclusion(s)Our data indicate that the AURKC c.144delC mutation has a relatively high carrier frequency in the Moroccan population; thus, we recommend screening for this deletion in infertile men with a high percentage of large-headed and multiflagellar spermatozoa. To evaluate the carrier frequency of the pathogenic c.144delC mutation in AURKC gene and the contribution of this mutation in male infertility in a Moroccan population. Sanger sequencing of exon 3 in AURKC gene in infertile and control patients in Morocco. Research institute. A total of 326 idiopathic infertile patients, and 450 age-related men. The incidence of AURKC c.144delC mutation was determined in men with unexplained spermatogenic failure and a control cohort of normospermic fertile men. Genomic DNA was extracted from peripheral blood lymphocytes and the screening of the c.144delC mutation in AURKC gene performed by polymerase chain reaction and sequencing. The c.144delC mutation in AURKC gene was found in patients at homozygous and heterozygous states, with an allelic frequency of 2.14%, whereas in controls this mutation was found only in the heterozygous state, with lower frequency (1%). Homozygous patients were characterized by macrocephalic and multiflagellar spermatozoa. Our data indicate that the AURKC c.144delC mutation has a relatively high carrier frequency in the Moroccan population; thus, we recommend screening for this deletion in infertile men with a high percentage of large-headed and multiflagellar spermatozoa.

Abstract Two agonadic sisters, one with a 46,XY and the other with a 46,XX karyotype, both with normal female external genitalia and hypoplastic Müllerian derivatives, born to a consanguineous marriage, were studied from a clinical, endocrinological, histological, and genetic perspective. Using PCR amplification, Southern hybridization, and DGGE analysis, it was found that the XY patient had no mutations in the conserved sequence of the SRY gene, the putative testis‐determining gene in mammals, whereas her XX affected sister is SRY‐negative. To our knowledge, this is the first report of XY and XX sibs in familial gonadal agenesis without other somatic abnormalities. The involvement of an autosomal locus impeding gonadal development in both sexes is discussed. © 1994 Wiley‐Liss, Inc.

Molecular deletions of the Y chromosome long arm are a frequent cause of male infertility. Because these deletions are thought to be inherited from fathers without Y chromosome deletions, the question arises as to whether their relatively high incidence in the male population could be due to the existence of a mosaicism in somatic and/or germinal paternal cells. This study included a total of 181 infertile men, among whom 18 were found to have an abnormal karyotype. In the other 163, polymerase chain reaction (PCR) analysis detected nine (5.5%) Y chromosome microdeletions. Blood, spermatozoa or testicular cells from 47 men (27 oligozoospermia, 20 azoospermia), including six Y-deleted patients, were screened for mosaicism using double target fluorescence in-situ hybridization (FISH) with Y centromeric and deleted in azoospermia (DAZ) gene-specific probes. Results indicated that: (i) percentages of double (intact Y chromosome) or single (deleted Y chromosome) fluorescent signals by FISH were in agreement with PCR data, thus demonstrating the reliability of the method; and (ii) a weak germ cell mosaicism was found in only two oligozoospermic patients, carrying 1.97 and 4.13% respectively of spermatozoa with a deleted Y chromosome. Further studies on larger populations are needed to evaluate precisely the incidence of Y deletion mosaicisms in infertile men.

ObjectiveTo assess genetic mutations and associated somatic anomalies in a series of patients with 46,XY gonadal dysgenesis (GD).DesignSingle center retrospective study.SettingUniversity pediatric hospital.Patient(s)Fourteen patients with 46,XY GD.Intervention(s)None.Main Outcome Measure(s)Genotype-phenotype relationship.Result(s)The presenting symptom was disorders of sex development (6 patients), primary amenorrhea (2 patients), discordance between 46,XY karyotype and female external genitalia (3 patients), discovery of Müllerian structures at surgery (2 patients), or diagnosed in the evaluation of a gonadal tumor (1 patient). Müllerian structures were shown by ultrasound evaluation in 7 of 13 patients, genitography in 3 of 6 patients and/or surgery in 8 of 10 patients (3 not seen at imaging), or only by histologic examination (1 patient). Three patients had gonadoblastoma and/or seminoma. A mutation was found in 7 patients of whom 2 had family history of reproductive problems and 5 had associated somatic anomalies. The mutations were FOG2/ZFPM2 (1 patient), SRY (2 patients), WT1 (1 patient), or deletions of distal chromosome 9p (3 patients). Among the three other patients with associated anomalies and no mutation, two had ectodermal dysplasia and one had leukemia.Conclusion(s)Mutations were observed in half of the patients with 46,XY GD with Müllerian structures. We also describe for the first time the association between GD and ectodermal dysplasia. Müllerian structures can be found in some cases only by histologic examination, which should be coupled to preventive gonadectomy because of the risk of tumor formation. To assess genetic mutations and associated somatic anomalies in a series of patients with 46,XY gonadal dysgenesis (GD). Single center retrospective study. University pediatric hospital. Fourteen patients with 46,XY GD. None. Genotype-phenotype relationship. The presenting symptom was disorders of sex development (6 patients), primary amenorrhea (2 patients), discordance between 46,XY karyotype and female external genitalia (3 patients), discovery of Müllerian structures at surgery (2 patients), or diagnosed in the evaluation of a gonadal tumor (1 patient). Müllerian structures were shown by ultrasound evaluation in 7 of 13 patients, genitography in 3 of 6 patients and/or surgery in 8 of 10 patients (3 not seen at imaging), or only by histologic examination (1 patient). Three patients had gonadoblastoma and/or seminoma. A mutation was found in 7 patients of whom 2 had family history of reproductive problems and 5 had associated somatic anomalies. The mutations were FOG2/ZFPM2 (1 patient), SRY (2 patients), WT1 (1 patient), or deletions of distal chromosome 9p (3 patients). Among the three other patients with associated anomalies and no mutation, two had ectodermal dysplasia and one had leukemia. Mutations were observed in half of the patients with 46,XY GD with Müllerian structures. We also describe for the first time the association between GD and ectodermal dysplasia. Müllerian structures can be found in some cases only by histologic examination, which should be coupled to preventive gonadectomy because of the risk of tumor formation.

