Vol.3, No.9, 566-570 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Gonadotropin and Testosterone horm one’s serum levels
and partial deletions in the AZFc region in Iranian
oligozoospermia infertile males
Nasser Salsabili1*, Reza Mirfakhraei2, Maryam Montazeri3, Mitra Ataei2, Paricheher Yaghmaei2,
Gholamreza Pourmand4
1IVF Department of Mirza Kouchak Khan Hospital, Rehabilitation Faculty, Tehran University of Medical Sciences, Tehran, Iran;
*Corresponding Aut hor: dr.salsabili56@yahoo.com
2Department of Biol o gy, Islamic Azad University (IAU), Science and Research Branch, Tehran, Iran;
3Department of Med i c a l Genetic, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran;
4Urology Research Center, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Received June 12th, 2011; revised July 26th, 2011; accepted August 10th, 2011.
To investigate the relation of luteinizing hor-
mone (LH), follicle-stimulating hormone (FSH)
and Testosterone serum levels with partial dele-
tions in the AZFc region in Iranian oligozoo-
spermia males. Material and methods: thirty
infertile oligozoospermia and 52 Iranian fertile
men included. The h ormonal assays were meas-
ured by the Radioimmunoassay (RIA). Multiplex
polymerase chain reaction (M-PCR) using eight
sequence-tagged site (STS) markers were mea-
sured on the Yq11 chromosome. Results: The
mean of FSH and LH levels in all oligozoo-
spermia males were higher than fertile men (p <
0.001) and testosterone was lower significantly
(p < 0.001). Five p atient s showed partial deletions
in AZFc region (four had gr/gr and one had
b2/b3 deletions). Six fertile men showed partial
deletions (five gr/gr and one b2/b3) with higher
level of FSH, LH in their group (p < 0.05). Con-
clusion: According to high incidence of partial
deletions in the AZFc region among Iranian oli-
gozoospermia males, hormonal assay and mo-
lecular screening should be advised before
considering for ART treatments.
Keywords: Oligozoospermia; Gonadotropin
Hormone; AZFc Partial Deletion
Male fertility is the end results of numerous and var-
ied processes which all must act in perfect concert to
allow the deposition of healthy sperm in to the female
reproductive tract. If any of these co-dependent steps is
faulty, infertility may result.
At least 30% - 50% of co uples with p rimary infertility
will have a male related problem as the sole or partial
reason for their infertility. Germ cell development is
dependent on the balanced endocrine interplay of the
hypothalamus, the failure of pituitary to secret FSH and
LH will result in disruption of testicular function lead ing
to infertility [1-3]. In addition, Yq, long arm of Y chro-
mosome, contains at least one gene that is responsible
for spermatogenesis [4]. Several genes are located in the
azoospermia factor region (AZF) on four major intervals
defined as AZFa, AZFb, AZFc and AZFd [5-8]. Partial
deletions of the AZFc region are reported as a significant
risk factor for oligozoospermia. AZFc, the most fre-
quently known genetic causes of impaired spermato-
genesis, is mapped between deletion intervals 6C and 6E
on Y chromosome.
Deletion in this region is found in 2% - 10% of azoo-
spermic or severely oligozoospermic men [5,8]. The
AZFc region removes 3.5 Mb and contains seven gene
families which have an important role in spermatogene-
sis [9]. The existence of the three subdeletions; namely
gr/gr, b1/b3 and b2/b3 have been confirmed by using the
new molecular markers [10].
The AZFc region is occupied halfly by the gr/gr type,
which spans 1.6 Mb; it is thought to be a risk factor for
spermatogenic failure [11]. It is found that the direct
homologous recombination causes this kind of the sub-
deletion which also accounts for another type, b1/b3
deletion (1.6 Mb) [10,11]. An inversion event is respon-
sible for two other types which are belonging to the 2nd
class. The gr/rg inversion is fo llowed by a b2/b3 deletio n
(1.8 Mb), while the b2/b3 inversion is followed by a gr/gr
deletion (1.8 Mb) [12] or g1/g3 delet i o n (2. 2 Mb ) [1 1] .
