Gonadotropin and Testosterone hormone’s serum levels and partial deletions in the AZFc region in Iranian oligozoospermia infertile males

doi:10.4236/health.2011.39097

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Vol.3, No.9, 566-570 (2011)

doi:10.4236/health.2011.39097

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.

ABSTRACT

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

1. INTRODUCTION

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

567567

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.

2. MATERIAL & METHODS

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

Assay.

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-

trols.

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.

3. RESULTS

The FSH and LH levels in oligozoospermic group

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568

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-

tions.

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 >

0.05).

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).

4. DISCUSSION

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.

STS

AZF Production Size(bp) sequence

sY1191 AZFc 385 5’-CCAGACGTTCTACCCTTTCG-3

5’-GAGCCGAGATCCAGTTACCA-3'

sY1291 AZFc 527 5’-TAAAAGGCAGAACTGCCAGG-3'

5’-GGGAGAAAAGTTCTGCAACG-3'

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

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may be due to limitation in sampling or oligozoo spermia

classification.

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-

eration.

5. ACKNOWLEDGEMENTS

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.

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