Aromatase deficiency (AD) is a very rare disorder resulting from mutations in the CYP19A1 gene encoding aromatase, a cytochrome P450 enzyme that plays a pivotal role in androgen conversion to estrogens. AD is inherited in an autosomal recessive trait, and to date only 35 cases have been described in the literature. Herein, we depict a new patient reared as a male, who presented at the age of 21 years with no palpable testis, hypoplastic scrotum, penis-like phallus (3 cm), and penoscrotal hypospadias. The patient was born to consanguineous parents, his karyotype was 46,XX, and SRY was negative. Pelvic sonar showed a small hypoplastic uterus, and no testis could be identified. Serum testosterone was within the reference range of females along with high gonadotropins. Pathology of gonadal biopsy showed ovarian stroma negative for oocytic follicle consistent with streak gonads. All these data were suggestive of AD, which was subsequently confirmed by molecular investigation of the CYP19A1 gene. A homozygous splice site mutation in the donor splice site of exon 9 was identified, c.1263 + 1G>T. This is the first report of such a rare disorder in an Egyptian patient. Our results reinforce the importance of considering AD in patients with 46,XX disorders of sex development after ruling out congenital adrenal hyperplasia.

Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, and AZFc) that are recurrently deleted in infertile males. In a blind study we screened 131 infertile males (46 idiopathic and 85 nonidiopathic) for Y chromosome microdeletions. Nineteen percent of idiopathic males, with an apparently normal 46,XY chromosome complement had microdeletions of either the AZFa, AZFb, or AZFc region. There was no strict correlation between the extent or location of the deletion and the phenotype. The AZFb deletions did not include the active RBM gene. Significantly, a high frequency of microdeletions (7%) was found in patients with known causes of infertility and a 46,XY chromosome complement. These included deletions of the AZFb and AZFc regions, with no significant difference in the location or extent of the deletion compared with the former group. It is recommended that all males with reduced or absence sperm counts seeking assisted reproductive technologies be screened for deletions of the Y chromosome.

CONTENTS – Sex determination in the fruit fly and nematode – Somatic sex determination – Dosage compensation – Germ‐line sex determination – Mammalian sex determination signifies testis determination – The mammalian sex‐determining master‐regulatory gene, SRY – XX and XY sex reversal in man – Sex‐determining region Y chromosome (SRY) – Is SRY the primary testis determinant?– Biochemical properties of SRY – Sequence versus structure recognition – SRY: positive or negative regulation?– Evidence for other mammalian sex‐determining genes – Murine autosomal sex‐determining genes – Wilms' tumour gene WT1 – Chromosomal abnormalities associated with sex reversal – Müllerian inhibiting substance: a target of SRY ?– Perspectives

The pathogenesis of 46 XX true hermaphroditism is uncertain and the role of the SRY gene in ovotestis development has not been thoroughly evaluated. We ascertained the presence of the SRY gene and SRY protein in the ovotestis.We evaluated 8 ovotestes by cytogenetic analysis of fibroblast cell culture and analysis of gonadal tissue by polymerase chain reaction to detect the SRY gene and by immunohistochemistry with a monoclonal antibody to human recombinant SRY protein.Fibroblast culture of the ovotestes demonstrated a 46XX karyotype. By polymerase chain reaction all 8 ovotestes demonstrated the SRY gene at low levels. By immunohistochemistry SRY protein was detected in all ovotestes, predominantly in Sertoli and germ cells.The SRY gene has a role in ovotestis genesis. Mosaicism with a Y bearing cell line in the gonad is a possible explanation and further study is warranted.

