F&S 2829 revised
Lardone et al. AZFc subdeletions in infertile Chilean men
AZFc partial deletions in Chilean men with severe spermatogenic failure
María C. Lardone, B.S.,a Daniela A. Parodi, M.Sc.,a Mauricio Ebensperger, M.D.,b Paulina
Peñaloza, M.D.,b Valeria Cornejo, M.D.,b Raúl Valdevenito, M.D.,c Ricardo Pommer, M.D.,a and
Andrea Castro, M.Sc.a
a
Institute of Maternal and Child Research, School of Medicine, University of Chile, Santiago,
Chile; bSan Borja Arriarán Clinical Hospital, Santiago, Chile; cJosé Joaquín Aguirre Clinical
Hospital, School of Medicine, University of Chile, Santiago, Chile.
Received August 17, 2006; revised January 4, 2007; accepted January 5, 2007.
This study was supported by grant no. 1030984 from the National Fund for the Scientific and
Technological Development (FONDECYT), , Chile,.
Reprint requests: Andrea Castro, Institute of Maternal and Child Research, School of Medicine,
University of Chile, Santa Rosa 1234, Santiago, Chile (FAX: 56-2-4247240; E-mail:
acastro@med.uchile.cl).
0015-0282/07/$32.00
doi:10.1016/j.fertnstert.2007.01.038
Fertility and Sterility® Vol.##, No.##, Month 2007
Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
Lardone et al.
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CAPSULE
The analysis of DAZ-SNVs and STS-specific AZFc markers in Chilean men with severe
spermatogenic failure, and controls with normal spermatogenesis, showed that AZFc
subdeletions are not associated with severe impairment of spermatogenesis.
Objective: To determine the prevalence of AZFc subdeletions in infertile Chilean men with
severe spermatogenic impairment.
Design: Prospective analysis.
Setting: University infertility clinic.
Patient(s): Ninety-five secretory azo/oligozoospermic men without AZFc Y chromosome
microdeletions: 71 whose testicular histology showed severe spermatogenic impairment and 24
who exhibited reduced testicular volume and elevated serum FSH levels. As controls, we studied
77 men (50 fertile and/or normozoospermic, and 27 with azoospermia and normal
spermatogenesis).
Intervention(s): Peripheral blood was drawn to obtain genomic DNA for polymerase chain
reaction (PCR) digestion assays of DAZ-sequence nucleotide variants and for AZFc-STS PCR
after a complete testicular characterization (biopsy, hormonal, and physical evaluation).
Main Outcome Measure(s): DAZ genes and AZFc subdeletion types.
Result(s): In cases we observed two “gr/gr” subdeletions (2.1%), one with absence of
DAZ1/DAZ2 (g1/g2 subtype), and the other with absence of DAZ3/DAZ4 (r2/r4 subtype).
Additionally, we found a g1/g3 subdeletion in a patient with Sertoli-cell-only syndrome. In
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controls, we observed two gr/gr subdeletions with absence of DAZ1/DAZ2 (2.6%) in a
fertile/normozoospermic and in an obstructive azoospermic man.
Conclusion(s): AZFc subdeletions do not seem to cause severe impairment of spermatogenesis.
Moreover, gr/gr-DAZ1/DAZ2 subdeletions do not appear to affect fertility in Chilean men.
Key Words: AZFc partial deletion, male infertility, spermatogenic impairment, Y chromosome,
DAZ gene.
Y chromosome microdeletions constitute an important etiological factor in spermatogenic
failure. Several studies indicate a prevalence of 5% to 20% in subjects with azoospermia or
severe oligozoospermia (1, 2), and 25% to 55% in patients with severe testiculopathies, such as
hypospermatogenesis, maturation arrest (MA), and Sertoli-cell-only syndrome (SOCS) (3, 4).
These deletions were described in three nonoverlapping regions in proximal (AZFa), middle
(AZFb), and distal (AZFc) Yq11.2 (5, 6). AZFc is the most frequently deleted region, and men
with AZFc deletions have a variable testicular phenotype that may be compatible with sperm
recovery at testicular sperm extraction (4, 5).
