Surgical indispensable part of clinical andrology. Sperms can

procedures of sperm retrieval in azoospermia


towards treatment of male infertility was revolutionised by the introduction of
novel method of ICSI in  1992 . (1 2). The use of surgical sperm retrieval from the
testis or epididymis associated to ICSI has given the chance for azoospermic
patients of fathering their own genetic children.  Asingle embryo can be injected into
an oocyte which resulted in normal fertilization, embryonic development and implantation.
source of sperm can be preferentially by ejaculation or from epididymis or
testis in azoospermic males irrespective of obstructive or non obstructive aetiology.
This process opened up an unique opportunity for azoospermic male for a
successful parenthood putting an end for their never ending agony of being
childless for the rest of their life. Surgical sperm recovery for ICSI has
become an indispensable part of clinical andrology.

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can still be retrieved in some cases of non obstructive azoospermia as the
testis persists to possess some isolated foci of active spermatogenesis. Pregnancies
resulting from surgically retrieved sperms were first published by 1993 and 1995


and azoospermia- A clinical dilemma.

pregnancy is plausible only when intravaginal ejaculation is successful.
Primarily this is possible by an intact ejaculatory mechanism which is a  neurologic reflex arc  which can be
disrupted through any type of trauma or disease causing damage to the CNS
and/or peripheral nerves. Ejaculation may be psychogenic or may result from spinal
cord injury or retroperitoneal lymph node dissection. These include 95% of
aetiology followed by diabetic neuropathy , multiple sclerosis, Parkinson
disease, bladder neck surgeries are less encountered causes.Ocassionally drugs
such as antidepressants ,antipsychotics and antihypertensive may cause
anejaculation. Since the outcome of medical treatment for anejaculation is
guarded penile vibratory stimulation or electroejaculation is considered the
first line management than offering surgical sperm retrieval since they are non-invasive
and does not require anaesthesia and they are successful ion 80% of the time 5.often epidydimal or
testicular sperm retrieval are offered initially as facilities of EVS or EJ may
not be available at all centres . Scrotal hematoma and risk of iatrogenic epidydimal
obstruction may preclude surgical sperm retrieval being offered as the first
line and should only be indicated when first line non-invasive management fail.
It is reasonably good to refer anejaculatory 
patients especially with spinal cord injuries to tertiary care centres
where assisted ejaculation and semen cryopreservation facilities are present.

sperm retrieval may be a treatment option for men with:


1.     An obstruction preventing sperm
release, due to injury or infection.

2.     Congenital absence of the vas deferens

3.     Vasectomy

4.     Non-obstructive azoospermia – the
testicles are producing such low numbers of sperm that they don’t reach the

Relative indictions:

1.     Sperms with increased DFI (DNA
fragmentation Index).

2.     Severe Oligoasthenoteratozoospermia(SOAT)

3.     Intractable Leucocytospermia.

In the first three conditions, sperm are  produced by the testes, but are unable to be
ejaculated primarily due to obstruction of transport  or congenital absence of the vasdeference.
They  can still ejaculate seminal fluid
but this fluid will not contain any sperm. It is possible to collect sperm
directly from the epididymis. most azoospermic patients suffer from primary
testicular ailure(60%).because these subset of patients do not show any clinical
signs of obstruction and they are often referred to as non obstructive
azoospermia NOA. However in few cases of azoospermia ensues due to hypogonadotropic
hypogonadism and not due to obstruction. These patients have an early
maturation arrest in spermatogenesis and adequate treatment with FSH and human
chorionic gonadotrophins might restore spermatogenesis.

Different types of SSR

1.     Percutaneous epididymal sperm
aspiration (PESA).

2.     Microsurgical epididymal sperm
aspiration (MESA).

3.     Testicular sperm aspiration (TESA).

4.     Testicular sperm extraction (TESE) –
single or multi-site.

5.     Microscope-assisted testicular sperm
extraction (MicroTESE).

All these
procedures can be safely  performed  as an outpatient basis with effective
preparation by local anesthesia or under general anesthesia.