Abstract 46,XY gonadal dysgenesis is characterized by abnormal testicular determination. We describe a large kindred in which various disorders of sexual development were observed, ranging from completely female phenotype without ambiguities of the external genitalia (five cases) to men with isolated penile or perineal hypospadias (four cases), including two cases with moderate virilization and one case with ambiguity of the external genitalia. Histologic examination of gonadal tissue was performed on seven subjects. These findings were suggestive of complete gonadal dysgenesis in one patient, partial gonadal dysgenesis in three patients, and mixed gonadal dysgenesis in three patients. Four patients developed gonadal tumors (two gonadoblastoma, two dysgerminoma, and one immature teratoma, i.e., one patient had a dysgerminoma with some areas of gonadoblastoma). All affected subjects had no other congenital anomalies or dysmorphic features. Analysis of families with several affected individuals with 46,XY gonadal dysgenesis implied an X‐linked mode of inheritance because of the apparent absence of male‐to‐male transmission. However, a sex‐limited autosomal dominant mode of inheritance affecting only XY individuals could not be ruled out. Analysis of the pedigree we report indicated an autosomal dominant mode of inheritance because of male‐to‐male transmission. This kindred supports the involvement of at least one autosomal gene in non‐syndromic 46,XY gonadal dysgenesis. © 2002 Wiley‐Liss, Inc.

Analysis of mtDNA and Y-chromosome variation in the Indo-Gangetic plains shows that it was a region where genetic components of different geographical origins (from west, east and south) met. The genetic architecture of the populations now living in the area comprise genetic components dating back to different time-periods during the Palaeolithic and the Neolithic. mtDNA data analysis has demonstrated a number of deep-rooting lineages of Pleistocene origin that may be witness to the arrival of the first settlers of South and Southwest Asia after humans left Africa around 60,000 YBP. In addition, comparisons of Y-chromosome and mtDNA data have indicated a number of recent and sexually asymmetrical demographic events, such as the migrations of the Parsis from Iran to India, and the maternal traces of the East African slave trade.

Cryptorchidism is the most common genital anomaly in men. The INSL3/LGR8 system is involved in testicular descent via gubernacular development. INSL3 binds with high affinity to its receptor LGR8 and receptor activation is associated with cAMP signaling. Analysis of human INSL3 and LGR8 mutations confirms that some cases of cryptorchidism are caused by mutations in these genes. The T222P mutation is the only one within the LGR8 gene associated with the cryptorchidism phenotype. A strong association of the T222P mutation with cryptorchidism was found in an Italian population. Due to the same mutation being found in patients within the Mediterranean area, a possible founder effect of this mutation is supposed.We screened 109 patients with cryptorchidism and 250 controls in a Moroccan population.We found that 3 of the 109 patients tested carry the T222P mutation and 4 individuals in the control group also carry the mutation.Our results show in fact that the same mutation is present in the Moroccan population, but an association between cryptorchidism and the T222P mutation was not found.

We screened 100 individuals with anomalies of testicular development or function for mutations in the TSPYL1 gene. A 46,XY female with complete gonadal dysgenesis carried a p.K320R mutation in the highly conserved NAP domain, and a 46,XY male with idiopathic azoospermia harbored a p.R89H mutation, and this data supports the hypothesis that mutations in TSPYL1 may contribute to anomalies of testicular development/function.

Temozolomide is an oral alkylating agent with proven efficacy in recurrent high-grade glioma. The antitumour activity of this molecule is attributed to the inhibition of replication through DNA methylation. However, this methylation may also perturb other DNA-dependent processes, such as spermatogenesis. The ability to father a child may be affected by having this treatment. Here we report a pregnancy and a baby born after 6 cures of temozolomide.The quality of gametes of the father has been studied through these cures and after the cessation of treatment. Sperm parameters, chromosomal content and epigenetic profiles of H19, MEST and MGMT have been analysed.Sperm counts decrease significantly and hypomethylation of the H19 locus increase with time even staying in the normal range.This is the first report of an epigenetic modification in sperm after temozolomide treatment suggesting a potential risk for the offspring. A sperm cryopreservation before the initiation of temozolomide treatment should be recommended.

Located at the cross-road between Europe and Africa, Tunisia is a North African country of 11 million inhabitants. Throughout its history, it has been invaded by different ethnic groups. These historical events, and consanguinity, have impacted on the spectrum and frequency of genetic diseases in Tunisia. Investigations of Tunisian families have significantly contributed to elucidation of the genetic bases of rare disorders, providing an invaluable resource of cases due to particular familial structures (large family size, consanguinity and share of common ancestors). In the present study, we report on and review different aspects of consanguinity in the Tunisian population as a case study, representing several features common to neighboring or historically related countries in North Africa and the Middle East. Despite the educational, demographic and behavioral changes that have taken place during the last four decades, familial and geographical endogamy still exist at high frequencies, especially in rural areas. The health implications of consanguinity in Tunisian families include an increased risk of the expression of autosomal recessive diseases and particular phenotypic expressions. With new sequencing technologies, the investigation of consanguineous populations provides a unique opportunity to better evaluate the impact of consanguinity on the genome dynamic and on health, in addition to a better understanding of the genetic bases of diseases.