N. Salsabili et al. / Health 3 (2011) 566-5 70
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
The aim of this study is to investigate Testosterone,
FSH, LH serum levels and the prevalence of partial de-
letions in the AZFc region (gr/gr, b1/b2, and b2/b3) in
Iranian oligozoosperm ia males.
Thirty infertile oligozoospermia with at least 3 year’s
duration of infertility referred from Mirza Kouchek
Khan hospital and Koasar fertility an d impotency center.
In all the oligozoospermia cases sperm concentration
was (<20 mil/ml) with normal karyotypes. Female part-
ners were examined for their fertility using hormonal
evaluation for ovarian function and laparoscopy for the
confirmation of tubal patency. Only those couples where
the male partner was found to be oligozoospermia and
the female was normal selected for the study. Fifty two
normal fertile men with normal karyotypes and sperm
concentration (<20 million/ml) were selected as control
group. The hormonal assays were measured by the Ra-
dioimmunoassay (RIA), and for the presence of eight
sequence-tagged site (STS) markers on the Yq11 chro-
mosome, Multiplex polymerase chain reaction (M-PCR)
was used. An informed written consent was obtained
from all subjects for molecular screening and hormonal
2.1. Specimen Collection
10 ml of peripheral blood was collected in a sterile
tube containing ethylene diamine tetra acetate (EDTA)
as an anticoagulant and divided into two parts:
1) 7 ml was centrifuged (2500 g rpm) for 5 minutes
and serum was stored at –20˚C for hormonal assay.
2) 3 ml was used for DNA extraction.
2.2 Assay Hormones
Serum levels of luteinizing hormone (LH), follicle-
stimulating hormone (FSH) and testosterone were col-
lected early in the morning and evaluated at the Mirza
kouch khan hospital pathology refere nce services.
In all subjects, serum levels of LH were measured by
radioimmunoassay kit (Amersham Corp., Arlington
Heights, Illinois, USA).Reference ranges were 4 to 23
mIU/ml. Similarly, serum levels of FSH were deter-
mined by radioimmunoassay kit. Reference ranges were
10 to 76 mIU/ml. and for serum levels of testosterone
were determined by radioimmunoassay kit (Amersham
Corp., Arlington Heights, Illinois, USA) with reference
ranges were 0.2 to 11 ng/ml.
2.3. Molecular Screening
Genomic DNA was extracted from peripheral blood
using Tadbir Azma DNA extraction kit (Tadbir Azma,
Tehran, Iran). First of all, we investigated classical AZF
microdeletions; consequently, the polymerase chain re-
action was used to detect 8 different sequence tag sites
(STS) corresponding to different AZF loci according to
the European Academy of Andrology (EAA), the Euro-
pean Molecular Genetics Quality Network (EMQN) (13 ).
These included sY84 and sY86 (AZFa); sY127and
sY134 (AZFb); sY254 and sY255 (AZFc); and sY145
and sY153 (AZFd). These STSs were analyzed using
three different M-PCRs:
Multiplex 1: sY86, sY127, sY254, and ZFY;
Multiplex 2: sY84, sY134, sY255, and SRY;
Multiplex 3: sY145, SY154, and SRY.
Two primers (ZFY, SRY) were used as internal con-
trols for M-PCR. Primers sequences with additional de-
tails are provided in a previous study [14]. The reaction
was comprised of 50 - 100 ng o f genomic DNA in 25 ul
PCR reactions, 10 mM Tris HCl (pH 8.3), 50 mM KCl,
1.5 mM MgCl2, 2 mmol dNTPs, 10 pmol of each prim-
ers and 1unit of Taq DNA polymerase. Initial denature-
tion step at 94˚C for 5min, followed by 28 cycles of
94˚C for 30 sec, 56˚C for 30 sec and 72˚C for 45 sec
ending with a final extension for 5 min at 72˚C.
