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Schematic representation of the human Y chromosome with the position of the sequence-tagged sites (STS) and the Y chromosome DNA probes used in our analysis.
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Defects in spermatogenesis have been found associated with deletions of different portions of Y chromosome long arm (Yq), suggesting the presence of the azoospermia factor in the control of spermatogenesis. We studied 67 men with idiopathic azoospermia and severe oligozoospermia, cytogenetically normal, for the presence of microdeletions on Yq chro...
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... 3. Representative Southern blots using as probe: ( A ) the purified polymerase chain reaction (PCR) products sY254, sY277, sY283, as indicated in figure; ( B ) the DNA fragment p49f; ( C ) the DNA fragment p50f2. Genomic DNA was digested with Eco RI in ( A ) and ( C ) and with Taq I in ( B ). The size of the Y-specific fragments in ( A ) is ~1.7 kbp for sY254, 3.0 kbp for sY283 and 1.7–1.8 kbp for sY277. The sizes of the visible bands in panels B and C corresponded to those previously described for ( B ) (Saxena et al. , 1996) and ( C ) (Ma et al. , 1992). The control fertile male and female allow discrimination between Y-specific and X autosomal bands. In ( B ) and ( C ) the azoospermic patient SP1 with no Yq microdeletions is also included. The arrow in ( C ) indicates the band corresponding to locus DYS7-C in the AZFc region of Yq (see Figure 1) found deleted in patient SP8. Probe p50f2 recognizes additional bands (A, B, D and E) corresponding to locus DYS7-A and -B in Yp, DYS7-D in the centromere, and DYS7-E in the AZFb region of Yq.
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... infertility in subjects with azoospermia or severe oligozoospermia with unknown aetiology was first associated with a possible genetic cause by Tiepolo and Zuffardi (1976), in a report of six azoospermic patients carrying a deletion of the distal portion of Y chromosome long arm. On the basis of this finding they proposed the existence of a spermatogenesis gene, the ‘azoospermia factor’ ( AZF ) on Yq. Confirmatory data for the actual presence of genes of spermatogenesis in Yq had to wait until molecular maps of Y chromosome and molecular tools for mapping microdeletions were made available. DNA probes specific for Y chromosome loci (Vergnaud et al ., 1986) or polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS) (Vollrath et al ., 1992) have allowed the identification of microdeletions in Yq regions known not to pair or recombine with the X chromosome. Up to now, three non-overlapping loci have been identified on Yq11; and their deletion is associated with sterility (azoospermia or severe oligospermia) (Vogt et al ., 1996). Two have been localized to interval 6 of Yq11 and are called AZFb and AZFc , and one is more proximal to the centromere in Yq11 and is called AZFa . In interval 6 of Yq11, two genes have been identified that are deleted in a number of infertile men; one is the Y chromosome RNA- recognition motif ( YRRM/RBM ) identified by Ma et al . (1993), the other is the Deleted in AZoospermia gene ( DAZ ) identified by Reijo et al . (1995). The first gene RBM , has 1116 been tentatively linked with AZF (Ma et al ., 1993) and is a member of an RNA-binding protein gene family, which is represented in multiple copies in different regions of the Y chromosome, and at least one copy is localized to the AZFb region (Elliott et al ., 1997). The second gene, DAZ , is at present the strongest candidate for AZF , and its deletion has been found to be associated with a wide range of spermatogenic defects from bona fide Sertoli cell-only (SCO) syndrome to reduced spermatogenesis resulting in severe oligozoospermia (Reijo et al ., 1995, 1996). In the proximal region of Yq11 the gene DFFRY has been recently linked to AZFa . This gene is the Y-linked homologue of the DFFRX ( Drosophila fat-facets related X gene), and it has been shown to be deleted in three azoospermic males (Brown et al ., 1998). However it must be stressed that the rare deletions in this locus (from 1–5% in different surveys) generally associate with a SCO phenotype (Ma et al ., 1992; Vogt et al. , 1996), with the exception of one published case showing reduced spermatogenesis (Qureshi et al. , 1996), thus suggesting that this locus is important for early steps of spermatogenesis, possibly in prenatal development. As a whole the data available would indicate that AZF is a multi- gene complex preferentially located in Yq11, whose members might act at specific differentiative steps of spermatogenesis. In the present report we analysed 67 azoospermic and severe oligozoospermic patients for the presence of microdeletions in Yq11 using 18 STS and two DNA probes. We found four subjects with deletions in AZFc , including the DAZ gene, and one patient with deletions in AZFa . The 67 patients with azoospermia or severe oligozoospermia, selected as described in Methods, were screened by PCR amplification and Southern blot analysis for potential deletions of 20 loci on Yq11. STSs and Y chromosome DNA probes used in this study, and their localization on Yq11 are shown in Figure 1. Most of the STS map to interval 6 of Yq11, which includes the DAZ gene in AZFc locus (sY254, sY277, sY283). Four STS (sY83, sY84, sY86, sY87) map to AZFa , and another STS (sY117) maps to AZFb . We detected deletions on Yq11 in five patients by PCR amplification. Figure 2 shows the Y-specific STS found deleted: patients SP8, FO3, FA16, and FA45 showed deletions in 1117 AZFc involving the DAZ locus and patient FA14 showed microdeletions in AZFa . Patients SP8 and FO3 showed deletions with the same extension including STS sY152, sY242, sY254, sY277 and sY283 but differed for STS sY155, which was deleted in FO3 and present in SP8. Patient FA16 showed a slightly shorter deleted region including STS from sY155 to sY277. Patient FA45 has a unique pattern of deletion in our screening, limited to the DAZ locus (sY277, sY254, sY283). The absence of the DAZ locus was confirmed in patients SP8, FO3 and FA16 by Southern blot analysis, using as a probe the same PCR product found deleted. Figure 3A shows a representative Southern blot experiment using STS sY254, sY277, sY283 as probes. DNA from the same patients was also analysed by a hybridization technique using the Y chromosome DNA probes p49f and p50f2 (see map in Figure 1), showing deletions of both loci DYS1 (detected by p49f) and DYS7C (detected by p50f2) (Figure 3B,C). The absence, in these patients, of DYS1 sequences, previously demonstrated to be 1118 the DAZ gene cluster (Saxena et al ., 1996), agrees with PCR data. The available DNA from the other DAZ -deleted patient FA45 was not sufficient for Southern analysis. Three more patients showed microdeletions of a single STS in AZFc by PCR amplification, being sY283 negative in two cases and sY157 in the third one. Although the PCR amplification was repeatedly negative in separate reactions, we demonstrated that they were false negatives, by a Southern blot study using as probe the same PCR product found deleted. The FA14 patient showed microdeletions only in AZFa , including the STS sY84, sY86, sY87, and sY83 (Figure 2). All other STS analysed in patient FA14 were normally amplified. Southern blot analysis using these STS as probes did not give conclusive results due to cross-reactivity with autosomal DNA. The clinical parameters of the five patients with microdeletions are summarized in Table I. Four patients were azoospermic and only the DAZ -deleted FO3 patient showed very rare and aberrant spermatozoa. Testicular histology was available in three deleted patients. FA14, carrying microdeletions in AZFa , had Sertoli cell-only syndrome with complete lack of germ cells. Patients carrying deletion of the DAZ gene (SP8 and FA45) showed different spermatogenic defects including Sertoli cell-only syndrome and maturation arrest. Testicular histology of patient FA45 carrying a DAZ deletion was associated with a SCO phenotype and is shown in Figure 4. Spermatogenesis is a complex event leading to the formation of mature spermatozoa, occurring under the control of a wide variety of factors acting from prenatal to adult life (Vogt, 1997). An important role in the regulation of spermatogenesis is certainly played by the genetic programming of germ cell itself based both on autosomal and sex chromosomal genes. A group of idiopathic infertility has been, in fact, associated with genetic causes linked to the Y chromosome and many studies have recently shown deletions on Y chromosome long arm in idiopathic azoospermic