R-spondin proteins are secreted agonists of canonical WNT/β-catenin signaling. Homozygous &lt;i&gt;RSPO1&lt;/i&gt; mutations cause a syndrome of 46,XX disorder of sexual development (DSD), palmoplantar keratoderma (PPK), and predisposition to squamous cell carcinoma. We report exome sequencing data of two 46,XX siblings, one with testicular DSD and the other with suspected ovotesticular DSD. Both have PPK and hearing impairment and carried a novel homozygous mutation c.332G&gt;A (p.Cys111Tyr) located in the highly conserved furin-like cysteine-rich domain-2 (FU-CRD2). Cysteines in the FU-CRDs are strictly conserved, indicating their functional importance in WNT signaling through interaction with the leucine-rich repeat-containing G-protein-coupled receptors. This is the first &lt;i&gt;RSPO1&lt;/i&gt; missense mutation reported in association with human disease.

The human testis-determining gene was recently isolated from a 35 kb region on the human Y chromosome which was present in four sex-reversed individuals, three XX males and one true hermaphrodite. One of the XX males and the true hermaphrodite were sibs. A more detailed molecular analysis of these two patients and their family for Y-DNA sequences including the testis-determining gene, SRY was performed. The father was found to harbor two copies of SRY, one on his Y chromosome and the other on his X chromosome located at Xp22 determined by in situ hybridization. Somatic cell hybrids were generated from peripheral blood lymphocytes. Analysis of Y chromosome-negative somatic cell hybrids from the XX male, the true hermaphrodite and their father, revealed that both the X and Y pseudo-autosomal boundaries were present. The present of both boundaries suggests than an unequal interchange of X and Y material occurred with the cross-over breakpoint located within the X pseudo-autosomal region. The paternal SRY-bearing X chromosome was transmitted to two of his children, a 46 XX true hermaphrodite and a 46,XX male. The presence of SRY on an X chromosome associated with two sex phenotypes strongly suggests that the phenotypic variability was caused by differential inactivation of the SRY-bearing X chromosome, thereby influencing SRY expression.

Recent advances in the evolutionary genetics of sex determination indicate that the only molecular similarity in sex determination found so far among phyla is between the fly doublesex, worm mab-3 and vertebrate DMRT1(dsx- and mab3-related transcription factor 1) /DMY genes. Each of these factors encodes a zinc-finger-like DNA-binding motif, DM domain. Insights into the evolution and functions of human DMRT1 gene could reveal evolutionary mechanisms of sexual development. Here we report the identification and characterization of multiple isoforms of human DMRT1 in the testis. These transcripts encode predicted proteins with 373, 275 and 175 amino acids and they were generated by alternative splicing at 3' region. Expression level of DMRT1a is higher than those of both DMRT1b and c, and the DMRT1c expression was the lowest in testis, based on comparisons of mean values from real-time fluorescent quantitative RT-PCR analysis. Both DMRT1b and c result from exonization of intronic sequences, including the exonization of an Alu element. A further search for Alu elements within the DMRT1 gene demonstrated that all 99 Alu elements are non-randomly distributed among the non-coding regions on both directions. These new characteristics of DMRT1 would have an important impact on the evolution of sexual development mechanisms.

The human Y chromosome is essential for human sex determination and spermatogenesis. The long arm contains the azoospermia factor (AZF) region. Microdeletions in this region are responsible for male infertility. The objective of this study was to determine the frequency of Y microdeletions in Algerian infertile males with azoospermia and oligoasthenoteratozoospermia syndrome (OATS) and to compare the prevalence of these abnormalities with other countries and regions worldwide. A sample of 80 Algerian infertile males with a low sperm count (1-20 × 10&lt;sup&gt;6&lt;/sup&gt; sperms/ml) as well as 20 fertile male controls was screened for Y chromosome microdeletions. 49 men were azoospermic and 31 men had OATS. Genomic DNA was isolated from blood and polymerase chain reaction was carried out with a set of 6 AZFa, AZFb and AZFc STS markers to detect the microdeletions as recommended by the European Academy of Andrology. Among the 80 infertile men screened for microdeletion, 1 subject was found to have microdeletions in the AZFc (sY254 and sY255) region. The deletion was found in azoospermic subjects (1/49, 2%). The overall AZF deletion frequency was low (1/80, 1.3%). AZF microdeletions were observed neither in the OATS group nor in the control group. The frequency of AZF microdeletions in infertile men from Algeria was comparable to those reported in the literature. We suggest analyzing 6 STS in the first step to detect Y microdeletions in our population.