The PCR products were separated on 2% agarose gels
stained with ethidium bromide, or 10% acrylamide gel
prepared in 0.5X TBE. In all reactions, DNA from a
normal woman and a normal man were included as con-
The samples without classical AZF deletions were in-
vestigated in order to find gr/gr, b1/b2, and b2/b3 poly-
morphisms. The presence or absence of these subdele-
tions was analyzed using M-PCR. Two STS markers
were detected for the study and were tested in one
M-PCR: SRY, SY1191, and SY1291.The sequence of
the primers is shown in Tab le 1. Reaction condition and
PCR program was similar to the program used for detec-
tion of classical AZF. The PCR products were separated
on 2% agarose gel. The absence of amplification of
sY1291 represented a gr/gr deletion and the absence of
sY1191 product showed b2/b3 deletion. Deletion of the
two markers was defined as b1/b3 deletion.
2.4. Statistical Analysis
Independent Student’s t-Test was used to compare the
numerical values of blood plasma hormonal assay in
both groups. All procedures were carried out using SPSS
for Windows V11.5. The type 1 error was set at 0.05 for
testing the hypothesis.
The FSH and LH levels in oligozoospermic group
N. Salsabili et al. / Health 3 (2011) 566-5 70
Copyright © 2011 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
men were significantly increased (p < 0.001). The mean
value of testosterone levels significantly decreased in
oligozoospermic men in compare with fertile men (p <
0.001) (Table 2).
Totally, only 1 ou t of 30 oligozoo spermic men (3.3%)
with spermatogenesis impairment was found to have
classical AZFb deletion. The gr/gr, b2/b3 and b1/b3 sub-
deletions in all subjects were determined using M-PCR.
After having eliminated 1 classical AZFb deletions
from 30 oligozoospermic men, subdeletions in the AZFc
region; was found in 4cases (13.8%) gr/gr subdeletions,
1(3.4%) b2/b3 subdeletions and 0(0%) b1/b3 subdele-
In contrast, screening of 52 control fertile men re-
vealed 5(9.6%) gr/gr subdeletions, 1(1.9%) b2/b3 sub-
deletions and 0(0%) b1/b3 subdeletions. There was no
significant difference in the frequency of the gr/gr and
b1/b3 subdeletions between the patients and controls (p >
The mean value for FSH, LH and testosterone con-
centration in normal fertile men was 7.48 mIU/ml, 6.73
mIU/ml and 3.93 ng/ml respectively (Table 2). In 4 oli-
gozoospermia infertile men with gr/gr subdeletion; FSH,
LH was higher and clearly subnormal testosterone is
shown (Table 3).
Hormonal assay of the FSH, LH and testosterone is
useful in the management of the male in fertility, and for
initiation of spermatogenesis and maturation of sper-
matozoa [3,15]. In the oligozoospermic men higher con-
centration of FSH is considered to be a reliable indicator
of germinal epithelial damage or primary testicular fail-
ure [7,8]. Moreover, different studies have reported that
serum FSH and LH were significantly above the mean
value in infertile oligozoospermic men with Y-chromo-
some microdeletion [6,16].
In the present study, the same results were obtained
and FSH, LH in oligozoospermia patients were signify-
cantly higher than fertile group and testosterone values
was lower (p < 0.001), this indicates primary testicular
failure in our oligozoospermia men. Five oligozoosper-
mia patients showed partial deletions in AZFc region (4
had gr/gr and one had b2/b3 deletions).
Combination of gr/gr subdeletion and sub normal tes-
tosterone serum level was not explained by any other
investigator s [17,20]. In opposite of some pr evious stud-
ies [17,18,20], these values were significantly different
in infertile oligozoospermia males with AZFc subdele-
tions. Different results obtained from the present study
Table 1. STS markers and primer sequences used for screening gr/gr, b1/b2, and b2/b3 polymorphisms.
