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- Clinical Experience -
Seminal plasma anti-Müllerian hormone level correlates with
semen parameters but does not predict success of testicular
sperm extraction (TESE)
Taymour Mostafa1, Medhat K.
Amer1,2, Guirgis
Abdel-Malak2,3, Taha Abdel
Nsser1,2, Wael Zohdy1,2, Shedeed
Ashour1, Dina El-Gayar4, Hosam H.
Awad1
1Andrology & Sexology Department, Faculty of Medicine, Cairo University, Cairo 11553, Egypt
2Adam International Clinic, Mohandesseen, Giza 12411, Egypt
3Artificial Insemination and Embryo Transfer Department, Reproductive Research Institute, Alharam, Giza 12111, Egypt
4Chemical Pathology Department, Faculty of Medicine, Cairo University, Cairo 11553, Egypt
Abstract
Aim: To assess seminal plasma anti-Müllerian hormone (AMH) level relationships in fertile and infertile
males. Methods: Eighty-four male cases were studied and divided into four groups: fertile normozoospermia
(n = 16), oligoastheno-teratozoospermia
(n = 15), obstructive azoospermia (OA) (n =
13) and non-obstructive azoospermia (NOA) (n =
40). Conventional semen analysis was done for all cases. Testicular biopsy was done with histopathology and
fresh tissue examination for testicular sperm extraction (TESE) in NOA cases. NOA group was subdivided according to
TESE results into unsuccessful TESE (n = 19) and successful TESE
(n = 21). Seminal plasma AMH was estimated
by enzyme linked immunosorbent assay (ELISA) and serum follicular stimulating hormone (FSH) was estimated in
NOA cases only by radioimmunoassay (RIA).
Results: Mean seminal AMH was significantly higher in fertile group than
in oligoasthenoteratozoospermia with significance (41.5 ± 10.9 pmol/L
vs. 30.5 ± 10.3 pmol/L, P < 0.05). Seminal AMH
was not detected in any OA patients. Seminal AMH was correlated positively with testicular volume
(r = 0.329, P = 0.005), sperm count
(r = 0.483, P = 0.007), sperm motility percent
(r = 0.419, P = 0.021) and negatively with sperm abnormal
forms percent (r = _0.413, p
= 0.023). Nonsignificant correlation was evident with age
(r = _0.155, P = 0.414) and plasma FSH (
r = _0.014, P = 0.943). In NOA cases, seminal AMH was detectable in 23/40 cases, 14 of them
were successful TESE (57.5%) and was undetectable in 17/40 cases, 10 of them were unsuccessful TESE (58.2%).
Conclusion: Seminal plasma AMH is an absolute testicular marker being absent in all OA cases. However, seminal
AMH has a poor predictability for successful testicular sperm retrieval in NOA
cases. (Asian J Androl 2007 Mar; 9: 265_270)
Keywords: seminal plasma; anti-Müllerian hormone; spermatogenesis; azoospermia; testicular sperm extraction
Correspondence to: Dr Taymour Mostafa, Andrology & Sexology
Deptment, Faculty of Medicine , Cairo University, Cairo 11553,
Egypt.
Tel: +20-1051-50297 Fax: +20-2363-2297
E-mail: taymour1155@link.net
Received 2006-04-08 Accepted 2006-11-06
DOI: 10.1111/j.1745-7262.2007.00252.x
1 Introduction
Anti-Müllerian hormone (AMH) or Müllerian
inhibiting substance (MIS) is produced by the Sertoli cells of
the prenatal testis and lasts throughout life [1]. In the
male embryo, it is responsible for regression of the
Müllerian duct. Its production does not cease after the
fetal period and it is present in the serum of adult men
[2]. Fénichel et al. [3] showed that high concentrations
of AMH were detectable in the seminal fluid of fertile
men but absent in all obstructive azoospermic specimens.
Fujisawa et al. [4] found that seminal AMH is a good
marker for Sertoli cell development and that it correlated
significantly with sperm concentration, testicular volume,
serum luteinizing hormone (LH) but not with serum
follicular stimulating hormone (FSH), testosterone or
estradiol. Al-Qahtani et al. [5] found that seminal AMH
concentrations in male factor infertility were not
significantly different from fertile men, where serum AMH was
lower in male factor infertility than that in fertile cases.
