AZFb Regions Research Articles

Characterization of HSFY, a novel AZFb gene on the Y chromosome with a possible role in human spermatogenesis.

HSFY (heat shock transcription factor, Y chromosome) has been mapped in the AZFb region of the Y chromosome, whose deletion results in severe male infertility. HSFY belongs to the heat shock factor family that has been shown to be implicated in spermatogenesis both in animals and humans. We report the characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene. By comparison of data obtained from expression analysis and from GenBank cDNA sequences, seven exons were identified. Alternative splicing generates three different transcripts and proteins, each containing an HSF domain typical of HSF proteins. Two identical and functional full-length copies of HSFY map in palindrome P4 of AZFb, whereas four similar sequences mapping in two clusters in palindrome P1 of AZFc and P3 seem to represent pseudogenes. Sequences similar to few HSFY exons are also located in the short arm of chromosomes Y, X and 22. Expression analysis revealed that the three HSFY transcripts are differentially expressed, transcript 1 being present in many tissues including testis and ejaculated sperm, and transcripts 2 and 3 being testis-specific. These data suggest that HSFY could have an important role in human spermatogenesis.

Sex chromosome alignment at meiosis of azoospermic men with azoospermia factor microdeletion.

Deletions in the q arm of the Y chromosome result in spermatogenesis impairment. The aim of the present study was to observe the X and Y chromosome alignment in the spermatocytes of men with Y chromosome microdeletion of the azoospermia factor (AZF) region. This was performed by multicolor fluorescence in situ hybridization probes for the centromere and telomere regions. Testicular biopsies were performed in a testicular sperm extraction-intracytoplasmic sperm injection set-up in 11 azoospermic men: 8 (nonobstructive) with AZF deletions and 3 (obstructive) controls. Histological sections, cytology preparations of the testicular biopsies, and evaluation of the meiosis according to the percentage of XY and 18 bivalents formation were assessed. Spermatozoa were identified in at least one location in controls and specimens with AZFc-deleted Y chromosomes. Complete spermatocyte arrest was found in those with a deletion that included the entire AZFb region. Bivalent formation rate of chromosome 18 was high in all samples (81%-99%). In contrast, the rate of bivalent X-Y as determined by centromeric probes was lower but in the range favorable with spermatozoa findings in controls and patients with the AZFc deletion (56%-90%), but not in those with AZFb-c deletions (28%-29%). A dramatic impairment in the normal alignment of X and Y telomeres in the specimen with AZFb-c deletion was shown (29%), compared to the specimens with AZFc deletion (70%-94%). It is suggested that the absence of sperm cells in specimens with the entire AZFb and with AZFb-c deletions is accompanied by meiosis impairment, perhaps as a result of the extent of the deletion or because of the absence of genes that are involved in the X and Y chromosome alignment.

Expression pattern of the Y-linked PRY gene suggests a function in apoptosis but not in spermatogenesis.

In this study, we aimed at analysing the expression of the PRY (PTPN-13 like on the Y chromosome) gene, located on the Y chromosome, in order to define the function of this gene. Active copies of the PRY gene (PRY1 and PRY2) are located in the AZFb region. PCR amplification of PRY cDNA indicated that the PRY gene is expressed in testicular tissue and ejaculated sperm, but not in Percoll-treated sperm. Furthermore, immunocytochemistry on testicular tissue showed the expression of the PRY gene in a small number of spermatozoa and spermatids. In the ejaculate of the male partner of 18 infertile couples, the PRY protein was found in 1.5-51.2% of spermatozoa and in most of the sperm precursor cells. The percentage of spermatozoa showing DNA fragmentation was also determined in 13 of these samples, by using the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) reaction. These data correlated with the percentage of PRY-positive cells. When double labelling for PRY and DNA fragmentation was performed to assess whether PRY-positive cells also show DNA fragmentation, we saw that 27-48% of the PRY-positive spermatozoa were also positive for the TUNEL reaction. The overall data of RNA analysis, immunocytochemistry and the TUNEL reaction indicate that the role of the PRY gene in spermatogenesis can be questioned, but suggest its involvement in apoptosis of spermatids and spermatozoa.