males. The frequencies of deletions of Yq, reported in different studies, range between 3 and 18% of males with non-obstructive azoospermia or severe oligozoospermia (Reijo et al ., 1995; Stuppia et al. , 1996; Vogt et al ., 1996; Girardi et al ., 1997; Pryor et al ., 1997; Qureshi et al ., 1997; Simoni et al. , 1997; Van der Ven et al ., 1997). In our screening of 67 infertile males we have found five patients carrying microdeletions, corresponding to a frequency of 7.5%, that perfectly agrees with the statistical value obtained averaging all the surveys reported to date (Simoni et al ., 1998). The frequency of microdeletions reported here is the result of a correction of the PCR data, made on the basis of Southern blot analyses. In fact, we could exclude three more patients showing a single STS deletion by PCR amplification, not confirmed by Southern blotting, thus lowering the microdeletion rate from ~12% to 7.5%. Even though it is not possible from our result to evaluate statistically whether some STS are more prone to give false negatives, or whether this technical problem might be relevant in explaining the higher frequency of microdeletions found by others, in our opinion Southern blot analysis has to be performed at least in those cases where PCR reaction fails to amplify a single STS. However, we cannot exclude the possibility that the failure of PCR amplification in Southern blot positive cases is due to mutations or small deletions occurring in the genomic sequence matching the STS ...
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... infertility in subjects with azoospermia or severe oligozoospermia with unknown aetiology was first associated with a possible genetic cause by Tiepolo and Zuffardi (1976), in a report of six azoospermic patients carrying a deletion of the distal portion of Y chromosome long arm. On the basis of this finding they proposed the existence of a spermatogenesis gene, the ‘azoospermia factor’ ( AZF ) on Yq. Confirmatory data for the actual presence of genes of spermatogenesis in Yq had to wait until molecular maps of Y chromosome and molecular tools for mapping microdeletions were made available. DNA probes specific for Y chromosome loci (Vergnaud et al ., 1986) or polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS) (Vollrath et al ., 1992) have allowed the identification of microdeletions in Yq regions known not to pair or recombine with the X chromosome. Up to now, three non-overlapping loci have been identified on Yq11; and their deletion is associated with sterility (azoospermia or severe oligospermia) (Vogt et al ., 1996). Two have been localized to interval 6 of Yq11 and are called AZFb and AZFc , and one is more proximal to the centromere in Yq11 and is called AZFa . In interval 6 of Yq11, two genes have been identified that are deleted in a number of infertile men; one is the Y chromosome RNA- recognition motif ( YRRM/RBM ) identified by Ma et al . (1993), the other is the Deleted in AZoospermia gene ( DAZ ) identified by Reijo et al . (1995). The first gene RBM , has 1116 been tentatively linked with AZF (Ma et al ., 1993) and is a member of an RNA-binding protein gene family, which is represented in multiple copies in different regions of the Y chromosome, and at least one copy is localized to the AZFb region (Elliott et al ., 1997). The second gene, DAZ , is at present the strongest candidate for AZF , and its deletion has been found to be associated with a wide range of spermatogenic defects from bona fide Sertoli cell-only (SCO) syndrome to reduced spermatogenesis resulting in severe oligozoospermia (Reijo et al ., 1995, 1996). In the proximal region of Yq11 the gene DFFRY has been recently linked to AZFa . This gene is the Y-linked homologue of the DFFRX ( Drosophila fat-facets related X gene), and it has been shown to be deleted in three azoospermic males (Brown et al ., 1998). However it must be stressed that the rare deletions in this locus (from 1–5% in different surveys) generally associate with a SCO phenotype (Ma et al ., 1992; Vogt et al. , 1996), with the exception of one published case showing reduced spermatogenesis (Qureshi et al. , 1996), thus suggesting that this locus is important for early steps of spermatogenesis, possibly in prenatal development. As a whole the data available would indicate that AZF is a