The AZFc region encompasses a complex of three palindromes, the largest spanning 3 Mb with
99.97% identity between its arms. The palindromes are constructed from six distinct families of
amplicons, and may have resulted from tandem duplication and inversion during primate
evolution. The palindromic complex contains 11 families of transcription units, all expressed in
testis. In between these transcription units DAZ genes are organized in two cluster groups of
genes in orientation 3′5′:: 5′3′ (7, 8).
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Deletion of AZFc removes all four copies of the DAZ gene spanning a 3.5-Mb segment bounded
by b2 and b4 amplicons, which serve as substrates for homologous recombination producing
complete AZFc deletion (8). DNA sequence alignments within AZFc also reveal the possibility
for the formation of subdeletions or partial deletions. Detailed analysis of the AZFc region using
new molecular nonrepeated STS markers has confirmed the existence of three such subdeletions,
namely, gr/gr, b1/b3, and g1/g3 (also known as b2/b3) (9–11). The most prevalent partial
deletion, called gr/gr (12), is caused by recombination between amplicons g and r (g1, r1, and/or
r2, with their respective homologous amplicons g2, r3, and/or r4). This subdeletion produces the
loss of two of the four copies of the DAZ gene (DAZ1/DAZ2 or DAZ3/DAZ4), one of three
copies of the BPY2 gene, and one of two copies of the CDY1 gene (CDY1a). Similarly, others
are the outcome of b1/b3 and g1/g3, leading to retention of two DAZ gene copies, two or one
BPY2 genes, and two or one CDY1 gene copies, respectively. Several investigators have found
an association between gr/gr subdeletions and spermatogenic failure (9, 13–16), whereas others
have not (17–20).
To determine the contribution of these AZFc subdeletions to male infertility, we assessed their
prevalence and associated testicular phenotype in a population of infertile men in Chile with
severe impairment of spermatogenesis. We used a combined methodological approach that
included the detection of the different DAZ gene copies by SNVs analysis (21) and AZFcspecific STSs (12). Our results showed that the prevalence of partial AZFc deletions was not
statistically different between cases and controls.
MATERIALS AND METHODS
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Patients
This study was approved by the Institutional Review Board of the School of Medicine at the
University of Chile, San Borja Arriarán Clinical Hospital, and José Joaquín Aguirre Hospital,
and all subjects gave their informed consent. We studied 95 Chilean infertile patients with
impairment of spermatogenesis, who sought consultation for infertility at the Institute of
Maternal and Child Research or at the José Joaquín Aguirre Hospital. The study included 77
controls: 19 healthy and fertile donors with unknown seminal parameters, 31 normozoospermic
donors with unknown fertility, and 27 obstructive azo/oligozoospermic men with normal
spermatogenesis assessed by testicular biopsy. Donors were Chilean men from the same centers,
and their fertility status was presumed by the fact that they reported to have fathered one or more
children.
All participants underwent an evaluation that included complete physical examination, hormonal
studies, and karyotype. Testis volume was measured by ultrasonography and/or Prader
orquidometer. Serum testosterone was determined using radioinmunoassay (RIA). LH and FSH
were measured by immunoradiometric assay (IRMA). Patients with hypogonadotrophic
hypogonadism, hypoandrogenism, chronic diseases, or AZFc Y chromosome microdeletions
were excluded. The Y chromosome microdeletion standard analysis was performed using a set of
34 STSs including AZFc markers (PRY, sY254, sY221, sY255, sY202, BPY2, sY283, CDY1,
and sY158) as previously described (22).
Semen Analysis
Semen analysis was performed according to normal standard parameters using the World Health
Organization (WHO) criteria (23). The diagnosis of azoospermia was based on the absence of
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sperm in at least two separate semen analyses and after centrifugation of semen samples (1,000 
g, 5 minutes). The selected infertile men were classified as azoospermic or severe
oligozoospermic patients (sperm count 5 million/mL).
