international journal of andrology, 24:31±36 (2001)

Molecular detection of Y chromosome microdeletions:

an Irish study

A. FRIEL*à, J. A. HOUGHTON*à, M. MAHER*, T. SMITH*, S. NOEÈL*,

A. NOLAN , D. EGAN and M. GLENNON*
*National Diagnostics Centre, BioResearch Ireland, National University of Ireland, Galway,
Fertility Unit, University College Hospital, Galway, and àMicrobiology Department, National
University of Ireland, Galway

Summary
The region of the Y chromosome most critical for male fertility is called the
azoospermia factor (AZF) region and it is located within subintervals ®ve 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 con®rmed that each deletion encompassed
the respective AZF region including the AZF candidate gene.

Keywords: AZF genes, azoospermia, Irish patients, microdeletions, oligozoospermia,

Y chromosome-long arm

Introduction
Infertility is commonly de®ned as the failure of concep- infertility accounts for approximately half of these cases and a
tion after at least 12 months of unprotected intercourse large proportion of these men suffer from idiopathic
(Rowe et al., 1993). Approximately 15% of couples trying to infertility. Recent studies report that DNA microdeletions
achieve pregnancy will experience some form of infertility on the long arm of the Y chromosome may be responsible
(Bhasin et al., 1994). It is generally accepted that male for some cases of idiopathic male infertility (Chandley &
Cooke, 1994; Reijo et al., 1995; Stuppia et al., 1996; Vogt
Correspondence: M. Glennon, National Diagnostics Centre, et al., 1996; Foresta et al., 1997; Simoni et al., 1997). The
BioResearch Ireland, National University of Ireland, Galway, most important region is located within intervals ®ve and six
Ireland. on the long arm of the Y chromosome and has been
E-mail: [email protected] designated the AZF region (Vogt et al., 1996, 1997). To

Ó 2001 Blackwell Science Ltd.