multi- gene complex preferentially located in Yq11, whose members might act at specific differentiative steps of spermatogenesis. In the present report we analysed 67 azoospermic and severe oligozoospermic patients for the presence of microdeletions in Yq11 using 18 STS and two DNA probes. We found four subjects with deletions in AZFc , including the DAZ gene, and one patient with deletions in AZFa . The 67 patients with azoospermia or severe oligozoospermia, selected as described in Methods, were screened by PCR amplification and Southern blot analysis for potential deletions of 20 loci on Yq11. STSs and Y chromosome DNA probes used in this study, and their localization on Yq11 are shown in Figure 1. Most of the STS map to interval 6 of Yq11, which includes the DAZ gene in AZFc locus (sY254, sY277, sY283). Four STS (sY83, sY84, sY86, sY87) map to AZFa , and another STS (sY117) maps to AZFb . We detected deletions on Yq11 in five patients by PCR amplification. Figure 2 shows the Y-specific STS found deleted: patients SP8, FO3, FA16, and FA45 showed deletions in 1117 AZFc involving the DAZ locus and patient FA14 showed microdeletions in AZFa . Patients SP8 and FO3 showed deletions with the same extension including STS sY152, sY242, sY254, sY277 and sY283 but differed for STS sY155, which was deleted in FO3 and present in SP8. Patient FA16 showed a slightly shorter deleted region including STS from sY155 to sY277. Patient FA45 has a unique pattern of deletion in our screening, limited to the DAZ locus (sY277, sY254, sY283). The absence of the DAZ locus was confirmed in patients SP8, FO3 and FA16 by Southern blot analysis, using as a probe the same PCR product found deleted. Figure 3A shows a representative Southern blot experiment using STS sY254, sY277, sY283 as probes. DNA from the same patients was also analysed by a hybridization technique using the Y chromosome DNA probes p49f and p50f2 (see map in Figure 1), showing deletions of both loci DYS1 (detected by p49f) and DYS7C (detected by p50f2) (Figure 3B,C). The absence, in these patients, of DYS1 sequences, previously demonstrated to be 1118 the DAZ gene cluster (Saxena et al ., 1996), agrees with PCR data. The available DNA from the other DAZ -deleted patient FA45 was not sufficient for Southern analysis. Three more patients showed microdeletions of a single STS in AZFc by PCR amplification, being sY283 negative in two cases and sY157 in the third one. Although the PCR amplification was repeatedly negative in separate reactions, we demonstrated that they were false negatives, by a Southern blot study using as probe the same PCR product found deleted. The FA14 patient showed microdeletions only in AZFa , including the STS sY84, sY86, sY87, and sY83 (Figure 2). All other STS analysed in patient FA14 were normally amplified. Southern blot analysis using these STS as probes did not give conclusive results due to cross-reactivity with autosomal DNA. The clinical parameters of the five patients with microdeletions are summarized in Table I. Four patients were azoospermic and only the DAZ -deleted FO3 patient showed very rare and aberrant spermatozoa. Testicular histology was available in three deleted patients. FA14, carrying microdeletions in AZFa , had Sertoli cell-only syndrome with complete lack of germ cells. Patients carrying deletion of the DAZ gene (SP8 and FA45) showed different spermatogenic defects including Sertoli cell-only syndrome and maturation arrest. Testicular histology of patient FA45 carrying a DAZ deletion was associated with a SCO phenotype and is shown in Figure 4. Spermatogenesis is a complex event leading to the formation of mature spermatozoa, occurring under the control of a wide variety of factors acting from prenatal to adult life (Vogt, 1997). An important role in the regulation of spermatogenesis is certainly played by the genetic programming of germ cell itself based both on autosomal and sex chromosomal genes. A group of idiopathic infertility has been, in fact, associated with genetic causes linked to the Y chromosome and many studies have recently shown deletions on Y chromosome long arm in idiopathic azoospermic males. The frequencies of deletions of Yq, reported in different studies, range between 3 and 18% of males with non-obstructive azoospermia or severe oligozoospermia (Reijo et al ., 1995; Stuppia et al. , 