Tra2α and hnRNP K might be functional partners of Rbm for regulation of RNA processes during spermatogenesis

Rbm is a male infertility gene located in the AZFb region of the Y chromosome. Expression pattern of Rbm indicates that Rbm is critical for early phase of male germ cell development. It shares strong structural homology with hnRNP G, suggesting a function as an RNA processing factor. In order to gain a clue on the molecular mechanisms of Rbm on male germ cell development, we examined interactions of Rbm with selected proteins in yeast. The results revealed specific interactions between Rbm, hnRNP K and Tra2α. These results suggest that hnRNP K and Tra2α may be functional partners of Rbm in male germ cells. We propose a model in which hnRNP K may play a role as a platform for Rbm and Tra2α.

AZF microdeletions on the Y chromosome of infertile men from Turkey

Intervals V and VI of Yq11.23 regions contain responsible genes for spermatogenesis, and are named as “azoospermia factor locus” (AZF). Deletions in these genes are thought to be pathogenetically involved in some cases of male infertility associated with azoospermia or oligozoospermia. The aim of this study was to establish the prevalence of microdeletions on the Y chromosome in infertile Turkish males with azoospermia or oligozoospermia. We applied multiplex polymerase chain reaction (PCR) using several sequence-tagged site (STS) primer sets, in order to determine Y chromosome microdeletions. In this study, 61 infertile males were enrolled for the molecular AZF screening program. In this cohort, one infertile male had 46,XX karyotype and the remaining had 46,XY karyotypes. Forty-eight patients had a diagnosis of azoospermia and 13 had oligozoospermia. Microdeletions in AZFa, AZFb and AZFc (DAZ gene) regions were detected in two of the 60 (3.3%) idiopathic infertile males with normal karyotypes and a SRY translocation was determined on 46,XX male. Our findings suggest that genetic screening should be advised to infertile men before starting assisted reproductive treatments.

Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions.

Y chromosome microdeletions are associated with severe male factor infertility. In this study, the success rate of testicular sperm retrieval was determined for men with deletions of AZF regions a, b or c. AZF deletions were detected by PCR of 30 sequence-tagged sites within Yq emphasizing the AZFa, b and c regions. Semen analysis and diagnostic testis biopsy or testicular sperm extraction (TESE) findings were correlated with the specific AZF region deleted. A total of 78 men with AZF deletions included three with AZFa deletion, 11 with AZFb, 42 with AZFc, 16 with AZFb+c and six with Yq (AZFa+b+c). All men with AZFa, AZFb, AZFb+c and Yq deletions were azoospermic and no sperm were found with TESE or biopsy. Of men with isolated AZFc deletion, sperm were found in 75% (9/12) by TESE and 45% (9/20) on biopsy (56% overall); 62% (26/42) were azoospermic and 38% (16/42) severely oligozoospermic. A total of 7 patients with deletion patterns that included the complete AZFa region and 23 that included the complete AZFb region who underwent TESE or biopsy did not have sperm detected by these surgical measures. Microdeletion of the entire AZFa or AZFb regions of the Y chromosome portends an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm within the semen or testes available for use in IVF/ICSI.

Y chromosome deletions in azoospermic men in India.

Genetic factors cause about 10% of male infertility. Azoospermia factors (AZFa, AZFb, AZFc) are considered to be the most important for spermatogenesis. We therefore made an attempt to evaluate the genetic cause of azoospermia, Y chromosome deletion in particular, in Indian men. We have analyzed a total of 570 men, including 340 azoospermic men and 230 normal control subjects. DNA samples were initially screened with 30 sequence-tagged site (STS) markers representing AZF regions (AZFa, AZFb, AZFc). Samples, with deletion in the above regions were mapped by STS walking. Further, the deletions were confirmed by Southern hybridization using the probes from both euchromatic and heterochromatic regions. Of the total 340 azoospermic men analyzed, 29 individuals (8.5%) showed Y chromosome deletion, of which deletion in AZFc region was the most common (82.8%) followed by AZFb (55.2%) and AZFa (24.1%). Microdeletions were observed in AZFa, whereas macrodeletions were observed in AZFb and AZFc regions. Deletion of heterochromatic and azoospermic regions was detected in 20.7% of the azoospermic men. In 7 azoospermic men, deletion was found in more than 8.0 Mb spanning AZFb and AZFc regions. Sequence analysis at the break points on the Y chromosome revealed the presence of L1, ERV, and other retroviral repeat elements. We also identified a approximately 240-kb region consisting of 125 bp tandem repeats predominantly comprised of ERV elements in the AZFb region. Histological study of the testicular tissue of the azoospermic men, who showed Y chromosome deletion, revealed complete absence of germ cells and presence of only Sertoli cells.