Preoperative assessment.Pre-operative evaluation consists of recording   the
patient’s health and social history, conducting a physical examination,
developing a plan of anesthesia care and developing a safe plan for discharge
to home from the practice after recovery from the procedure.

1.Percutaneous approaches-(PESA / TESA).


1.Minimal training

2. No need for microscopic instruments

3.Percutaneous route more approachable

4.No need for sedation.

5.Lower complication rate




Low yield of sperm retrieved compared with open

1. Percutaneous epididymal sperm
aspiration (PESA)

PESA is a needle aspirate of the head of the
epididymis for attempted retrieval of more mature, motile sperm. Glina and
colleagues reported a sperm retrieval rate of 82% patients who underwent PESA,
while no complications were reported 6, while
the  complication rate of PESA was 3.4%
and included pain, hydrocele, infection, and swelling.(7)






Under local anaesthesia
scrotum is initially painted with antibiotic solution followed by repeat
painting with normal saline to remove any residual antibiotic solution. On
dependant hand supports the testis and the head of the epididymis is palpated
and stabilised with thumb and forefinger. Aspiration of epididymis is performed with a 27. G needle
mounted with tuberculin syringe containing culture medium. With the needle still
within the epididymis the syringe is advanced in different direction while
maintaining continuous suction. The needle is gently withdrawn from the
epididymis while the suction is released. The aspirate is then emptied into the
dish containing the sperm wash media to be examined under microscope for the
presence of any sperms which can be eventually cryopreserved for future use
during an ICSI. A repeat attempt at an aspiate is made in case of negative
aspiration but at a different location along the epididymal head and repeated
on the contralateral testis. Since this is a blind procedure sometimes several
attempts are required before good quality sperm are found.




and low cost.
morbidity, repeatable.
microsurgical expertise required.
instruments and materials.
open surgical exploration.

sperm retrieved.
number of sperm for cryopreservation.
and obstruction at the aspiration site.
of hematoma/spermatocele .


epididymal sperm aspiration (PESA)


Testicular sperm aspiration

TESA is a needle aspirate of seminiferous tubules
most often containing only non-motile or immature sperm first described in Israel by Lewin and
colleagues in 1996 ( 8 ) . It can be considered “therapeutic” for cases of elective
cryopreservation of retrieved sperms or can be coordinated and concurrent with their
female partner’s egg retrieval for ICSI. Occasionally, TESA doesn’t provide
enough tissue/sperm and an open testis biopsy is needed. TESA is also of “diagnostic”
utility when the diagnosis of azoospermia is not certain of obstructive or non- obstructive aetiology and
a focussed counselling and plan  of
treatment can be laid down for subsequent sperm retrieval. Jensen et al. reported a sperm
retrieval rate of 100% in OA, with a complication rate of 3% ( 9


Under spermatic cord block of short general
anesthesia scrotum is initially painted with antibiotic solution followed by
repeat painting with normal saline to remove any residual antibiotic solution.Testicular
sperm aspiration can be done either by 18G butterfly needle attached to a 20 ml
syringe  containing 1 ml of culture media
. Multi-quadrant testicular tissue aspiration can be done by applying
continuous suction and aspirating the testicular fluid and tissue. The aspirated tissue is then
processed in the embryology laboratory and the sperm cells extracted are used
for ICSI. The aspirated tissue is then processed in the embryology laboratory
and the sperm cells extracted are used for ICSI or cryopreservation.In case of
sample collection stress syndromes or anejacualtion TESA is a quick way for
sperm retrieval in cases of known spermatogenesis on the day of oocyte

Testicular sperm aspiration




and low cost.
morbidity, repeatable.
microsurgical expertise required.
instruments and materials.
open surgical exploration.

to tunical blood vessel and hematocele formation.
sperm retrieved.
number of sperm for cryopreservation.
and obstruction at the aspiration site.
of hematoma/spermatocele .