1996; Vogt et al ., 1996; Girardi et al ., 1997; Pryor et al ., 1997; Qureshi et al ., 1997; Simoni et al. , 1997; Van der Ven et al ., 1997). In our screening of 67 infertile males we have found five patients carrying microdeletions, corresponding to a frequency of 7.5%, that perfectly agrees with the statistical value obtained averaging all the surveys reported to date (Simoni et al ., 1998). The frequency of microdeletions reported here is the result of a correction of the PCR data, made on the basis of Southern blot analyses. In fact, we could exclude three more patients showing a single STS deletion by PCR amplification, not confirmed by Southern blotting, thus lowering the microdeletion rate from ~12% to 7.5%. Even though it is not possible from our result to evaluate statistically whether some STS are more prone to give false negatives, or whether this technical problem might be relevant in explaining the higher frequency of microdeletions found by others, in our opinion Southern blot analysis has to be performed at least in those cases where PCR reaction fails to amplify a single STS. However, we cannot exclude the possibility that the failure of PCR amplification in Southern blot positive cases is due to mutations or small deletions occurring in the genomic sequence matching the STS primers that we used. This should be investigated by sequence analysis of wider genomic regions spanning the involved STS. We could not obtain control DNA of fathers or brothers of the patients carrying Yq microdeletions, thus the possibility that some of the deletions that we have identified simply reflect polymorphisms cannot be excluded. However, larger studies have shown that most of Yq microdeletions are not paternally inherited (Reijo et al ., 1995), therefore they must occur de novo at some stage of paternal germ cell differentiation (at stages compatible with completion of sperm cell differentiation and fertilization even in the lack of the affected gene) or possibly in fertilized eggs (Edwards and Bishop, 1997). As for the mechanism of the appearance of microdeletions, it has been suggested the occurrence of an intrachromosomal recombination event between repeated sequences flanking the affected gene, causing a loop that is deleted (Edwards and Bishop, 1997). If this is the case, the possibility exists of more such events in the same chromosome causing discontinuous deletions, e.g. the one observed in patient SP8. Many efforts have been made to identify the genes on Yq that are important for a normal ...
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... 67 patients with azoospermia or severe oligozoospermia, selected as described in Methods, were screened by PCR amplification and Southern blot analysis for potential deletions of 20 loci on Yq11. STSs and Y chromosome DNA probes used in this study, and their localization on Yq11 are shown in Figure 1. Most of the STS map to interval 6 of Yq11, which includes the DAZ gene in AZFc locus (sY254, sY277, sY283). ...
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... (B) and (C) the azoospermic patient SP1 with no Yq microdeletions is also included. The arrow in (C) indicates the band corresponding to locus DYS7-C in the AZFc region of Yq (see Figure 1) found deleted in patient SP8. Probe p50f2 recognizes additional bands (A, B, D and E) corresponding to locus DYS7-A and -B in Yp, DYS7-D in the centromere, and DYS7-E in the AZFb region of Yq. ...
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... 3A shows a representative Southern blot experiment using STS sY254, sY277, sY283 as probes. DNA from the same patients was also analysed by a hybridization technique using the Y chromosome DNA probes p49f and p50f2 (see map in Figure 1), showing deletions of both loci DYS1 (detected by p49f) and DYS7C (detected by p50f2) (Figure 3B,C). The absence, in these patients, of DYS1 sequences, previously demonstrated to be 1118 the DAZ gene cluster (Saxena et al., 1996), agrees with PCR data. ...
Citations
... However, such correlations are not evident in most of the studies reported. Reviewing papers related to Y chromosomal microdeletions published between 1994 and 1998 (Kobayashi et al, 1994;Reijo et al, 1995;Qureshi et al, 1996;Vogt et al, 1996;Girardi et al, 1997;van der Ven et al, 1997;Foresta et al, 1998;Grimaldi et al, 1998;Oliva et al, ...