In non-obstructive azoospermia (NOA), minute foci
of spermatogenesis if present could be used
successfully for intracytoplasmic sperm injection (ICSI).
However, testicular sperm extraction (TESE) may not
always be successful in all NOA cases. Therefore,
determination of factors that can predict a successful sperm
recovery procedure can offer realistic expectations for
both the couple and the physician [6]. Different criteria
were suggested as predictive markers of TESE outcome;
clinical, laboratory or histopathological [7_8]. Several
markers have already been proposed, e.g. transferrin [9],
lactate dehydrogenase [10], insulin-like growth factor
(IGF)-1 [11] and inhibin B [12]. However, no one could
foretell about the presence of a spermatogenic focus
inside an NOA testis regarding TESE/ICSI processes,
especially in border-line conditions such as small-sized
testicles and aged female partner.
In NOA, Fénichel et al.
[4] found that undetectable seminal AMH was associated mostly with lack of
testicular spermatozoa retrieval, while detectable seminal AMH
was associated with persistent spermatogenesis in 70%
of cases with a negative predictive value of 83%.They
suggested that seminal AMH may represent a non-invasive
marker of spermatogenesis in NOA, which may indicate
the likley success of testicular sperm recovery before ICSI.
In the present work, we assessed the diagnostic value
for seminal plasma AMH estimation in different groups
of male infertility.
2 Materials and methods
2.1 Patients
Eighty four male cases (mean age 35.5 ± 7.6 years,
range 25_37 years) were recruited consecutively, after
consent and Institutional Review Board (IRB) approval,
from the Andrology & Sexology Department, Faculty
of Medicine Cairo University Hospital (Cairo, Egypt)
and Adam International Clinic (Giza, Egypt). They were
divided into four groups: fertile normozoospermia
(n=16), oligoasthenoteratozoo-spermia
(n = 15), obstructive azoospermia (OA) (n
= 13) and NOA with normal 46 XY karyotype (n
= 40). They were subjected to history taking,
general and genital examinations and semen analysis.
Serum FSH assays and testicular biopsy were performed for
NOA cases. OA cases were selected from those
scheduled for epididymo-vasostomy operations or
post-vasectomy cases. The NOA group was further subdivided
according to TESE trials into unsuccessful and
successful TESE subgroups.
2.2 Samples
Semen samples were collected after a period of sexual
abstinence for 4 to 5 days. All samples were retrieved in
a specimen container and allowed to liquefy for 30
minutes over a slide warmer at 37ºC. A routine semen
analysis was then performed according to World Health
Organization (WHO) guidelines [13], azoospermia was
verified after two semen analyses and centrifugation.
Semen samples were centrifuged at 100 × g
for 15 min and the supernatant seminal plasma was stored in
polypropylene tubes at _80ºC until used.
2.3 Testicular biopsy and histopathology
Under anesthesia, a small incision (0.5 cm) was done
in the least apparent vascular area to expose testicular
tissue. Gentle pressure was applied to extrude a piece of
testicular tissue (0.5 × 0.5 cm) microsurgically, excised
with sharp scissors and added directly to Bouin's
fixative solution. Histological sections were prepared from
the paraffin blocks and stained by hematoxylin and eosin
stain, then examined under the light microscope.
Testicular sections were classified according to the
prevailing pattern of spermatogenic cells into: normal
spermatogenesis pattern (> 50% of tubules had full
spermatogenesis up to spermatozoa), hypospermatogenesis (little
number of full spermatogenesis tubules), spermatogenic
arrest (none of the tubules with sperms but with
spermatids or primary spermatocytes), Sertoli cell only (SCO)
(tubules had no germ cells and lined with Sertoli cells
only) and tubular atrophy (no seminiferous epithelial cells
with tubular sclerosis).