Genes and male infertility: what can go wrong?

Genes and male infertility: what can go wrong?

A simplified method for the detection of Y chromosome microdeletions in infertile men using a multiplex sequence-tagged site-based amplification.

Sixteen sequence-tagged sites (STSs) were combined in five amplification reactions, to screen for deletions of DNA fragments located within the AZFa, AZFb, and AZFc regions of the Y chromosome. This multiplex strategy is fast and reliable, and most of the azoospermia-associated deletions reported so far are detected with this simplified method. Internal control STSs are included that allow discrimination between deletion and failure of amplification.

Best practice policies for male infertility

University of California, San Francisco, San Francisco, California; Johns Hopkins University School of Medicine, Baltimore, Maryland; University of Louisville School of Medicine, Louisville, Kentucky; Baylor College of Medicine, Houston, Texas; Brown University, Providence, Rhode Island; Cleveland Clinic Foundation, Cleveland, Ohio; New York Presbyterian Hospital-Cornell, New York, New York; University of Virginia School of Medicine, Charlottesville, Virginia; Mayo Medical School, Rochester, Minnesota; University of Pennsylvania School of Medicine, York, Pennsylvania; University of California, Davis, Davis, California; and SUNY Health Science Center at Brooklyn, Brooklyn, New York

The Y chromosome

The Y chromosome has evolved to provide sex determination in mammals. In association with its evolution, genes important for spermatogenesis have been sequestered on this chromosome. Further, X chromosome inactivation has developed as a mechanism to prevent over-expression of genetic factors important for somatic function in females, with maintenance of their activity in males. The multi-repeat organization of the Y chromosome and limited regions of crossover with other chromosomes predisposes it to internal recombination and loss of genes that may be important for spermatogenesis. Y chromosome microdeletion testing of infertile men with non-obstructive azoospermia provides prognostic information useful for management of these patients. In the presence of a complete deletion of the azoospermic factor a (AZFa) or AZFb regions, sperm retrieval is highly unlikely. Recent advances in our understanding of the organization and function of the Y chromosome are likely to enhance further the role of the Y chromosome in normal spermatogenesis and fertility.

Y-chromosome microdeletions and cytogenetic findings in unselected ICSI candidates at a Danish fertility clinic.

To determine the frequency and type of microdeletions on the Y chromosome, and to evaluate cytogenetic findings in unselected ICSI candidates at a Danish Fertility Clinic. Genomic DNA was extracted from blood samples, which were collected prospectively from 400 ICSI candidates attending the Fertility Clinic at Aarhus University Hospital, Denmark. Twenty-five sequence tagged sites (STSs) spanning the azoospermia factor (AZF) regions of the Y chromosome were amplified in 5 multiplex sets to investigate Y microdeletions. Semen analysis, karyotype analysis, and histological evaluation of testicular biopsies were also performed. Y microdeletions were detected in 3 (0.75%) of 400 unselected ICSI candidates. The frequency of Y microdeletions was found higher in azoospermic men (2%) than in oligozoospermic men (0.6%). Two patients having oligozoospermia had Y microdeletions in the AZFc region only, whereas the patient having azoospermia had Y microdeletions spanning the AZFb and AZFc regions. No microdeletion was detected in the AZFa region. Chromosomal anomalies were found in 6.1% of azoospermic men and in 2.7% of oligozoospermic men. A high frequency of cytogenetic abnormalities was found in normozoospermic men with fertilization failure (7.4%). The frequency of Y microdeletions both in the unselected ICSI candidates and subgroups classified as azoospermic and oligozoospermic seems rather low compared to results of previous studies, which have been quite varying. It is possible that in addition to patient selection criteria, ethnical and geographical differences may contribute to these variations. Cytogenetic evaluation of normozoospermic men with fertilization failure seems indicated because of a high frequency of cytogenetic abnormalities.