... 1998), we have corroborated that 76 out of 81 (93.83%) microdeletions reported in the papers should be unequivocally detected by our fast microdeletion test. Two cases, corresponding to unique STS deletion, could be not detected by us (Qureshi et al, 1996; van derVen et al, 1997), and the 3 remaining cases are not accurately delineated (Kobayashi et al, 1994;Grimaldi et al, 1998), thus we are not sure that we would be able to detect them. The rate of microdeletions in the 127 individuals reported in the reviewed papers is 6.44%; using our set of 9 STSs we would have a 6.05% rate of mirodeletions. ...
Recent investigations have pointed to a high prevalence of Y chromosome submicroscopic deletions in men with severely impaired spermatogenesis. We report on the incidence in 128 infertile men, in whom karyotype, sperm count, and hormonal parameters were evaluated. Patients with abnormal karyotype (other than an abnormal Y chromosome) or sperm concentration of more than 2 million/mL were excluded. Genomic DNA was extracted from the peripheral leukocytes of 57 men with azoospermia and 71 with severe oligospermia. Molecular analysis was performed by 3 multiplex polymerase chain reactions using a set of 9 sequence tagged sites (STSs) from 3 different regions of the Y chromosome: AZFa, AZFb, and AZFc. In 7% of the studied patients Yq microdeletions were detected, with a high prevalence in men with azoospermia (14%). No deletions were detected in the AZFa region. Deletions were present in AZFb, AZFc, or both regions. The deletion observed in 1 patient that did not overlap with the DAZ region demonstrates that genes other than DAZ may also be involved in the pathogenesis of some subsets of male infertility. Furthermore, common Yq deletions present different testicular pictures, suggesting that some unknown factors may be disturbing spermatogenesis. Because men with severe infertility suffer a high risk of Y chromosome deletion, screening for these men is recommended prior to treatment with assisted reproduction.
The Y chromosome has been a very rich area for beginning the study of the genetic control of spermatogenesis. The Y chromosome contains 60 multicopy genes composed of 9 different gene families concentrated in regions of multiple repeat sequences called amplicons arranged in mirror images called palindromes. It contains many testis-specific spermatogenesis genes. This very complicated pattern is susceptible to deletions caused by homologous recombination with itself and can explain the presence of small numbers of sperm in otherwise azoospermic men. It is also the beginning of understanding the genetic control of spermatogenesis.
Objective: Study carried out between the years 2009 and 2010 in the cities of Barranquilla and Sincelejo that has as main objective to determine the microdeletions in the DAZ region of the Y chromosome in infertile men. Materials and Methods: study was developed with a sample of 33 men, selected in specialized consultation, previous confirmation of infertility through spermograms studies. Seventeen STSs were analyzed from different azoospermia regions in addition to the SRY, the DS271 and the Kaly gene. DNA extraction from peripheral blood through Bio-mol protocol, PCR multiplex master mix of Promega and electrophoresis in agarose gel 4% was performed for data analysis and the images were visualized through photo-documenter. Results: Findings showed 3.03% microdeletions in Y chromosome in STSs: SY242 (DAZ) SY208 (DAZ), SY254 (DAZ), SY255 (DAZ) and SY157 (DYS240), compromising the whole AZFc region in the STSs studied. Conclusions: was determined microdeletion of AZFc region of Y chromosome in a proportion of 3.03%, with evident absence in the genes of the internal control, the bands studied for the set of the master indicators D and E are presented, confirming reproducibility of the product obtained through PCR. Patients with presence of locus SRY, secretory azoospermia, left varicocele, normal sexual organs, testicular biopsy with absence 80% of germ cells and 20% without maturation. The proportion of cases located in the range found in various studies globally.
PRY (PTP-BL related on the Y chromosome) has been proposed as a candidate spermatogenesis gene. 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 the cDNA sequence with the genomic sequence, five exons were identified. Analysis of GenBank-derived clones on the Y chromosome revealed the presence of two full-length copies in azoospermia factor region b (AZFb) (PRY1 and PRY2) and two shorter versions of the PRY gene containing exons 3, 4 and 5 in AZFc (PRY3 and PRY4). A clone containing sequences homologous to exons 3, 4 and 5 is located in area 5L (between AZFa and AZFb), a clone containing a sequence homologous to exon 5 is located in area 5M (in AZFb) and a clone containing a fragment homologous to exon 3 is located in 6F. A repeat structure of exons 1 and 2 is present on the short arm of the Y chromosome as well as on the long arm. PRY1 and PRY2, two gene copies that are located in AZFb, a region often deleted in patients with severe male infertility, were shown to be expressed in the testis. PRY may therefore play an important role in spermatogenesis.