Testicular Biopsy
In secretory or obstructive azo/oligozoospermic infertile men, the testicular biopsy was
performed for spermatic recuperation by TESE or for diagnosis. Among the controls, some
normozoospermic men (n = 10) were also subjected to testicular biopsy during genital surgery,
because of testicular cancer (n = 3) or for non-neoplasic reasons (2 hidrocele, 2 epididymal cysts,
and 3 varicocele), at the Urology Service of the San Borja Arriarán Hospital, and all had normal
testicular histology. Testicular histology assessment included a qualitative and quantitative
analysis of germinal epithelium in 25 tubules, and the modified Johnsen score was calculated
(24, 25). According to this score, the tissues were classified in SOCS, complete or incomplete
(some foci of spermatogenesis); MA, germ cells until spermatogonia, spermatocyte, or
spermatid, which may be complete or incomplete (some tubules show germ cells more developed
than the maturation detention stage); and hypospermatogenesis (proportional and quantitative
reduction of the different types of germ cells).
Combined Analysis of AZFc Partial Deletions
A two-step approach was employed to identify partial AZFc deletions. First, screening was
performed in all patients to discriminate between DAZ1-DAZ4 gene copies (21) using pairs of
primers specific to single nucleotide variants (SNVs)—SNV I, SNV II, SNV III, SNV IV, SNV
V (sY587) and sY581, and the STS Y-DAZ3 (Table 1)—and combined with the use of the STS
marker sY1291 specific to gr/gr subdeletion (12). A negative result was repeated for
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confirmation, and a second step with six additional pairs of primers was used for further
characterization: sY1258, sY1197, sY1161, sY1191, sY1206, and sY1201 (12).
Second,single PCR reactions were performed. The components of PCR were 1 μL aliquot of the
genomic DNA (140 g/mL), 1.5 μL of 10 buffer solution, 0.15 μL of 100 mmol/L of each
dNTP, 0.7 μL of each primer (10 μmol/L) and 0.2 μL of Taq DNA polymerase (5 U/μL) (MBI
Fermentas) in a total volume of 15 μL. PCR was performed according to the following protocol:
amplification by 30 cycles at 94°C for 45 seconds, annealing at 55°C (SNV IV), 58°C (SNV I,
SNV II, sY581, sY587, and Y-DAZ3), or 63°C (SNV III) for 60 seconds, and 72°C for 60
seconds. The program was preceded by a 3-minute denaturation step at 94ºC and followed by a
final extension step at 72°C for 10 minutes. PCR conditions for sY1291, sY1258, sY1197,
sY1161, sY1191, sY1206, and sY1201 were similar except that annealing temperature was 57°C
and the reaction consisted of 35 cycles. The PCR result was made visible on a 1.5% agarose gel
with ethidium bromide. In all PCRs, a female DNA and a water sample (no template) were
included as negative controls.
DAZ gene copy-specific restriction analysis was performed after digestion of PCR product.
Briefly, 7 μL of each PCR product was incubated with 5 U of the respective restriction enzyme
at 37 °C (except for Taq I that uses 63 ºC) during 3 hours in a final volume of 10 μL (Table 1).
The fragments were visible on a 3% agarose gel with ethidium bromide.
Statistical Analysis
Differences in hormonal levels and seminal parameters among groups were calculated with the
Kruskal-Wallis test. Differences among frequencies were calculated with both the 2 test and the
Fisher’s exact test. P<0.05 was considered statistically significant.
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RESULTS
By Y chromosome microdeletion standard analysis we diagnosed 8% (8/101) cases (secretory
azo/oligozoospermic group) with AZF deletions. We analyzed the presence of AZFc
subdeletions in 95 patients with no AZFc deletions, including one patient with AZFa and another
with AZFb deletions. No AZF deletions were observed in the 77 control males tested.
Semen analysis was performed in 95 cases and 58 controls. In the fertile men control group, no
semen analysis was available. Sperm counts are shown in Table 2. We observed a higher
percentage of azoospermia in patients with SOCS and complete MA. The hormonal analysis
revealed that FSH levels in case subgroups of SCOS, and in those without diagnosis by testicular
biopsy, were higher compared with complete or incomplete MA, with hypospermatogenesis, and
controls (Table 2). Similarly, cases with higher FSH levels had reduced testicular volumes in a
higher proportion than other subgroups.