vasal sperm aspiration (PVSA)


Not very popular among
reproductive endocrinologist is most widely utilised among cases of obstructive
lesions of vas deference resulting from herniorrhaphyp surgery , vasectomy or
ejaculatory duct obstruction. Because it is not widely performed, a standard technique of
PVSA is relatively unknown. First described by Qiu and colleagues(10) The vas deferens was held between the surgeon’s thumb and
forefinger and fixed under the scrotal skin or exposed by a paramedian incision
. The  vas deferens is then cannulated by
 by using a 21-gauge sharp needle, and a
23-gauge blunt tip needle with a central hole and a side hole was introduced
through the sharp needle in the direction of the epididymis. The blunt-tipped
needle was connected to a 10-mL syringe with 1.0 mL of sperm culture. Alternatively
vasotomy or partial vasectomy can be performed for successful cannulation. The
sperm preparation medium was pushed into the vas deferens approximately 0.2 to
0.3 mL, suction was applied to the syringe, and the needle was withdrawn gradually
to a point at which segments of fluid from the vas entered the sperm
preparation medium (i.e., the fluid became turbid). A small drop of fluid was
smeared onto a slide, and routine semen analysis was performed immediately.



Open surgical approaches to surgical sperm retrieval .

epididymal sperm aspiration (MESA)

First reported by Temple-Smith et al , MESA was an endeavour at
retrieving more quality sperms in consideration against testicular sperms. This
was conclusively proved by major studies which resulted in superior outcomes in
ICSI with epidydimal sperms against testicular sperms (11)  Several different
techniques for MESA that have been reported in literature (
12,13 14). Extracting sperm from their storage site
in the epididymis is an excellent way of getting sperm to use for IVF in
patients with a blockage in the reproductive tract.Under spinal anesthesia with
a median incision healthier testicle is delivered out while  testis and epididymis is exposed. With the use
of operating microscope and under magnification epididymotomy is performed ,
identifiying a suitable dialated epididymal tubule for sperm aspiration
preferably at distal end of epiidymis where the sperms have completed
maturation process and have acquired motility while their transport as a part
of normal sperm development. More frequently multiple foci aspiration may be
necessary to evaluate before motile sperm are identified.




epididymal sperm aspiration (MESA)






TESE (Testicular sperm extraction)

Most popular method of SSR is TESE. Because the
procedure is identical to a testicular biopsy as it does not use operative
microscope and requires extensive microsurgical training.
approximately, 1% of all men and 10%
of all male infertility men are because of NOA, as the result of testicular
failur14.  TESE combined with
ICSI is the first-line treatment in NOA patients. It may distinguish between OA and NOA, extract substantial
sperm for cryopreservation, while it supersedes testicular biopsy and the obtained
tissue can be subjected to histopathology examination contingent upon negative SSR. Using TESE-ICSI,
sperm retrieval leading to pregnancy and the delivery of healthy children is
possible for men with long-standing azoospermia after chemotherapy. Testicular
spermatozoa can be retrieved in some NOA males because of the existence of
isolated foci of active spermatogenesis with a retrieval rate approaching 50%.


TESE involves making a small incision in the testis and
examining the tubules for the presence of sperm. It is either done as a
scheduled procedure or is coordinated with their female partner’s egg
retrieval. TESE is usually performed in the operating room with sedation, but
can be performed in the office with local anesthesia alone. The retrieved tissue is then
finely minced with iris scissors within approximately 1 mL of sperm wash medium
in a glass petri dish, and a small drop is placed on a slide for evaluation by
the embryology staff. Hemostasis is achieved through bipolar electrocautery,
and the incision in the tunica albuginea is closed with the pre-placed suture
in running fashion. Multiple biopsy sites are often not necessary. The most appropriate number of biopsies to be
performed still remains controversial. To increase the chance of finding a
focus of sperm production, it is advisable to take multiple samples from
different sites of the testis. In addition, it has been reported that the
number of biopsies required is significantly higher in MA and SCO cases,
compared with hypospermatogenesis patterns, The tunica vaginalis, dartos fascia, and skin are
closed in running fashion with 3-0 chromic.