Azoospermia factor (AZF) subdeletions were reported to be significant risk factors for spermatogenesis. In this study, we screened classical and partial microdeletions of the Y-chromosome AZF region in a group of 261 infertile men. Partial deletions were also screened in a control group of fertile men (n=124). In addition, Y haplogroups were analyzed in 24 gr/gr deleted patients. Among the 261 studied infertile men, seven subjects were found to have classical microdeletions. The most common partial deletion of AZFc (gr/gr) was observed in 13.02% of infertile men and in 12.90% of fertile men. The b1/b3 deletion was identified in 4.98% of infertile men and in 2.41% of fertile men. In addition, the b2/b3 deletion was identified in 1.53% of infertile patients but not in the control group. Our results suggest that partial AZFc deletions are not associated with spermatogenic failure in the Tunisian population.

Nuclear receptor subfamily 5 group A member 1/Steroidogenic factor 1 (NR5A1; SF-1; Ad4BP) mutations cause 46,XY disorders of sex development (DSD), with phenotypes ranging from developmentally mild (e.g., hypospadias) to severe (e.g., complete gonadal dysgenesis). The molecular mechanism underlying this spectrum is unclear. During sex determination, SF-1 regulates SOX9 (SRY [sex determining region Y]-box 9) expression. We hypothesized that SF-1 mutations in 46,XY DSD patients affect SOX9 expression via the Testis-specific Enhancer of Sox9 core element, TESCO. Our objective was to assess the ability of 20 SF-1 mutants found in 46,XY DSD patients to activate TESCO. Patient DNA was sequenced for SF-1 mutations and mutant SF-1 proteins were examined for transcriptional activity, protein expression, sub-cellular localization and in silico structural defects. Fifteen of the 20 mutants showed reduced SF-1 activation on TESCO, 11 with atypical sub-cellular localization. Fourteen SF-1 mutants were predicted in silico to alter DNA, ligand or cofactor interactions. Our study may implicate aberrant SF-1-mediated transcriptional regulation of SOX9 in 46,XY DSDs.

Abstract Objective SRY ‐negative 46,XX testicular and ovotesticular disorders/differences of sex development (T/OTDSD) represent a very rare and unique DSD condition where testicular tissue develops in the absence of a Y chromosome. To date, very few studies have described the phenotype, clinical and surgical management and long‐term outcomes of these patients. Particularly, early blockade of the gonadotropic axis in patients raised in the female gender to minimize postnatal androgenization has never been reported. Design Retrospective description of sixteen 46,XX T/OTDSD patients. Results Sixteen 46,XX SRY ‐negative T/OTDSD were included. Most (12/16) were diagnosed in the neonatal period. Sex of rearing was male for six patients and female for ten, while the clinical presentation varied, with an external masculinization score from 1 to 10. Five patients raised as girl were successfully treated with GnRH analog to avoid virilization during minipuberty. Ovotestes/testes were found bilaterally for 54% of the patients and unilaterally for the others (with a contralateral ovary). Gonadal surgery preserved appropriate tissue in the majority of cases. Spontaneous puberty occurred in two girls and one boy, while two boys required hormonal induction of puberty. One of the girls conceived spontaneously and had an uneventful pregnancy. DNA analyses (SNP‐array, next‐generation sequencing and whole‐exome sequencing) were performed. A heterozygous frameshit mutation in the NR2F2 gene was identified in one patient. Conclusions This study presents a population of patients with 46,XX SRY ‐negative T/OTDSD. Early blockade of gonadotropic axis appears efficient to reduce and avoid further androgenization in patients raised as girls.

Recently, a number of genes have been identified that are associated with a failure of human sex determination, including WT1, DAX-1, SOX9, ATRX, and the Y-linked testis determination gene, SRY. Most cases of human sex reversal, XY females and XX males, do not, however, appear to be caused by mutations in these genes. This review highlights recent advances in this field and discusses the prospects of identifying genes in the sex-determining pathway.

Twelve Y-chromosomal short tandem repeats (STRs), DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS388, DYS426 and DYS439 were typed in Berber-speaking (n=49) and Arabic-speaking (n=60) population samples from Morocco.

No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital facial defects, with an incidence of 1 in 700-1,000 live births among individuals of European descent. Several linkage and association studies of NSCL/P have suggested numerous candidate genes and genomic regions. A genomewide linkage analysis of a large multigenerational family (UR410) with NSCL/P was performed using a single-nucleotide-polymorphism array. Nonparametric linkage (NPL) analysis provided significant evidence of linkage for marker rs728683 on chromosome 18q21.1 (NPL=43.33 and P=.000061; nonparametric LOD=3.97 and P=.00001). Parametric linkage analysis with a dominant mode of inheritance and reduced penetrance resulted in a maximum LOD score of 3.61 at position 47.4 Mb on chromosome 18q21.1. Haplotype analysis with informative crossovers defined a 5.7-Mb genomic region spanned by proximal marker rs1824683 (42,403,918 bp) and distal marker rs768206 (48,132,862 bp). Thus, a novel genomic region on 18q21.1 was identified that most likely harbors a high-risk variant for NSCL/P in this family; we propose to name this locus "OFC11" (orofacial cleft 11).