DAZ-SNVs Copy Analysis
The results of DAZ gene copy analysis in cases and controls are shown in Table 3 and Table 4,
respectively. We observed absence of one or two DAZ genes in 14 out of 77 (18%) controls and
in 18 out of 95 (19%) cases. DAZ2 deletions (negative result with SNV III and SNV IV) were
observed in 9 cases (9.5%) and 5 controls (6.5%). DAZ3 deletions were detected in 1 (1.3%)
control patient and in 2 (2.1%) cases. DAZ4 deletion was found in 2 cases (2.1%) and 4 controls
(5.2%). No isolated DAZ1 deletion was found in either cases or controls. Simultaneous DAZ2
and DAZ3 deletions were observed in 1 (1.1%) case and 2 (2.6%) controls. DAZ1/DAZ2 cluster
deletion was found in 2 (2.6%) controls and 2 (1.1%) cases. DAZ3/DAZ4 cluster deletion was
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observed in 2 (1.1%) cases, but not in controls. Significant differences between case and control
groups or subgroups were not observed.
DAZ-SNVs and AZFc-STS Combined Analysis
The amplification of sY1291 in cases and controls did not show any negative result in patients
with absence of DAZ2, DAZ3, DAZ4, or DAZ2/DAZ3 by DAZ-SNVs analysis. In contrast, the
absence of sY1291 was associated with the loss of DAZ1/DAZ2 cluster. Similarly, the absence
of sY1191 was observed only in patients with DAZ3/DAZ4 deletion.
Figure 1 shows the result of AZFc subdeletions analysis in cases and controls with the
corresponding complete AZFc region structure of the Y chromosome. Overall, we identified 4
gr/gr subdeletions with two different deletion patterns: one with absence of sY1291 and
DAZ1/DAZ2 (subtype g1/g2), and the other with absence of sY1291 and DAZ3/DAZ4 (subtype
r2/r4). We also found a patient with a g1/g3 deletion. In addition, in a patient with AZFb
deletion, we found an AZFc subdeletion with absence of the sY1258, sY1161, sY1197, sY1191,
and sY1291 STSs markers and the DAZ1/DAZ2 gene cluster. The STS sY1258 localizes in the
distal end of AZFb, whereas the absence of sY1291 suggests that the distal breakpoint localizes
in a region within gr1 and Y1. Deletions involving AZFb and part of AZFc have been named
“P5/proximal P1” (26).
The frequency of partial AZFc deletions among the secretory azo/oligozoospermic group was
4.2%, and 2.6% in the control group (Table 5). The prevalence of partial AZFc deletions was not
statistically different between cases and controls, among the different subdeletions, or among
case subgroups.
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The normozoospermic man (P1012) who presented a gr/gr-DAZ1/DAZ2 subdeletion had
fathered two children, each with a different woman. The boy had the same subdeletion. Further
investigation showed that his father exhibited the same gr/gr subdeletion and reported a normal
fertility history (currently 63 years old with poor seminal parameters). He had reportedly
fathered three more children. We studied the patient’s brother and nephews, who also showed the
same subdeletion. Investigation of the family of the azoospermic obstructive control (P195)
showed that one of his four brothers carried the same gr/gr subdeletion. This subject had fathered
two children, but no semen analysis was available. The other brothers had normal fertility, but no
semen or DNA samples were available.
DISCUSSION
Complete deletion of the AZFc region of the Y chromosome includes the loss of the DAZ gene
family and is responsible for most cases of spermatogenic impairment in infertile men. Although
this deletion has been associated with a variable testicular or seminal phenotype, it might be
compatible with the development of sperm, and therefore has a better prognosis than AZFa or
AZFb deletions. In this study, we found a high percentage of Y chromosome microdeletions that
included the AZFc region. Three of four patients with AZFc deletions had sperm in their
ejaculate or in their testicular extraction procedure. Therefore, our results agree with the clinical
characteristics of patients with AZFc deletion described in the literature (1–5, 27).
The main contribution of our study was determining the prevalence of AZFc partial deletions in
Chilean men carefully characterized by testicular histology. In this study we first applied a DAZSNV-specific gene copy and sY1291 (specific gr/gr subdeletion) analysis in 93 secretory
azo/oligozoospermic patients with normal karyotypes and without Y chromosome
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microdeletions by a standard assay. We subsequently characterized these subdeletions by
investigating six additional AZFc-specific STSs (including sY1191, specific for g1/g3
subdeletion) (9–11). In addition, our study included the analysis of AZFc subdeletions in one
patient with AZFb (complete MA) and another with an AZFa deletion (SCOS).