The most appropriate number of biopsies to be performed still
remains controversial. To increase the chance of finding a focus of sperm
production, it is advisable to take multiple samples from different sites of
the testis. In addition, it has been reported that the number of biopsies
required is significantly higher in MA and SCO cases, compared with hypo
spermatogenesis patterns ( 15 )



Testicular sperm extraction



Microdissection TESE (microTESE)

technique was originally described in 1999 ( 16
). MicroTESE is a procedure performed
for men who have a sperm production problem and are azoospermic. MicroTESE is
performed in the operating room with general anaesthesia under the operating
microscope. MicroTESE is carefully coordinated with the female partner’s egg
retrieval, and is performed the day before egg retrieval. This allows for each
partner to be there for the other’s procedure. Patients frequently have donor
sperm backup in case sperm are not found in the male partner. MicroTESE has
significantly improved sperm retrieval rates in azoospermic men, and is a safer
procedure since less testicular tissue is removed. Patients cryopreserve sperm
during this procedure for future IVF/ICSI. Conventional
testicular sperm extraction (c-TESE) in patients with NOA has been partially
replaced by micro-TESE. It is still under debate the problem regarding the
higher costs related to micro-TESE when compared with c-TESE. 

anaesthesia testis is opened widely in an equatorial plane along the mid
portion. This allows wide exposure of seminiferous tubules in a physiological
approach that follows intratesticular blood flow. Due to the heterogeneity of
sperm production in the testicle, microdissection must permit examination of
all seminiferous tubules. Use of the operating microscope with magnification
allows identification of the seminiferous tubules that are most likely to
contain sperm. If sperm production is present within a seminiferous tubule,
then the tubule appears larger and more opaque . Targeting the larger
tubules, improves the yield of sperm retrieval and limits the amount of
testicular tissue that needs to be removed. A comparative study including 116 men found a significantly
higher SRR with micro-TESE 47% vs. 30% in conventional TESE (17) .


        Microdissection TESE (microTESE)

Minimal testicular damage is the rule
since only seminiferous tubules are evaluate and thereby increasing chances of
successful sperm retrieval as wide area of testis is examined under
magnification. Though only a small amount of tissue is removed, the large
tunical incision and the dissection of the testicular tissue can cause devascularization
and fibrosis of the testis.



Comparison of conventional (c) and
microdissection (m) testicular sperm extraction (TESE) sperm retrieval rates
(SRR) ( 18 )



Single seminiferous
tubule – biopsy



Open conventional biopsy versus
microsurgical biopsy techniques

If a single biopsy shows sperm
then the method of biopsy does not make a difference. However, in men with
testicular failure, in whom multiple biopsies have to be done, microsurgical
biopsy techniques – which minimize testicular damage while allowing extensive
sampling – are preferred to the conventional open testicular biopsy.


Percutaneous versus open
testicular biopsy

The percutaneous  technique gives adequate tissue in most cases
and is psychologically more acceptable to patients. The open methods (microdissection
and SST) allow for extensive sampling and give the best chances of sperm
recovery, especially when the testis is small and fibrotic.