Specific localization of RBM1a in the nuclei of all cell types except elongated spermatids within seminiferous tubules of the human.

Recent studies have indicated that at least three regions (AZF a-c) on the long arm of the Y-chromosome code for factors are involved in spermatogenesis. One of the candidate genes in the AZFb region is RBM1a, coding for a protein with an RNA binding motif. In this study, poly clonal antibodies raised against a 15 amino acid peptide, corresponding to residues 263-304 of the deduced amino acid sequence of RBM1a, has been used to localize the RBM1a protein in the human testis. Immunohistochemistry on normal human testis using this RBM1a antibody, localized the antigen to the nuclei of spermatogonia, primary spermatocytes, and round spermatids but not to the nuclei of elongated spermatids. The antibody also specifically identified the nuclei of Sertoli cells, although the fluorescence was not as strong as in the germ cell nuclei it identified. No specific fluorescence was seen in the nuclei of either peritubular, endothelial or Leydig cells. Western blot of normal human testicular tissue using the anti-RBM1a antibody gave rise to a single specific band of approximately 55 kDa, corresponding to the expected size of RBM1a. In view of its expression in germ cells, and because RBM1a has an RNA binding domain, RBM1a may be involved in RNA processing, such as RNA splicing or RNA export which are events necessary for normal spermatogenesis.

The recovery of full-length DAZ mRNA from mature, ejaculated human spermatozoa.

Objective: The Deleted in AZoospermia (DAZ) gene has been implicated as important in spermatogenesis. Reduced fertility in human males has been associated with spontaneous deletions of the portions of the Y-chromosome including regions that contain the DAZ gene and/or other genes of the AZFa, AZFb, AZFc, and AZFd (AZoospermia Factor) regions. Most current molecular diagnostics focus on detecting microdeletions in the genomic complement of DNA. We speculate that point mutations, like microdeletions, can frequently occur spontaneously in the DAZ gene located on the human Y-chromosome and contribute to reduced spermatogenesis. Alternatively, transcripts of the chromosome 3 copy of the DAZ gene may salvage spermatogenesis in the absence of non-expressed DAZ transcripts from the Y locus. To test this hypothesis, we explored methods to extract full-length DAZ mRNA transcripts from ejaculated sperm cells of individual males.

Molecular diagnosis of Y chromosome microdeletions in Europe: state-of-the-art and quality control.

The polymerase chain reaction (PCR) screening of microdeletions of the Y chromosome has become an important diagnostic step in the work-up of male infertility. However, there is no agreement about how this diagnosis should be performed. There are suggestions that the large variation in deletion frequency reported in the literature could be due to the various selection criteria of the patients analysed, although methodological aspects may play a role as well. As for other genetic diseases, molecular diagnosis of Y chromosome microdeletions should be controlled by adopting strict internal quality control measures and by participating in external quality assessment schemes. Such an external quality assessment project is presently being organized jointly by the European Academy of Andrology and the European Molecular Genetics Quality Network. Three preliminary trials have given a state-of-the-art picture of the diagnostic performance in various European laboratories, showing an overall rate of misdiagnosis of approximately 5% for both AZFb and AZFc regions, and providing data useful in the generation of guidelines for the molecular diagnosis of Y chromosome microdeletions.

Molecular detection of Y chromosome microdeletions: an Irish study.

The region of the Y chromosome most critical for male fertility is called the azoospermia factor (AZF) region and it is located within subintervals five and six on the long arm of the Y chromosome. Several genes, all residing here, contribute to spermatogenesis and deletions in these genes are thought to be pathogenetically involved in some cases of male infertility associated with azoospermia or oligozoospermia. The aim of this study was to establish the prevalence of microdeletions in the AZF region of the Y chromosome in an Irish male population undergoing fertility treatment. To do this, we applied and compared two independent polymerase chain reaction (PCR) based screening methods, namely, a PCR protocol using several sequence-tagged site (STS) primer sets and a recently published multiplex PCR Y chromosome screening protocol. A total of 78 patients, attending the IVF unit at University College Hospital, Galway, were included in this study. Of them, 56 suffered from idiopathic azoospermic/oligozoospermic infertility. The remaining 22 patients had various conditions, which may have contributed to their infertility. A total of 50 age-matched normospermic men were included as controls. Two microdeletions were found; one in the AZFa region and one in AZFb region. These deletions were observed among the truly idiopathic cases. Further analysis was performed to study the extent of the deletions and it was confirmed that each deletion encompassed the respective AZF region including the AZF candidate gene.