By DAZ-SNVs analysis we observed a similar percentage of absence of DAZ copy genes in
patients with severe impairment of spermatogenesis, and in controls with normal
spermatogenesis and/or fertility. When we studied the presence of STS sY1291 or the other
AZFc-STS (sY1258, sY1197, sY1161, sY1191, sY1206, sY1201), we found negative
amplifications only in DNA from patients who presented absence of DAZ1/DAZ2 or
DAZ3/DAZ4 (Fig. 1). Therefore, these patients with DAZ cluster deletions may represent true
AZFc subdeletions. This agrees with the mechanism of homologous recombination between
amplicons proposed previously where DAZ genes are deleted in doublets (9–11).
In the cases of DAZ2, DAZ3, DAZ4, and DAZ2/DAZ3 presumed deletions, we found no
associations with absence of any AZFc-STS, indicating that these SNVs negative results in
controls and/or cases may represent gene conversion events and not true deletions (21).
Moreover, when we compared the prevalence of each of these negative results, we observed
similar results in cases and controls, suggesting that our cases and controls represent a similar
Chilean subpopulation, which is mainly of Spanish origin (28). According to that, it has been
suggested that some DAZ deletion haplotypes might be polymorphic events associated with
specific Y chromosome haplogroups (29). In addition, the Y chromosome sequence described
and proposed as reference (RPCI-11) belongs to haplogroup R; consequently, the different DAZ
haplotypes should be regarded in the context of certain haplogroups.
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We usually found consistent results using DAZ-SNVs combined with AZFc-STS analysis in our
cases and controls. However, in cases with deletions of DAZ3/DAZ4, predicted by the combined
analysis of SNV I, Y-DAZ3, and sY1291 or sY1191, we found an incongruous result with sY587
attributable to the presence of the 196 bp fragment. Moreover, a nonspecific fragment of 196 bp
is amplified in female DNA and in DNA from patients with AZFc microdeletion. Therefore,
according to our PCR conditions, it should not discriminate the DAZ3/DAZ4 gene cluster.
However, sY587 is a good marker for deletion of DAZ1/DAZ2 cluster since it was congruent
with the result obtained by SNV II, SNV III/IV, and sY1291. Additionally, the inconsistent
results found with SNV III and SNV IV might be related to the variability in DAZ sequences
among individuals (polymorphisms in DAZ-SNVs) or gene conversion events (7, 30).
In three cases where we did not detect any negative result by DAZ-SNVs analysis, we found a
negative result with the pairs of primers specific to sY1291. This result was considered false
after a positive amplification of sY1291 with a second DNA sample. A French study (18), which
used DAZ-SNVs and AZFc-STS analysis, found one fertile man who failed to amplify sY1291.
The investigators do not report that the negative result was confirmed by a second DNA sample,
and explained that it may have a low frequency of Y chromosomes (<0.2%) that lack this
evolutionary junction fragment.
We did not find an association between gr/gr subdeletions and severe spermatogenic failure.
Similarly to other studies (18, 19), we found gr/gr subdeletion with loss of DAZ1/DAZ2 in two
men with normal spermatogenesis. This subdeletion was also present in the fertile male relatives
studied. Nevertheless, many studies found a positive correlation between gr/gr subdeletions and
spermatogenic failure (9, 13–16), but others did not (17–20). This disagreement may be
explained by different factors. The study of Lynch et al. (14) would not have found an
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association between gr/gr subdeletion and infertility if they had studied only secretory
azo/oligozoospermic men and had not included infertile men with sperm count of >20  106
sperm/mL.
In contrast to other studies (13–15) where most patients were oligozoospermic, the azoospermic
subjects in our study represent approximately 70% of cases. In agreement with this, the
quantification of DAZ genes by real-time polymerase chain reaction in a population of 90
infertile men and 50 fertile men concluded that partial deletions of the DAZ genes are associated
with oligozoospermia but not with azoospermia (31). In that study, the testicular histology of the
oligozoospermic men was not evaluated, and hormonal and testicular volume parameters were
not documented. Thus, the population studied would not represent true secretory
oligozoospermia but rather obstructive oligozoospermia with normal spermatogenesis.