2.4 Fresh testis examination
During testis biopsy procedure, a piece of tissue was
put in a Petri dish (Falcon cat no. 300, Becton Dickson,
Lakes, NJ, USA) containing 1 mL
N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) buffered
Earl's salt solution and transferred immediately into the
adjacent laboratory. The tissues were minced under
laminar flow and then examined under an inverted
microscope × 400 for the presence of testicular spermatozoa.
If no spermatozoa were seen after checking the whole
dish, the contents were transferred into a 5-mL falcon
tube after removing the testicular debris and centrifuged
at 1 800 × g for 5 min, then the supernatant was
removed and the pellet was rechecked for spermatozoa
[14]. The result was interpreted when sperms were seen
or not as successful or unsuccessful TESE trials.
2.5 AMH and FSH estimation
AMH was measured (pmol/L) in seminal plasma after centrifugation by ELISA method (DSL, Webster,
TX, USA). In the assay, standards, controls, and
serum samples were incubated in microtitration wells
coated with anti-AMH antibody. After incubation and
washing, the wells were treated with secondary
anti-AMH detection antibody labeled with biotin. After a
second incubation and washing step, the wells were
incubated with streptavidin-horseradish peroxidase
(HRP). After a third incubation and washing step, the
wells were incubated with the substrate
tetramethylben-zidine (TMB). An acidic stopping solution was then
added and the degree of enzymatic turnover of the
substrate was determined by dual wavelength absorbance
measurement at 450 and 620 nm. The minimum detection limit was 3.0 pmol/L with an intra-assay coefficient
of variation (CV) 2.4_4.6%, and an inter-assay CV
4.8_8.0%. Serum FSH (mIU/mL) was estimated using
radioimmunoassay (RIA) with sensitivity of 0.06 mIU/mL, an
intra- and inter-assay CV < 10%.
2.6 Statistical analysis
Numerical data were expressed as mean ± SD and
range. Comparisons were performed by paired
t-test. Correlations were tested by Spearman's test.
Comparisons and correlations were considered statistically
significant if P < 0.05.
3 Results
Normozoospermic fertile men showed mean testicular volume of 16.8 ± 1.6 mL, mean sperm
concentration 66.2 ± 45.1 ×
106/mL, mean sperm motility 56.9 ± 6.0%, mean sperm abnormal forms 26.3 ±
5.0%. oligoasthe-noteratozoospermic men had mean
testicular volume of 13.1 ± 1.6 mL, mean sperm
concentration 3.9 ± 2.2 ×
106/mL, mean sperm motility 20.0 ± 8.5%,
mean sperm abnormal forms 74.7 ± 9.9%. Mean
seminal AMH in the fertile group was significantly higher
than that in oligoasthenoteratozoospermia (41.5 ± 10.9
pmol/L vs. 30.5 ± 10.3 pmol/L,
P < 0.05). Seminal AMH was not detected in
any OA case and was detected in 23/40 (57.5%) of NOA cases (Table 1).
Seminal AMH was correlated positively with testicular
volume (r = 0.32, P = 0.005), sperm count
(r = 0.483, P = 0.007), sperm motility percent
(r = 0.419, P =0.021) and negatively with sperm abnormal forms percent
(r = _0.413, P = 0.023). Nonsignificant correlation
was evident with age (r = _0.155, P = 0.414) and plasma
FSH (r = _0.014, P = 0.943).
NOA unsuccessful TESE cases (n = 19) had a mean
testicular volume of 9.9 ± 4.6 mL, mean serum FSH of
17.3 ± 9.2 mIU/mL and mean seminal AMH of 14.2 ±
13.5 pmol/L. AMH was detectable in 9/19 cases (47.4%)
(Table 2). NOA successful TESE cases (n = 21) had a
mean testicular volume of 12.6 ± 4.6 mL, mean serum
FSH 13.1 ± 10.9 mIU/mL and mean seminal of 23.2 ±
20.6 pmol/L. AMH was detectable in 14/21 cases (66.7%)
(Table 3).
5 Discussion
The testes express a high level of AMH, produced by
Sertoli cells, from early fetal life driven by the
transcription factors SOX9, SF1, WT1 and GATA4, until puberty,
when it is downregulated by testosterone and meiosis.