32 A. Friel et al.

date, three non-overlapping spermatogenesis loci have been Screening for mutations in the cystic ®brosis gene CFTR
identi®ed in this region, namely, AZFa, AZFb and AZFc. was performed where appropriate. An in-house PCR
Candidate genes have been isolated for each AZF locus, protocol was used to screen for DF508, DI507, G551D,
USP9Y (Homo Sapiens chromosome Y ubiqutin speci®c R553X, R560T/K, G542X, R117H and 1717±1G®A
protease 9) (Brown et al., 1998), for AZFa, the RBMY mutations (Cystic ®brosis mutation database: HTTP://
(RNA binding motif) gene family for AZFb (Ma et al., 1993) www.genet.sickkids.on.ca/cftr/). Based on these tests, it
and the DAZ (deleted in azoospermia) gene cluster for AZFc was concluded that 56 patients were suffering from
(Reijo et al., 1995). Intracytoplasmic sperm injection (ICSI) idiopathic infertility. Within this group, 39 patients were
is now used frequently to facilitate pregnancy in many azoospermic, seven were oligozoospermic and 10 were
couples suffering from primary infertility and it has been oligoasthenoteratozoospermic. In the remaining 22 non-
shown that a patient with a Y chromosome deletion idiopathic patients, 11 had a past history of genital infection
undergoing ICSI can transmit that deletion to male offspring or biological abnormalities suggesting partial obstruction or
(Jiang et al., 1999; Page et al., 1999). As a result of this, PCR damage to the genito-urinary tract, two displayed several
analysis of the Y chromosome is being introduced as part of abnormal hormone readings, eight were carriers for the
the clinical work-up for the infertile male in our laboratory cystic ®brosis gene mutation, DF508 and one was suffering
and many laboratories world-wide. According to recent from secondary infertility. A total of 50 age-matched
studies, the prevalence of microdeletions varies from 1% normospermic men, who had all fathered children, were
(Van der Ven et al., 1997) to 55.5% (Foresta et al., 1998). included as controls.
Differences in deletion frequency and, in some cases,
location, are likely to be related to study design but they DNA extraction
may also re¯ect population variances or environmental Blood samples from each patient were collected and
in¯uences (Krausz et al., 1999a). The purpose of this study stored in EDTA vacuum tubes and genomic DNA was
was to determine the frequency, status and extent of Y obtained from peripheral leucocytes using the CF(12)-PCR
chromosome microdeletions in a population of Irish men method (Ortho-Clinical Diagnostics, Amersham, UK).
undergoing IVF treatment. Although the majority of papers
report microdeletions in idiopathic azoospermic and oligo- PCR based screening for Y chromosome microdeletions
zoospermic men, they have also been detected in patients Simplex PCR. In an initial study, 50 patients were
with other known causes of infertility (Kremer et al., 1997; screened using 22 different Y chromosome speci®c sequence
Pryor et al., 1997; Krausz et al. 1999b). We have included tagged site primer sets which spanned the AZFb and AZFc
men suffering from idiopathic and non-idiopathic infertility regions of the Y chromosome: sY138, sY139, sY143, sY153,
along with normospermic men in this study. For the sY152, sY150, sY155 sY147, sY149 (Vollrath et al., 1992);
purposes of comparison, 50 patients were initally screened sY220, sY245, sY242, sY262, sY257, sY254, sY255, sY272,
using 22 primer sets which spanned the AZFb and AZFc sY273, sY269, sY243 (Reijo et al., 1995); RBMY and
regions of the Y chromosome, these patients were re- SPGY (Vogt et al., 1996). These primers were manufactured
screened along with a further 28 patients and 50 controls by Genosys Biotechnologies (Europe) (Pampisford, UK).
using the multiplex PCR Y chromosome screening protocol PCR was carried out in duplicate in a 50 lL reaction
(Simoni et al., 1999). volume containing 150 ng of genomic DNA, 1.5 U Taq
polymerase, 0.2 mM each of dTTP, dCTP, dGTP, dATP,
100 ng each of oligonucleotide primers, 1.5 mM MgCl2
Materials and methods (3 mM for sY147 and 2 mM for sY272 and sY269) made up
in a ®nal concentration of 1 ´ PCR reaction buffer (10 mM
Patients
Tris-HCl pH 9.0, 50 mM KCl and 0.1% Triton X-100). All
A total of 78 men attending our fertility unit were
reagents were obtained from Promega (Madison, WI, USA).
analysed in this study. For each patient at least two complete
For all STS primers (except sY243, sY138, sY254, sY255,
semen analyses were performed based on the World Health
RBMY and SPGY) ampli®cation conditions commenced
Organisation (WHO, 1992) guidelines for semen analysis.
with an initial denaturation step of 3 min at 94 °C, followed
The patients were classi®ed as azoospermic (no sperm in the
by 35 cycles of 1 min denaturation at 94 °C, 1 min
ejaculate), severely oligozoospermic (sperm count <5 ´
annealing at temperatures ranging from 59 to 63 °C and
106 mL), oligozoospermic (sperm count <20 ´ 106 mL)
1 min extension at 72 °C and completed with a ®nal
and oligoasthenoteratozoospermia (sperm count <20 ´
elongation step at 72 °C for 5 min and cooling to 4 °C. For
106 mL, motility <40% and normal forms <40%). The
the remaining primer sets, the initial denaturation was 96 °C
karyotype was determined for each patient and those who
for 5 min and the denaturation step during each cycle was
were not 46XY were excluded from this study. Serum
96 °C for 1 min. PCR reaction products (15 lL) were
follicle-stimulating hormone (FSH), luteinizing hormone
separated on a 2% agarose gel (Roche-Boehringer Mann-
(LH) and testosterone levels were measured for each patient.
heim, Germany) by electrophoresis in 1X TBE