The cause of isolated gonadotropin-independent precocious puberty (PP) with an ovarian cyst is unknown in the majority of cases. Here, we describe 11 new cases of peripheral PP and, based on phenotypes observed in mouse models, we tested the hypothesis that mutations in the GNAS1, NR5A1, LHCGR, FSHR, NR5A1, StAR, DMRT4 and NOBOX may be associated with this phenotype.11 girls with gonadotropin-independent PP were included in this study. Three girls were seen for a history of prenatal ovarian cyst, 6 girls for breast development, and 2 girls for vaginal bleeding. With one exception, all girls were seen before 8 years of age. In 8 cases, an ovarian cyst was detected, and in one case, suspected. One other case has polycystic ovaries, and the remaining case was referred for vaginal bleeding. Four patients had a familial history of ovarian anomalies and/or infertility. Mutations in the coding sequences of the candidate genes GNAS1, NR5A1, LHCGR, FSHR, NR5A1, StAR, DMRT4 and NOBOX were not observed.Ovarian PP shows markedly different clinical features from central PP. Our data suggest that mutations in the GNAS1, NR5A1, LHCGR, FSHR StAR, DMRT4 and NOBOX genes are not responsible for ovarian PP. Further research, including the identification of familial cases, is needed to understand the etiology of ovarian PP.

Biological assessment of abnormal genitalia is based on an ordered sequence of endocrine and genetic investigations that are predicated on knowledge obtained from a suitable history and detailed examination of the external genital anatomy. Investigations are particularly relevant in 46,XY DSD where the diagnostic yield is less successful than in the 46,XX counterpart. Advantage should be taken of spontaneous activity of the pituitary-gonadal axis in early infancy rendering measurements of gonadotrophins and sex steroids by sensitive, validated assays key to assessing testicular function. Allied measurement of serum anti-Müllerian hormone completes a comprehensive testis profile of Leydig and Sertoli cell function. Genetic assessment is dominated by analysis of a plethora of genes that attempts to delineate a cause for gonadal dysgenesis. In essence, this is successful in up to 20% of cases from analysis of SRY and SF1 (NR5A1) genes. In contrast, gene mutation analysis is highly successful in 46,XY DSD due to defects in androgen synthesis or action. The era of next generation sequencing is increasingly being applied to investigate complex medical conditions of unknown cause, including DSD. The challenge for health professionals will lie in integrating vast amounts of genetic information with phenotypes and counselling families appropriately. How tissues respond to hormones is apposite to assessing the range of genital phenotypes that characterise DSD, particularly for syndromes associated with androgen resistance. In vitro methods are available to undertake quantitative and qualitative analysis of hormone action. The in vivo equivalent is some assessment of the degree of under-masculinisation in the male, such as an external masculinisation score, and measurement of the ano-genital distance. This anthropometric marker is effectively a postnatal readout of the effects of prenatal androgens acting during the masculinisation programming window. For investigation of the newborn with abnormal genitalia, a pragmatic approach can be taken to guide the clinician using appropriate algorithms.

Congenital heart diseases (CHDs) are the most common cause of all birth defects and account for nearly 25% of all major congenital anomalies leading to mortality in the first year of life. Extracardiac anomalies including urogenital aberrations are present in ∼30% of all cases. Here, we present a rare case of a 46,XY patient with CHD associated with ambiguous genitalia consisting of a clitoris-like phallus and a bifid scrotum. Exome sequencing revealed novel homozygous mutations in the &lt;i&gt;FGFR1 &lt;/i&gt;and&lt;i&gt; STARD3&lt;/i&gt; genes that may be associated with the phenotype.

Testicular dysgenesis syndrome encompasses low sperm quality, hypospadias, cryptorchidism and testicular cancer. Epidemiological studies and genetic data from familial cases suggest that testicular dysgenesis syndrome has a common etiology. The Y chromosome is known to encode genes that are involved in germ cell development or maintenance. We have therefore investigated if different classes of Y chromosomes in the general population (Y chromosome haplogroups) are associated with aspects of the testicular dysgenesis syndrome. We defined the Y chromosome haplogroups in individuals from different European counties who presented with either (i) oligo- or azoospermia associated with a Y chromosome microdeletion, (ii) unexplained reduced sperm counts (<20 x 10(6)/ml) or (iii) testicular cancer. We failed to find Y chromosome haplotype associations with either microdeletion formation or testicular cancer. However, in a study of the Danish population, we found that a specific Y chromosome haplogroup (hg26) is significantly overrepresented in men with unexplained reduced sperm counts compared with a Danish control population. The factors encoded by genes on this class of Y chromosome may be particularly susceptible to environmental influences that cause testicular dysgenesis syndrome. Our current data highlight the need for further analyses of clinically well-defined patient groups from a wide range of ethnic and geographic origins.