AZF Production Size(bp) sequence
Table 2. Serum FSH, LH and testosterone levels in fertile and oligozoosperic men.
Hormonal levels mean(SD)
Group No. subjects
FSH (miu/ml) LH (miu/ml) Testosterone (ng/ml)
Fertile men 52 7.48 ± 4.21
Range (2.50 - 18) 6.73 ± 3.73
Range (2.10 - 18.30)3.93 ± 1.09
Range (2.20 - 8.25)
Infertile men
(Oligozoospermia) 30 20.28 ± 11.59*
Range (4.50 - 40) 14.67 ± 7.29*
Range (3.80 - 35) 3.11 ± 1.67*
Range (0.80 - 8.50)
*p < 0.001.
Table 3. Clinical features of 4 oligozoospermia men with gr/gr subdeletion.
Patient No Sub deletion FSH mIU/mlLH mIU/mlTestosterone ng/mlSperm count mil/ml
7 gr/gr 36 15 2.60 1 × 106
8 gr/gr 37 20 2.60 1 × 106
22 gr/gr 40 17 1.50 5 × 106
26 gr/gr 37 22 2.50 3 × 106
N. Salsabili et al. / Health 3 (2011) 566-5 70
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
may be due to limitation in sampling or oligozoo spermia
The FSH producing by pituitary is affected by the In-
hibin B feed-back control [17], which is produced by the
sertoli cells. In the patients with the germinal cells func-
tion deficiency, the inhibin rate will be reduced while the
FSH rate will be increased. Indeed, this is a possible
interaction between the germinal and sertoli cells. The
serum FSH rate is related to the epithelium function of
the germinal cells [18]. Additionally, It is commonly
approved that complete deletion of Y chromosome is a
determining factor of sever spermatogenesis disorder.
And the relationship between partial deletions of AZFc
region and male sterility is to be discussed [6,19].
Furthermore many studies have been carried out for
detecting the relationship between subdeletions and ste-
rility in recent years [13,14,18,20]; however the results
in different populations were not iden tical. In the present
survey no significant differences were detected for the
frequencies of gr/gr and b2/b3 deletions in the studied
groups (p > 0.05).
According to the surveys carried out on a population
in East Asia the frequency of gr/gr deletion in terms of
prevalence percentage were estimated respectively as
10.3% and 10.1% [12,21], that is comparable with our
result. The only deletion which is created through gr/gr
recombination is the significant cause of spermatogene-
sis failure [14].
Men with AZFc subdeletion are somatically healthy
with most likely have useable sperm, stable sperm pro-
duction over time and a good chance to experience bio-
logical paternity. However, their sons would also be
AZFc subdeletion carrier [14,16,19,20]. In our study,
b2/b3 frequency equated to 3.4% showing no significant
difference in comparison to the frequency of these sub-
deletions in fertile po pulatio n. In all th e prev iou s studies,
no significant relationship was detected between the
prevalence of this deletio n and sterility wh ich can be due
to the racial differences among populations [19,21].
Furthermore the present study may be in conflict with
similar studies or other surveys [14,19]. The role of
b2/b3 deletions in male sterility is still unknown becau se
of its low frequency. It seems that the role of DAZ3/4
deletion in male sterility is limited to the b2/b3 subdele-
tion [14,22]. Therefore; more studies should be done for
detecting the role of gr/gr deletion and b2/b3 subdeletion
in the context of Y haplogroups. However, there are dif-
ferent factors which influence the results of the studies.