When the androgen negative effect is absent, FSH
increases the secretion of AMH. Serum AMH estimation
was found to be useful in evaluating children with
non-palpable gonads, with or without ambiguous genitalia. It
signals the existence of functional testicular tissue and
allows a distinction between gonadal dysgenesis and
dissociated tubular-interstitial dysfunction. Also, serum AMH
was found to be a useful marker in the follow-up of
males with precocious puberty or hypogonadotrophic
hypogonadism and patients with sex cord stromal tumours of the gonads
[15]. Isikoglu et al. [16] speculated that studying serum AMH might be more
advantageous than seminal plasma because of the presence of
seminal proteases. However, there was no difference in
serum of AMH levels between the studied groups.
In different studies, seminal AMH was related to
sperm motility and testicular spermatogenesis [3, 4, 17].
In the present study, fertile normozoospermic cases had
significantly higher AMH levels compared to infertile
oligoasthenozoospermia. In addition, seminal AMH was
positively correlated with testicular volume, sperm
concentration, sperm motility percent and negatively with
sperm abnormal forms percent. A nonsignificant
correlation was demonstrated with either the age or serum
FSH. Fujisawa et al. [4]
correlated seminal AMH significantly with sperm concentration, testicular volume,
serum LH but not with serum FSH, testosterone or estradiol.
Fallet et al. [17] suggested that AMH may have a
function in modulating motility.
In the obstructive azoospermia group (n =
13), AMH was not detectable in all cases confirming its testicular
origin. This corresponds with the study of Fénichel
et al. [3], who observed undetected seminal AMH in their nine
obstructive azoospermic studied cases. Total alpha
glucosidase was used for a long period to discriminate
epididymal obstruction, but it was not valid because of its
multi-producing sources [18]. Therefore, seminal AMH
may stand as one of the testicular markers. In addition,
AMH and alpha-glucosidase could be complementary in
their diagnostic values. Normal neutral glucosidase value
with no AMH would indicate no obstruction of the
epididymal tubule, which has a normal secretory function
(seminal vesicle secretes acidic glucosidase) showing
possibly a testicular defect or blockage of the efferent
ducts.
In NOA cases, seminal AMH was detectable in 23/40
cases, 14 of them were successful TESE, while it was
undetectable in 17 cases, 10 of them were unsuccessful
TESE. Fénichel et al. [3] detected seminal AMH in 9/23
NOA cases, 7 of them gave successful TESE, while it
was undetectable in 14 cases, 11 of them gave
unsuccessful TESE. This can give a clue as to why seminal
plasma AMH alone cannot be used to predict TESE results. Seminal AMH was detectable in cases with
advanced spermatogenesis, which corresponds with Baarends
et al. [19] who demonstrated that the presence
of developmentally more advanced spermatogenic cells
may enhance AMH secretion, related to the specific
stages of the seminiferous epithelium cycles.
NOA cases with unsuccessful TESE (n = 19) showed
no detection of seminal AMH in 10/19 (52.6%). Most of
these cases were of progressive testicular pathology, small
testicles size or with elevated FSH. Undetectable AMH
in these cases may follow failed spermatogenic activity
or hindered production by different antagonizing
testicular paracrine function. A definite threshold of AMH
secretion from intact Sertoli cells seems to be required in
order to appear in seminal plasma. Fénichel
et al. [3] showed that the dramatic decrease in seminal AMH was
associated with spermatogenic failure, suggesting a link
between AMH and spermatogenic hormones. In addition,
the possibility that decreased seminal AMH reflects a
primary alteration in Sertoli cell function that leads to
spermatogenic arrest must not be ruled out. Nine cases of
this group (47.4%) showed seminal AMH that may be because in unsuccessful TESE a lack of spermatozoa in
one or more sites does not guarantee a complete lack of
sperms in the whole testis [4, 20]. This sheds light on
the fact that seminal plasma AMH alone cannot foretell
the absence of spermatogenic focus inside the testis.
In conclusion, seminal plasma AMH may stand as one of the testicular markers. However, it has a poor
predictability for successful testicular sperm retrieval in
NOA cases.
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