46,XY gonadal dysgenesis was transmitted as an autosomal-dominant trait in a large family with multiple affected members. Expressivity of the trait was highly variable, ranging from pure to partial gonadal dysgenesis associated with normal female genitalia or sexual ambiguity, to mild hypospadias in otherwise normal males. The phenotypic features of this trait appeared to be confined to the genitourinary system. Multipoint parametric analysis using markers D5S664, D5S633, and D5D2102 yielded an LOD score of 4.47, assuming sex-limited, autosomal-dominant inheritance with a penetrance of 0.6. Because mutation in testis-determining genes leads to gonadal dysgenesis in 46,XY individuals, we postulate that the gene mapped by this study normally plays a role in gonadal differentiation.

ObjectiveTo determine the genetic cause of 46,XY primary amenorrhea in three 46,XY girls.DesignWhole exome sequencing.SettingUniversity cytogenetics center.Patient(s)Three patients with unexplained 46,XY primary amenorrhea were included in the study.Intervention(s)Potentially pathogenic variants were confirmed by Sanger sequencing, and familial segregation was determined where parents' DNA was available.Main Outcome Measure(s)Exome sequencing was performed in the three patients, and the data were analyzed for potentially pathogenic mutations. The functional consequences of mutations were predicted.Result(s)Three novel homozygous nonsense mutations in the luteinizing hormone receptor (LHCGR) gene were identified:c.1573 C→T, p.Gln525Ter, c.1435 C→T p.Arg479Ter, and c.508 C→T, p.Gln170Ter.Conclusion(s)Inactivating mutations of the LHCGR gene may be a more common cause of 46,XY primary amenorrhea than previously considered. To determine the genetic cause of 46,XY primary amenorrhea in three 46,XY girls. Whole exome sequencing. University cytogenetics center. Three patients with unexplained 46,XY primary amenorrhea were included in the study. Potentially pathogenic variants were confirmed by Sanger sequencing, and familial segregation was determined where parents' DNA was available. Exome sequencing was performed in the three patients, and the data were analyzed for potentially pathogenic mutations. The functional consequences of mutations were predicted. Three novel homozygous nonsense mutations in the luteinizing hormone receptor (LHCGR) gene were identified:c.1573 C→T, p.Gln525Ter, c.1435 C→T p.Arg479Ter, and c.508 C→T, p.Gln170Ter. Inactivating mutations of the LHCGR gene may be a more common cause of 46,XY primary amenorrhea than previously considered.

Sex is determined in mammals by the SRY gene, which is located on the non-recombining region of the Y chromosome. Although the presence of mutations in SRY associated with male to female sex reversal clearly indicates that this gene is essential for testis formation, little is known concerning other genes in this process or how the expression of SRY itself is controlled. This is mainly due to the absence of an appropriate in vitro cellular model. Previous studies have indicated that SRY coding sequences are undergoing rapid evolution in mammals. In this study, we cloned and compared a Y chromosome-specific region immediately 5′ to the SRY open reading frame in several primate species. The divergence of the region 5′ to SRY between primates was found to be comparable with that described for autosomal sequences. An alignment of sequences within the primate lineage, together with sequences from the cow and pig, revealed the presence of several highly conserved motifs. These domains may have a function in the control of SRY expression during fetal development.

PCR-based screening of microdeletions in the azoospermic factor (AZF) on the Yq chromosome is an accepted means of identifying a common genetic cause of male infertility, responsible for 5-15% of cases associated with a low sperm count (</=5 x 10(6) sptz/ml). Based on an extensive analysis of the literature, we have established a cost-effective preliminary PCR-based diagnostic screening test, with a set of six pairs of primers ("set-of-6") that have the capability of detecting up to 95% of the Y microdeletion cases already published. These primers are: sY84 in AZFa, sY114, sY129, sY143 in AZFb, and sY149, sY254 in AZFc. Initially, the set-of-6 was tested with 13 other pairs of primers covering the three AZF subregions. A sample of 114 infertile men was tested and 10 (8.8%) microdeletions were found, 3 of which were among the 26 (11.5%) idiopathic azoospermic men. These results showed that all detected microdeletions would be identified using the set-of-6 only. Another sample of 34 patients was subsequently tested using the set-of-6 and 3 (8.8%) microdeletions were found in this group. A comparison of our results with those reported in the literature showed similar microdeletion detection frequencies, demonstrating that the set-of-6 primers provides a reliable, simple and cost-effective way of detecting AZF deletions.