Observed differences in the deletions of AZFc region in
various studies can be due to diverse demographic com-
position, differences in criteria for selection of patients,
environmental imp acts and also the categorization method
applied for the definition of oligozoospermia due to dif-
ferent classification depend on the sperm count and
characteristics [22]. Vogt considered the oligozoospermic
criteria less than 2 mil/ml sperm in his study [7]. In
Other investigations oligozoospermic criteria was con-
sidered less than one mil/ml, which obviously can highly
influence the results of the studies [23,24]. In our study
less than 20 mil/ml was considered as oligozoospermia,
which may cause significant increase in the amount of
gonadotropin hormones and decreased testosterone se-
rum level in compare to normal fertile group. Our study
revealed that FSH, LH, and testosterone evaluation is
useful in the management of male infertileity, but the
couples with a high risk transferring of a main genetic
defect recommended performing molecular screening of
AZF partial or subdeletion before any decision for gen-
The authors are grateful to Sina Urology Centre of Tehran Univer-
sity of Medical Sciences and Koasar Fertility and Impotency Center for
their kind collaboration.
[1] Mittal, R.D., Singh, G., Srivastava, A., Pradhan, M., Ke-
sari, A., Makker, A. and Mittal, B.Y. (2004) Chromosome
micro-deletions in idiopathic infertility from Northern
India. Annales de Génétique, 47, 331-337.
[2] Lynch, M., Cram, D.S., Reilly, A., O’Bryan, M.K. and
Baker, H.W.G. (2005) The Y chromosome gr/gr subdele-
tion is associated with male infertility. Molecular Human
Reproduction, 11, 507-512. doi:10.1093/molehr/gah191
[3] Madhukar, D. and Rajender, S. (2009) Hormonal treat-
ment of male infertility: Promises and Pitfalls. Journal of
Andrology, 30, 95-112.
[4] Raicu, F., Popa, L., Apostol, P., Cimponeriu, D., Danl, L.,
Ilinca, E., et al. (2003) Screening for microdeletions in
human Y chromosome AZF candidate genes and male
infertility. Journal of Cellular and Molecular Medicine.,
7, 43-48. doi :10.1111 /j.1582-4934.2003.tb00201.x
[5] Tiepolo, L. and Zuffardi, O. (1976) Localization of fac-
tors controlling spermatogenesis in the nonfluorescent
portion of the human Y chromosome long arm. Human
Genetics, 34, 119-124. doi:10.1007/BF00278879
[6] Reijo, R., Lee, T.Y., Salo, P., Alagappan, R., Brown, L.G.,
Rosenberg, M., et al. (1995) Diverse spermatogenic de-
fects in humans caused by Y chromosome deletions en-
compassing a novel RNA-binding protein gene. Nature
Genetics, 10, 383-393. doi:10.1038/ng0895-383
[7] Vogt, P.H., Edelmann, A., Kirsch, S., Henegariu, O.,
Hirschmann, P., Kiesewetter, F., et al. (1996) Human Y
chromosome azoospermia factors (AZF) mapped to dif-
ferent subregions in Yq11. Human Molecular Genetics, 5,
933-943. doi:10.1093/hmg/5.7.933
[8] Kent-First, M., Muallem, A., Shultz , J., Pryor, J., Roberts,
N. Salsabili et al. / Health 3 (2011) 566-5 70
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
K., Nolten, W., et al. (1999) Defining regions of the
Y-chromosome responsible for male infertility and identi-
fication of a fourth AZF region (AZFd) by Y-chromosome
microdeletion detection. Molecular Reproduction and De-
velopment, 53, 27-41.
[9] Ferlin, A., Tessari, A., Ganz, F., Marchina, E., Barlati, S.,
Garolla, A., et al. (2005) Association of partial AZFc re-
gion deletions with spermatogenesis impairment and
male infertility. Journal of Medical Genetics, 42,
209-213. doi:10.1136/jmg.2004.025833
[10] Repping, S., Skaletsky, H., Brown, L., Van Daalen, S.K.,
Korver, C.M., Pyntikova, T., et al. (2003) Polymorphism
for a 1.6-Mb deletion of the human Y chromosome per-
sists through balance between recurrent mutation and
haploid selection. Nature Genetics, 35, 247-251.