Other aspects contributing to the discrepancy in AZFc subdeletions results may be that some
investigators (13, 16) did not use a combined approach to define DAZ gene copies. Instead they
used sY1291 and sY1191 as the first step for their AZFc-STS analysis; therefore, they were not
able to distinguish subtypes of gr/gr partial deletions, DAZ gene copies, and false negatives.
Among the gr/gr subdeletions described in the literature, the DAZ3/DAZ4-CDY1a deletion
(subtype r2/r4) showed an association with infertility (18). In our study, we found one gr/gr
subdeletion of r2/r4 subtype in one man with incomplete MA but none in the controls, suggesting
their possible participation in this type of less-severe spermatogenic impairment. However, this
result did not reach statistical significance. Another subdeletion called g1/g3 involving
DAZ3/DAZ4 is more frequent in individuals of the N haplogroup (common in northern Europe
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and Asia) and has been shown to have no spermatogenic impact (11, 27). In this study, we found
one patient with g1/g3 subdeletion, but we could not perform Y chromosome haplotyping.
The deletion referred as P5/proximal P1 was found in a patient with AZFb deletion previously
determined by the absence of sY113 and sY142 at the proximal and distal breakpoints,
respectively. Recently, with the availability of nonrepeated STS, it has been described that the
AZFb deletion may overlap AZFc by 1.5 Mb (30). Moreover, homologous recombination
between direct repeats (P5 in AZFb and P1.2 in AZFc) was proposed as a mechanism to explain
the deletions involving AZFb region and AZFc subdeletion (30). Although we were not able to
define the proximal AZFb breakpoint at palindrome P5, we speculated the occurrence of a sole
deletion event encompassing AZFb and part of the AZFc region.
In summary, we investigated the presence of subdeletions in Chilean azo/oligozoospermic men
with severe impairment of spermatogenesis. We used a combined approach of DAZ-SNVs and
AZFc-STS. We found gr/gr subdeletions with loss of DAZ1/DAZ2 (g1/g2 subtype) in two men
with normal spermatogenesis, and in one man with azoospermia and SCOS. Our results suggest
that gr/gr-DAZ1/DAZ2 subdeletions are not sufficient to cause severe spermatogenic
impairment. In addition, the participation of gr/gr subdeletion, which involves DAZ3/DAZ4
(r2/r4 subtype), may contribute to some spermatogenic defects, such as incomplete MA or lesssevere spermatogenic impairment.
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Acknowledgment: We are grateful to Eduardo Gazzo, Flor González, Eduardo Castro, Marcia
Madariaga, and Patricia López for their collaboration with some experimental protocols and the
contribution on sample semen analysis and karyotype.
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FIGURE 1
AZFc structure and results of AZFc-specific STSs and DAZ-specific SNVs analysis. The
ampliconic structure of the AZFc region is shown according to Kuroda-Kawaguchi et al. (8). STSs
utilized to detect deletions. Red stars 1 to 4: Position of the four DAZ genes. Black boxes: STS
present. Lines: STS absent. Gray boxes: STS that normally amplifies by PCR but assumed to be
absent in context of deletion pattern. (A) gr/gr subdeletions identified by absence of sY1291. DAZspecific SNVs analysis distinguishes between those with DAZ1/DAZ2 deletion (g1/g2) in patients
P136, P195, P1012, and those with DAZ3/DAZ4 deletion (r2/r4) in patient P171. Deletion
identified by absence of sY1258, sY1161, sY1191, sY1197, and sY1291 is referred to as
P5/proximal P1 (P10). (B) Blue letters: Ampliconic structure of the AZFc region shown after the
b2/b3 inversion. g1/g3 subdeletion identified by absence of sY1191. DAZ-specific SNVs analysis
shows absence of DAZ3/DAZ4 (P131).
FIGURE 2
Pedigree of the normozoospermic subject with gr/gr-DAZ1/DAZ2 deletion. Crossed rectangles:
affected patient (gray box) and relatives. OZ: oligozoospermia; NZ: Normozoospermia.
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