Prognostic factors for sperm retrieval in non-obstructive


Tough surgical sperm retrieval is practised extensively
still clinicians are unable to predict the percentage chances of successful
sperm recovery as there is still paucity of data derived from  clinical and laboratory prognostic factors. No reliable positive prognostic factors
guarantee sperm recovery for patients with non-obstructive azoospermia. The
only negative prognostic factor is the presence of AZFa and AZFb microdeletions ( 19 20 )



ROC curves for
inhibin B, FSH, and inhibin B/FSH. The table reflects the area under the curve,
the optimal cut-off point, the sensitivity and specificity of each of the


For non-obstructive azoospermia, these
three prognostic factors are typically considered alone or combined with
endocrinologic data. No parameters can reliably
predict whether sperm will be found on TESE. Sperm may be found in men with
very small testesand high FSH levels, irrespective of the general histological


Sperm retrieval
rate in azoospermic patients with genetic alterations




Sperm retrieval
rate in azoospermic patients with genetic alterations



3.Sperm retrieval rate in patients with nonobstructive
azoospermia depending on testicular size




use of surgical sperm retrieval from the testis or epididymis associated to
ICSI has given the chance for azoospermic patients of fathering their own
genetic children. NOA subjects may retrieve spermatozoa through TESE, giving
the chance for an assisted reproductive technology process.  The sperm
retrieval rate in OA patients is about 100%. In NOA, the most frequently
reported sperm retrieval rate is about 50% .




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2. Van Steirteghem AC1, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, Wisanto A, Devroey P. High fertilization and implantation rates after intracytoplasmic
sperm injection. Hum Reprod. 1993 Jul;8(7):1061-6.


3. Craft I, Bennett V, Nicholson N. Fertilising ability of testicular spermatozoa.Lancet. 1993 Oct 2;342(8875):864.


4. Devroey P1, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A, Silber S.Pregnancies after testicular sperm extraction
and intracytoplasmic sperm injection in non-obstructive azoospermia.
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5 . Brackett NL1, Ibrahim E, Iremashvili V, Aballa TC, Lynne CM. Treatment for ejaculatory dysfunction in men with spinal cord injury:
an 18-year single center experience. J Urol. 2010 Jun;183(6):2304-8.


6. Glina S,
Fragoso JB, Martins FG, et al. Percutaneous
epididymal sperm aspiration (PESA) in men with obstructive azoospermia. Int Braz J Urol 2003;29:141-5


7. Esteves SC, Lee
W, Benjamin DJ, et al. Reproductive potential of men
with obstructive azoospermia undergoing percutaneous sperm retrieval and
intracytoplasmic sperm injection according to the cause of obstruction. J Urol 2013;189:232-7.


8.  Lewin A, Weiss DB,
Friedler S, et al. Delivery following
intracytoplasmic injection of mature sperm cells recovered by testicular fine
needle aspiration in a case of hypergonadotropic azoospermia due to maturation
arrest. Hum
Reprod 1996;11:769-71.


9. Jensen
CF, Ohl DA, Hiner MR, et al. Multiple needle-pass
percutaneous testicular sperm aspiration as first-line treatment in azoospermic
men. Andrology 2016;4:257-62.


10. Qiu Y, Wang S, Yang D, et al. Percutaneous
vasal sperm aspiration and intrauterine insemination in the treatment of
obstructive azoospermia. Fertil Steril 1997;68:1135-8. 


11. Van Wely M, Barbey N, Meissner A, et al. Live birth rates after MESA or TESE in men with obstructive
azoospermia: is there a difference? Hum
Reprod 2015;30:761-6.


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13. Goldstein M, Tanrikut C. Microsurgical
management of male infertility. Nat Clin
Pract Urol2006;3:381-91.


14. Palermo GD1, Schlegel PN, Hariprashad
JJ, Ergün B, Mielnik A, Zaninovic N, Veeck LL, Rosenwaks Z.
Fertilization and pregnancy outcome with intracytoplasmic sperm injection for
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15.  Tournaye H1, Liu J, Nagy PZ, Camus M, Goossens A, Silber S, Van Steirteghem AC, Devroey P.Correlation
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using testicular spermatozoa. Hum Reprod. 1996 Jan;11(1):127-32.



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Testicular sperm extraction: impact of testicular histology on outcome, number
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18. Saccà,
A.; Pastore, A. L.; Roscigno, M.; Naspro, R.; Pellucchi, F.; Fuschi, A Conventional
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