[11] Fernandes, S., Huellen, K., Goncalves, J., Dukal, H.,
Zeisle, J., Souse, M., et al. (2002) High frequency of
DAZ1/DAZ2 gene deletions in patient with severe oli-
gozoospermia. Molecular Human Reproduction, 8,
286-298. doi:10.1093/molehr/8.3.286
[12] Repping, S., Saskia, K.M., Van Daalen, S.K., Korver , C.
M., Brown, L.G., Marszalek, J.D., et al. (2004) A family
of human Y chromosomes has dispersed throughout
northern Eurasia despite a 1.8-Mb deletion in the azoo-
spermia factor c region. Genomics, 83, 1046-1052.
[13] Simoni, M., Bakker, E. and Krausz, C. (2004)
EAA/EMQN best practice guidelines for molecular di-
agnosis of Y-chromosomal microdeletions. State of the
ART 2004. International Journal of Andrology, 27,
240-249. doi:10 .1111/j.1365-2605.2004.00495.x
[14] Mirfakhraie, R., Mirzajani, F., Mahdi Kalantar, S., Mon-
tazeri, M., Salsabili, N., Pourmand, G.R. and Houshmand,
M. (2010) High prevalence of AZFb microdeletion in
Iranian patients with idiopathic non-obstructive azoo-
spermia. Indian Journal of Medical Research, 132,
[15] Babu, S., Sadhnanl, M.D., Swama, M., Padmavathi, P.
and Reddy, P.P. (2004) Evaluation of FSH, LH and tes-
tosterone levels in different subgroups of infertile males.
Indian Journal of Clinical Biochemistry, 19, 45-49.
[16] Ferlin, A., Arre di, B.A., E Spe ltra., et al. (2007) Molecu-
lar and clinical characterization of Y chromosome mi-
crodeletions in infertile men: A 10-year experience in It-
aly. The Journal of Clinical Endocrinology & Metabo-
lism, 97, 762-770.
[17] Nowroozi, M.R. and Radkhah, K. (2008) Serum inhibin
B concentration as a prognostic factor for prediction of
sperm retrieval in testis biopsy of patients with azoo-
spermia. Archives of Iranian Medicine, 11, 54-56.
[18] Kumar, R., Dada, R. and Gupta, N.P. (2006) Serum FSH
levels and testicular histology in infertile men with non
obstructive azoospermia and Y chromosome microdele-
tions. Indian Journal of Urology, 22, 332-336.
[19] Claudia, C.M.B., Zuccherato, L.W., Bastos-Rodrigues, L.,
Santos, F.R. and Pena, S.D.J. (2006) No association
found between gr/gr deletions and infertility in Brazilian
males. Molecular Human Reproduction, 12, 269-273.
[20] Oates, R.D., Silber, S., Brown, L.G. and Page, D.C.
(2002) Clinical characterization of 42 oligospermic or
azoospermic men with microdeletion of the AZFc region
of the Y chromosome, and of 18 children conceived via
ICSI. Human Reproduction, 17, 2813-2824.
[21] Zhang, F., Li, Z., Wen, B., Jiang, J., Shao, M., Zhao, Y.,
et al. (2006) A frequent partial AZFc deletion does not
render an increased risk of spermatogenic impairment in
East Asians. Annals of Human Genetics, 70, 304-313.
[22] Olive, A., Spira, A. and Multigner, L. (2001) Contribu-
tion of environmental factors to the risk of male infertil-
ity. Human Reproduction, 16, 1768-1776.
[23] Carrara, R.C.V., Yamasaki, R., Mazucatto, L.F., Veludo,
M.A.L., et al. (2004) Somatic and grem cell cytogenetic
studies and AZF microdeletion screening in infertile men.
Genetics and Molecular Biology, 27, 477-482.
[24] Hellani, A., Al-Huellen, S., Iqbal, M. and Coskun, S.
(2006) Y chromosome microdeletion in infertile men
with idiopathic olig- or azoospermia. Journal of Experi-
mental & Clinical Assisted Reproduction, 3, 1.