Ó 2001 Blackwell Science Ltd, International Journal of Andrology, 24, 31±36

 Molecular detection of Y chromosome microdeletions 33

(Tris±borate/EDTA buffer). The products were visualized patient yielded a weak PCR product when RBMY1 primers
by ethidium bromide staining followed by exposure to were used. This was resolved when a more stringent PCR
ultraviolet light. DNA from a normal male control (positive assay was set up, indicating that the primers may have been
control) and female (negative control) were included in each amplifying pseudogenes or other family members. Many
experiment, along with a water control (no DNA) to ensure RBMY genes are present on both arms of the Y chromo-
accuracy of results. some, however, it is thought that the functional genes only
reside within AZFb (Elliott et al., 1997) and these are
Multiplex PCR. These patients were re-screened along with denoted RBMY1.
a further 28 patients and 50 normospermic men using the
multiplex PCR Y chromosome screening protocol described
by Simoni et al. (1999). Each multiplex reaction was carried Discussion
out in a 25 lL reaction volume containing 200 ng DNA, The AZFa deletion in patient A, included the genes
2 U Amplitaq (Roche Molecular Systems, Branchburg, NJ, USP9Y and DBY. The role of these genes in spermatogen-
USA), 5 pmol of each primer, 10% DMSO, 1.5 mM esis remains to be con®rmed and deletions in this area of the
dNTP's, 160 ng/lL BSA and the mulitplex buffer- Y chromosome have not been reported as often as DAZ and
16.6 mM (NH4)2SO4, 66.7 mM Tris-HCl pH 8.8, 6.7 mM RBMY associated deletions (Ferlin et al., 1999). The
MgCl2, 6.8 lM EDTA and 5 mM Beta-mercaptoethanol. microdeletion for patient A ranges from distal 5B to
Ampli®cation conditions (using PE 9600 thermocycler-PE, proximal 5D encompassing subinterval 5C on the long
Biosystems, CT) included an initial denaturation step of arm of the chromosome (Kent-First et al., 1999). A testicular
5 min at 94 °C, followed by 25 cycles of 30 sec denaturation biopsy sample was not available for this patient and therefore
at 94 °C, 30 sec annealing at 55 °C and 4 min elongation at we are unable to determine the presence or absence of
65 °C, ended by a last elongation step of 5 min and cooling spermatids. However, if we follow the hypothesis generated
to 4 °C. PCR products (15 lL) were separated on a 2% by recent experiments we would not expect to ®nd any
NuSieve (FMC BioProducts, Rockland, ME, USA) plus testicular germ cells, as it now appears that the loss of DBY
0.5% MP agarose gel in 1 X TBE. Again, a normal male and compounds the detrimental effects caused by the loss of
female DNA control plus a water control were included for USP9Y thereby leading to Sertoli cell only syndrome (Sun
each PCR experiment. et al., 1999; Foresta et al. 2000).
The AZFb deletion in patient B, included all the RBMY1
gene copies. Indeed, this AZFb deletion was found to range
Results from proximal 6A to distal 6D encompassing subinterval 6C
A total of 128 men (78 infertility patients and 50 controls) (Kent-First et al., 1999). As a result of the multicopy nature of
were screened for DNA microdeletions within the AZF this gene family, it has proved dif®cult to establish its precise
region of the Y chromosome. Two deletions were detected role in spermatogenesis. However, neither spermatozoa nor
in men with idiopathic azoospermia, one in AZFa region late spermatids were recovered from the biopsy sample taken
and one in AZFb region (Fig. 1). No deletions were found from this patient. This would suggest that this patients'
in those testiculopathies of known cause nor in the control testiculopathy could be directly related to the absence of
population. Patient A had a slightly elevated level of FSH, RBMY1 expression caused by the Yq deletion in AZFb.
whereas patient B had clinical parameters which were all Unfortunately, we were unable to obtain a blood sample from
within the normal range (Table 1). The AZFb deletion was the father or male sibling in either of these cases. Therefore,
detected in the original screening protocol and it was despite the probability that these are de novo deletions, we are
con®rmed using the multiplex protocol. The AZFa deletion unable to provide further data to support this theory. Frequent
was ®rst detected using the multiplex protocol. Following reports of DAZ deletions in infertile patients (Reijo et al.,
their detection, second round primers were used to con®rm 1995, 1996; Simoni et al., 1997), have led to it becoming the
both the deletions (Simoni et al., 1999). Further AZFa and strongest AZF candidate gene among the players. However, in
AZFb primers were also used to study the extent of the this study on Irish male infertility, no deletions were detected
deletions: sY81, sY89, sY165, sY85 (Vollrath et al., 1992); in the AZFc/DAZ region of the Y chromosome. Further-
`DEAD box' protein (DBY), ubiquitous TPR motif Y more, only two patients were found to possess a microdeletion
(UTY) (Kostiner et al., 1998) and USP9Y (Brown et al., in the AZF region of the Y chromosome. This percentage
1998) (Table 2). (3.6) of deletions in the idiopathic group is lower than many of
The AZFa deletion encompassed the AZFa candidate the previously published studies on selected patient groups
gene USP9Y. The deletion also includes another gene, (Foresta et al., 1998; Chang & Tsai, 1999). These results could
DBY, which resides in this region. USP9Y and DBY are be because of the small numbers in the study but they could
single copy genes and both have a homologue on the X also be a geographical circumstance.
chromosome (Lahn & Page, 1997). The deletion does not As Y chromosome screening is a relatively new area, there
encompass UTY, a further AZFa gene. The AZFb deleted has been much confusion as to which primer sets to include

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34 A. Friel et al.