Y chromosome microdeletion screening in infertile men.

Molecular analysis of Y-chromosomal microdeletions is routinely performed in the work-up of male infertility, in order to establish a diagnosis and for genetic counseling of the couple, since such microdeletions are transmitted to the male offspring. The review of published data shows that microdeletions are relatively common in patients with azoospermia or severe oligozoospermia, with wide variations in the reported deletion frequency depending mainly on the selection criteria. In general, patients with proximal deletions, involving the AZFa and/or the AZFb region show severe defects of spermatogenesis with a high prevalence of Sertoli cell only syndrome, while deletions of the distal AZFb and of the AZFc region can be compatible with residual spermatogenesis. Microdeletions have been only sporadically found in normozoospermic patients. For the time being the molecular analysis of microdeletions of the Y chromosome is indicated in infertile patients with sperm concentration <5 x 10(6)/ml and in men undergoing assisted reproduction techniques, since the genetic defect and, most probably, the related infertility problem will be transmitted to the sons.

RBMY genes and AZFb deletions.

Microdeletions of the AZFb region of the human Y chromosome usually result in severe consequences for spermatogenesis. AZFb contains at least four kinds of genes/gene families. These include a number of RBMY genes, which are clustered in the AZFb deletion interval. They are amongst the oldest genes on the mammalian Y chromosome, and are related to the gene encoding hnRNPG (RBMX) on the X chromosome. A retroposon-derived version of these genes is found on chromosome 11 that might replace the function of these genes during meiosis, during which time the X and Y chromosomes are transcriptionally inactivated. Each of these genes encodes proteins with an RNA binding motif, and interacts with more ubiquitously expressed proteins involved in pre-mRNA splice site selection. These findings imply that important pre-mRNA processing pathways might be disrupted in the germ cells of AZFb men.

The Prognostic Value of Y Chromosome Microdeletions Involving the AZFb Region in Testicular Sperm Extraction