Determination of microdeletion in chromosome Y in infertile men, candidates to assisted reproduction techniques Intracytoplasmatic spermatozoid Injection. Severe spermatic damage in infertile man can be the result of presence of deletion in chromosome Y and in AFZc region (Zoosperm factor Zone C). This study was made in 97 men with infertility and a frequency of 6% to the presence of deletion in the studied population. Of those men positive to deletion in Zone Yq (AZFc) were born three children by using intracytoplasmatic injection (ICSI), whom inherited mutation in chromosome Y, it established the importance to create a sifting program to detect the presence of deletion in this chromosome
The aim of the study was to investigate the hypothesis that Y chromosome microdeletions are directly implicated in testicular maldescent. Genomic DNA was extracted from the peripheral blood of 292 subjects. This population consisted of (i) 180 children with all phenotypes of isolated (non-syndromic) testicular maldescent from 174 index families, (ii) affected adult relatives available (n = 12) and (iii) 100 unrelated children with normal external genitalia (controls). The sequence-tagged site primer set and the conditions of conventional polymerase chain reaction amplification were based on the current laboratory guidelines for molecular diagnosis of Y chromosome microdeletions recommended by the European Academy of Andrology and the European Molecular Genetics Quality Network. Two multiplex reactions were designed to screen the regions of azoospermic factors a, b and c. Each multiplex reaction included adequate internal and external amplification controls. Amplification products were submitted to electrophoresis on 2% agarose gel impregnated with ethidium bromide dye solution for 80 volt-h and visualised under ultraviolet light. No microdeletions were detected in any subject. These results indicate that Y chromosome microdeletions are not directly implicated in the pathogenesis of testicular maldescent. Other factors should be investigated to potentially explain the genetic predisposition that seems to exist in at least a subgroup of these patients.
LEARNING OBJECTIVES 1. The ART physician should become thoroughly knowledgeable about the rates of various chromosomal anomalies in the embryos and offspring derived from ICSI. 2. The ART physician should be familiar with chromosomal studies of sperm in normal versus infertile men. 3. The ART physician should become familiar with the results of conventional IVF versus ICSI in various clinical groups and the genetic implications. 4. The ART physician and laboratory personnel should understand the indications for PGD (pre-implantation genetic diagnosis) for male factor IVF cases. 5. The ART physician should be familiar with comparative results of ICSI-IVF with obstructive and non-obstructive azoospermia and severe and moderate oligospermia.
Male factor infertility is present in up to 50% of infertile couples, making it increasingly important in their treatment. Although most research into the genetics of male infertility has focused on the Y chromosome, male factor infertility may result from other genetic factors. We utilized the whole genome array comparative genomic hybridization (CGH) to identify novel genetic candidate associated with severely impaired spermatogenesis. We enrolled 37 patients with severe male factor infertility, defined as severe nonobstructive type oligozoospermia (≤ 5 × 10(6)/ml) or azoospermia, and 10 controls. Routine cytogenetic analyses, Yq microdeletion PCR test and whole genome bacterial artificial chromosome (BAC)-array CGH were performed. Array CGH results showed no specific gains or losses related to impaired spermatogenesis other than Yq microdeletions, and there were no novel candidate genetic abnormalities in the patients with severe male infertility. However, Yq microdeletions were detected in 10 patients. Three showed a deletion in the AZFb-c region and the other 7 had deletions in the AZFc region. Although we could not identify novel genetic regions specifically associated with male infertility, whole genome array CGH analysis with higher resolution including larger numbers of patients may be able to give an opportunity for identifying new genetic markers for male infertility.