Figure 1. Multiplex PCR showing the AZFa and AZFb deletions. Lanes 1±5: Multiplex A. Lane 1: Patient B (AZFb deletion); Lane 2: Patient A (AZFa deletion);
Lane 3: Male control; Lane 4: Female control; Lane 5: No DNA control; Lanes 6±10: Multiplex B. Lane 6: Patient B; Lane 7: Patient A; Lane 8: Male control; Lane 9:
Female control; Lane 10: No DNA control. Molecular weight marker (100 bp) is shown. Multiplex A ± ZFY*: 495 bp, SRY*: 472 bp, sY254: 400 bp (AZFc), sY86:
320 bp (AZFa), sY127: 274 bp (AZFb). Multiplex B ± ZFY: 495 bp, SRY: 472 bp, sY84: 326 bp (AZFa), sY134: 301 bp (AZFb), sY255: 126 bp (AZFc). *ZFY
appli®es both male and female DNA and SRY ampli®es only male DNA.

in the initial screening programs. This study evaluated two sY155, sY272 and sY269 should not be included in Y
methods and during the course of the study, it became chromosome screening experiments (Simoni et al., 1999 &
apparent that several primer sets should not be used in Y private communication at EAA/EMQN Best Practice
chromosome screening experiments because of their repet- meeting on Y chromosome microdeletions. L'Aquila, Italy,
itive/polymorphic nature leading to non-speci®c ampli®ca- 2000). Although the initial in-house screening protocol did
tion products. Speci®cally, sY138, sY139, sY143, sY153, detect our AZFb deletion, it was a much more laborious

Table 1. Clinical data of patients with microdeletion in Yq11

Sperm conc. FSH LH Testosterone

Patient Age (106/mL) (mIU/mL) (mIU/mL) (nmol/L) Karyotype CFTR

A 36 Azoospermic 20.3 3.9 17.4 46XY Normal

B 37 Azoospermic 10.9 9.2 15.4 46XY Normal
Normal hormone 1±15 1±13 15±21
range

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 Molecular detection of Y chromosome microdeletions 35

Table 2. Characterization of Y chromosome deletions found in two Based on our experiments, we recommend that the
patients laboratory guidelines outlined by Simoni et al. (1999) should
be used as a ®rst step screening protocol. The use of this
STS Patient A STS Patient B protocol will enable the detection of over 90% of the
primer AZFa deletion primer AZFb deletion deletions in the three AZF regions. Once a deletion is
detected, the extent of the deletion can be determined using
sY81 + sY135 + the second choice primer sets. Finally, if one wishes to
sY82 + sY114 ± determine the presence/absence of the AZF candidate genes,
sY83 + sY127 ± primer sequences are available for each of the candidate
sY86 ± sY134 ) genes (Reijo et al., 1995; Vogt et al., 1996; Brown et al.,
sY85 ± sY139 ) 1998). This multiplex PCR system represents a rapid
sY84 ± sY143 ) screening test that can provide valuable information to a
uspq ± RBM1 ) cohort of infertile couples planning to undergo fertility
sY87 ± sY152 + treatment.
DBY ± sY220 +
UTY + sY150 +
sY165 + Acknowledgements
sY88 + We thank Dr David Barton and Dr David Bailey of the
sY89 + National Centre for Medical Genetics, Our Lady's Hospital
for Sick Children, Crumlin, Dublin, for providing us with
+PCR product is present. control DNA samples and for all their helpful advice. We
)PCR product not detected. also thank Sarah Cahill and Grainne Cashin for their help in
the establishment of the multiplex PCR in this laboratory.
method compared with the rapid and simple multiplex Many thanks to the European Academy of Andrology and
method which yielded the same result. the European Molecular Genetics Quality Network for their
help and support.

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