Objectives: Testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) is an effective treatment for men with non-obstructive azoospermia. Overall, approximately 35% of these men have no spermatozoa found in their testicular tissue. The success of TESE cannot be predicted based on preoperative hormonal profiles (FSH, testosterone), nor based on testicular volume. Even a prior diagnostic testis biopsy does not absolutely predict the success of TESE, due to the heterogeneous nature of sperm maturation within seminiferous tubules of the testicle. Preliminary data in recent literature suggest a correlation between microdeletions involving the AZFb of the Y-chromosome and the absence of spermatozoa with TESE (Brandell et al., Human Reprod., 1998). We hereby report our expanded experience of the relationship between microdeletions involving AZFb and TESE results in men with non-obstructive azoospermia.Design: A retrospective analysis of azoospermic men who underwent TESE from 1996 to 2000 was performed. The outcomes of men with AZFb microdeletion of the Y-chromosome were analyzed.Materials and Methods: All TESE were performed under local or general anesthesia by one surgeon. TESE were performed with either multiple biopsies or with a single large incision with multiple samples of the seminiferous tubules under microscopic vision (microdissection TESE). The procedures were performed in conjunction with planned in-vitro fertilization cycles with ICSI for the female partner. Y-chromosomal microdeletion was tested in all patients using DNA extracted from peripheral leukocytes with a series of 35 sequence-tagged sites on Yq using polymerase chain reaction.Results: Among 181 patients who undergone TESE, 10 (6%) were found to have microdeletion involving the AZFb regions and 5 of them (3%) have microdeletion in AZFb region only. For men with non-obstructive azoospermia and no detectable deletions of the AZFb region, 114/171 (67%) men had sperm retrieved with TESE, whereas none of the 11 patients with microdeletions involving the AZFb region had sperm found with TESE (p<0.001). The average duration of TESE in patients with deletions involving the AZFb region was 3.5 hours.Conclusions: Genetic studies may provide important prognostic information in the management of patients with severe male infertility, as illustrated by our observation that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. When counseling men with AZFb deletions, clinicians should apprise them of these results before attempting TESE-ICSI. Objectives: Testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) is an effective treatment for men with non-obstructive azoospermia. Overall, approximately 35% of these men have no spermatozoa found in their testicular tissue. The success of TESE cannot be predicted based on preoperative hormonal profiles (FSH, testosterone), nor based on testicular volume. Even a prior diagnostic testis biopsy does not absolutely predict the success of TESE, due to the heterogeneous nature of sperm maturation within seminiferous tubules of the testicle. Preliminary data in recent literature suggest a correlation between microdeletions involving the AZFb of the Y-chromosome and the absence of spermatozoa with TESE (Brandell et al., Human Reprod., 1998). We hereby report our expanded experience of the relationship between microdeletions involving AZFb and TESE results in men with non-obstructive azoospermia. Design: A retrospective analysis of azoospermic men who underwent TESE from 1996 to 2000 was performed. The outcomes of men with AZFb microdeletion of the Y-chromosome were analyzed. Materials and Methods: All TESE were performed under local or general anesthesia by one surgeon. TESE were performed with either multiple biopsies or with a single large incision with multiple samples of the seminiferous tubules under microscopic vision (microdissection TESE). The procedures were performed in conjunction with planned in-vitro fertilization cycles with ICSI for the female partner. Y-chromosomal microdeletion was tested in all patients using DNA extracted from peripheral leukocytes with a series of 35 sequence-tagged sites on Yq using polymerase chain reaction. Results: Among 181 patients who undergone TESE, 10 (6%) were found to have microdeletion involving the AZFb regions and 5 of them (3%) have microdeletion in AZFb region only. For men with non-obstructive azoospermia and no detectable deletions of the AZFb region, 114/171 (67%) men had sperm retrieved with TESE, whereas none of the 11 patients with microdeletions involving the AZFb region had sperm found with TESE (p<0.001). The average duration of TESE in patients with deletions involving the AZFb region was 3.5 hours. Conclusions: Genetic studies may provide important prognostic information in the management of patients with severe male infertility, as illustrated by our observation that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. When counseling men with AZFb deletions, clinicians should apprise them of these results before attempting TESE-ICSI.

Y Chromosome Microdeletion Analysis in Infertile and Fertile Danish Men Indicate That Microdeletions Are Specific for Spermatogenic Failure and Do Not Explain Reduced Sperm Counts in the General Danish Population

Objectives: Y chromosome microdeletions are frequently associated with male infertility. The incidence of Y deletions varies in a wide range between 1–55% in different studies. Study design and type of markers used for screening appear to be the main reason for these differences. We report a large double blind survey of microdeletions in DNA samples from infertile and healthy men with detailed semen analysis parameters of Danish origin. Design: We analyzed 124 consecutive ICSI patients (48 affected by idiopathic azoo/oligozoospermia), 100 fertile men and 107 young men from the general Danish population. Materials and Methods: The study population was screened for 15 anonymous STS markers on the Yq, including the AZFa, AZFb and AZFc regions and for the genes DBY, eIF-1AY, XKRY, CDY, BPY2 and DAZ Results: Deletions were identified 6 infertile patients affected by azoospermia and cryptozoospermia (<1 × 106/ml) (4 and 2 patients respectively). The deletions removed the whole AZFc region, including the genes BPY2 and DAZ. In two cases testicular histology were available showing spermatid arrest and SCOS. FSH levels were above the normal range with very low or undetectable levels of inhibin B in the 6 patients. In the infertile group we found a deletion frequency of 4.8%. Subdivision of the study population revealed a deletion incidence of 17.8% in idiopathic and 2.9% in non-idiopathic azoo/cryptozoospermic patients. No isolated gene-specific deletions were found. Conclusions: The lack of deletions among 207 normal controls gives further evidence that Y deletions are specific for reduced testicular function and suggests that Y chromosome microdeletions are not involved in the high incidence of reproductive abnormalities and relatively low sperm counts of Danish population in comparison with e.g. Finland.