Omphalocele is a congenital birth defect characterised by the presence of internal organs located outside of the ventral abdominal wall. The purpose of this study was to identify the underlying genetic mechanisms of a large autosomal dominant Caucasian family with omphalocele.A genetic linkage study was conducted in a large family with an autosomal dominant transmission of an omphalocele using a genome-wide single nucleotide polymorphism (SNP) array. The analysis revealed significant evidence of linkage (non-parametric NPL = 6.93, p=0.0001; parametric logarithm of odds (LOD) = 2.70 under a fully penetrant dominant model) at chromosome band 1p31.3. Haplotype analysis narrowed the locus to a 2.74 Mb region between markers rs2886770 (63014807 bp) and rs1343981 (65757349 bp). Molecular characterisation of this interval using array comparative genomic hybridisation followed by quantitative microsphere hybridisation analysis revealed a 710 kb duplication located at 63.5-64.2 Mb. All affected individuals who had an omphalocele and shared the haplotype were positive for this duplicated region, while the duplication was absent from all normal individuals of this family. Multipoint linkage analysis using the duplication as a marker yielded a maximum LOD score of 3.2 at 1p31.3 under a dominant model. The 710 kb duplication at 1p31.3 band contains seven known genes including FOXD3, ALG6, ITGB3BP, KIAA1799, DLEU2L, PGM1, and the proximal portion of ROR1. Importantly, this duplication is absent from the database of genomic variants.The present study suggests that development of an omphalocele in this family is controlled by overexpression of one or more genes in the duplicated region. To the authors' knowledge, this is the first reported association of an inherited omphalocele condition with a chromosomal rearrangement.

Families with 46,XY Disorders of Sex Development (DSD) have been reported, but they are considered to be exceptionally rare, with the exception of the familial forms of disorders affecting androgen synthesis or action. The families of some patients with anorchia may include individuals with 46,XY gonadal dysgenesis. We therefore analysed a large series of patients with 46,XY DSD or anorchia for the occurrence in their family of one of these phenotypes and/or ovarian insufficiency and/or infertility and/or cryptorchidism. A retrospective study chart review was performed for 114 patients with 46,XY DSD and 26 patients with 46,XY bilateral anorchia examined at a single institution over a 33 year period. Of the 140 patients, 25 probands with DSD belonged to 21 families and 7 with anorchia belonged to 7 families. Familial forms represent 22% (25/114) of the 46,XY DSD and 27% (7/26) of the anorchia cases. No case had disorders affecting androgen synthesis or action or 5 α-reductase deficiency. The presenting symptom was genital ambiguity (n = 12), hypospadias (n = 11) or discordance between 46,XY karyotyping performed in utero to exclude trisomy and female external genitalia (n = 2) or anorchia (n = 7). Other familial affected individuals presented with DSD and/or premature menopause (4 families) or male infertility (4 families) and/or cryptorchidism. In four families mutations were identified in the genes SRY, NR5A1, GATA4 and FOG2/ZFPM2. Surgery discovered dysgerminoma or gonadoblastoma in two cases with gonadal dysgenesis. This study reveals a surprisingly high frequency of familial forms of 46,XY DSD and anorchia when premature menopause or male factor infertility are included. It also demonstrates the variability of the expression of the phenotype within the families. It highlights the need to the physician to take a full family history including fertility status. This could be important to identify familial cases, understand modes of transmission of the phenotype and eventually understand the genetic factors that are involved.

Turner syndrome is a complex human disorder that generally associates a 45,X karyotype to a female phenotype presenting with gonadal dysgenesis, short stature and a number of characteristic somatic features. It has been hypothesized that this specific phenotype was the consequence of the haploinsufficiency of some X-linked genes having functional homologs on the Y chromosome. Here we describe four patients with deletions of the long arm of their Y chromosome and presenting with azoospermia and with or without Turner stigmata. Analysis of their breakpoints by Southern blotting and Y-specific sequence tagged sites (STS) allows us to delimit a region located in proximal interval 5 of the Y chromosome involved in skeletal development and growth.

The human Y chromosome encodes genes that are essential for male sex determination, spermatogenesis and protection against Turner stigmata. In recent years mutations have been identified in Y-chromosome genes associated with these phenotypes and a series of microdeletions of the long arm of the Y have been defined that are specifically associated with male infertility. In parallel, the discovery of polymorphic markers on the Y, comprising of both slow-mutating binary markers and rapidly-mutating microsatellites, has enabled the high resolution definition of a large number of paternal lineages (haplogroups). These Y-chromosome haplogroups have been extensively used to trace population movements and understand human origins and histories, but recently a growing number of association studies have been performed aimed at assessing the relationship between the Y-chromosome background and Y-linked phenotypes such as infertility and male-specific cancers. These preliminary studies, comparing haplogroup distributions between case and control populations, are promising and suggest an association between different Y-chromosome lineages, sperm counts and prostate cancer. However, we highlight the need to extend these studies to other world populations. Increased sample numbers and a better haplogroup resolution using additional binary markers in association studies are necessary. By these approaches novel associations between Y-chromosome haplotypes and disease may be revealed and the degree to which selection is acting on the human Y chromosome may be determined.