2010 THE AUTHORS; JOURNAL COMPILATION
Original Articles
2010 BJU INTERNATIONAL
OUTCOME OF INTRACYTOPLASMIC SPERM INJECTIONKALSI
et al.
BJUI
Analysis of the outcome of intracytoplasmic
sperm injection using fresh or frozen sperm
BJU INTERNATIONAL
Jas Kalsi, M. Yau Thum*, Asif Muneer*, John Pryor*, Hossam Abdullah* and
Suks Minhas*
Department of Andrology, University College London Hospitals NHS Foundation Trust and *The Lister Hospital,
London, UK
Accepted for publication 7 May 2010
Study Type – Diagnostic (retrospective
cohort)
Level of Evidence 2b
OBJECTIVES
• To compare the outcome of first-attempt
intracytoplasmic sperm injection (ICSI) ICSI–
embryo transfer (ET) cycles using frozenthawed testicular sperm (FTTS), fresh
testicular sperm (FTS), frozen-thawed
epididymal sperm (FTES) and fresh
epididymal sperm (FES) so as to determine
which of these has the most successful
ICSI outcome with respect to fertilization
rate (FR), pregnancy rate (PR) and birth
rate.
• To assess the outcomes according to the
underlying aetiology of azoospermia.
PATIENTS AND METHODS
• The records of 493 patients undergoing
first-attempt ICSI between 1993 and 2008
were reviewed retrospectively. FTS was used
in 112 cycles, FTTS in 43 cycles, FES in 279
cycles, and FTES in 59 cycles.
• Within each group, the aetiology of the
azoospermia was recorded according to
history, clinical examination and histological
analysis (n = 316).
INTRODUCTION
Intracytoplasmic sperm injection (ICSI),
which has been in use since 1994, has
revolutionized the management of male
factor infertility. With this technique,
azoospermic patients have been able to
father children after either epididymal or
11 2 4
What’s known on the subject? and What does the study add?
The results of ICSI using fresh or frozen sperm on the site of sperm retrieval remains
controversial with respect to outcome.
The results of this study showed no difference in outcome using ICSI either with respect
to the site of retrieval or whether the sperm used was fresh or frozen. It also showed that
the outcome of ICSI is not related to the underlying cause of the azoospermia.
• The FR, clinical PR and delivery rate were
calculated for each group with respect to the
type of sperm retrieval used.
RESULTS
• Analysis of the data showed no significant
differences between any of the four groups
in the FR, PR or delivery rate (P > 0.05).
• There were no significant differences seen
between fresh sperm (FTS and FES) and
frozen sperm (FTTS and FTES) or between
epididymal sperm (FES and FTES) and
testicular sperm (FTS and FTTS) in any of the
outcomes measured (P > 0.05). However,
sub-set analysis showed a statistically higher
FR and PR for FTTS over fresh sperm.
• When comparing aetiologies, there was
no significant difference in the FR, clinical PR
and delivery rate between obstructive
azoospermia (OA) and non-obstructive
azoospermia (NOA) groups. However, sub-set
analysis showed a higher PR and birth rate
for FTTS over fresh sperm in both OA and
NOA groups.
testicular sperm extraction. Previous studies
have shown that the success rates of ICSI
with fresh testicular or epididymal
spermatozoa are equivalent to those
achieved with in vitro fertilization (IVF) using
ejaculated spermatozoa [1–3]. However, the
ability to cryopreserve sperm before
undergoing ICSI allows more flexibility and
CONCLUSIONS
• The results of the present study suggest
that using frozen sperm in ICSI cycles is a
reliable and favourable method with the
same outcome as fresh sperm.
• Testicular and epididymal sperm have
similar ICSI outcomes for both fresh and
frozen samples. However, results suggest a
tendency for higher PRs and birth rates for
frozen than for fresh testicular sperm in
both OA and NOA aetiologies.
• The aetiology of azoospermia does not
significantly affect the outcome of firstattempt ICSI. The higher rates in the frozen
groups suggest that these patients have had
better quality semen when they were initially
harvested and frozen.
KEYWORDS
ICSI, Fresh, Frozen, Sperm, Aetiology
increases the range and number of
therapeutic options available.
Frozen sperm avoids the need for repeated
surgical sperm retrieval procedures. Moreover,
having frozen sperm on standby allows for
more effective treatment planning, meaning
that concurrent sperm and oocyte retrievals
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2 0 1 0 B J U I N T E R N A T I O N A L | 1 0 7 , 11 2 4 – 11 2 8 | doi:10.1111/j.1464-410X.2010.09545.x
OUTCOME OF INTRACYTOPLASMIC SPERM INJECTION
might not be required [4]. Previous studies
report that using cryopreserved epididymal
and testicular spermatozoa for ICSI yields
acceptable fertilization rates (FRs) and
pregnancy rates (PRs) [5,6]. However, it is
unknown whether using fresh or frozen
sperm is associated with better ICSI outcomes
[7,8], as previous reports have suggested that
cryopreservation reduces the fertilizing
capacity of sperm [9,10]. Also, the retrieval
site (testicular or epididymal) of the sperm
which is subsequently frozen has been
suggested to have an impact on the outcome
when used in subsequent cycles [8].
To address whether there is a significant
impact of cryopreservation and whether there
is a difference between testicular and
epididymal sperm on the outcomes of ICSI, we
retrospectively compared the outcomes of
first-attempt ICSI–embryo transfer (ET) cycles
when using frozen-thawed testicular sperm
(FTTS), fresh testicular sperm (FTS), frozenthawed epididymal sperm (FTES) and fresh
epididymal sperm (FES). We also determined if
outcomes were affected by the underlying
aetiology of the azoospermia.
PATIENTS AND METHODS
The hospital records of patients undergoing
first-attempt ICSI between 1993 and 2008
were retrospectively reviewed. In all, 493
couples were included in the study and all had
first-attempt ICSI cycles. FTS was used in 112
cycles, FTTS in 43 cycles, FES in 279 cycles, and
FTES in 59 cycles. The FR, clinical PR and
delivery rate were calculated for each group.
Within each group the aetiology of the
azoospermia was recorded (n = 316)
according to history, clinical examination or
biopsy result. The FR, clinical PR and delivery
rate were calculated for each group with
respect to the type of sperm retrieval used.
All patients with a history of azoospermia
were evaluated at the andrology clinic to
confirm their diagnosis before starting ICSI. If
no sperm were seen, these patients were
offered sperm retrieval after a full assessment
and counselling with a consultant urologist.
stimulation to ensure the ovaries were
quiescent. For the long protocol, patients
were down-regulated with either Nafarelin or
Buserelin at the mid-luteal phase. For the
Cetrotide protocol, GnRH antagonist was
started when the leading follicle reached
12 mm. When follicles reached the preovulatory size (18–22 mm), 10 000 IU of hCG
was administrated. Oocytes were aspirated
using trans-vaginal US guidance at 34–36
hours after hCG administration. Embryo
transfer was performed on day 2 or day 3
using a soft catheter with trans-abdominal
US guidance. All patients received
progesterone 400 mg pessaries as a
supplement throughout the luteal phase. A
pregnancy test was performed 2 weeks after
the embryo transfer.
RETRIEVAL OF TESTICULAR SPERMATOZOA
The technique of microdissection testicular
sperm extraction, as previously described [11],
was used to retrieve sperm after 2005. Before
this, patients underwent multiple biopsy
testicular sperm extraction (TESE). Using
high-powered ICSI microscopes, testicular
tubules were dissected immediately by an
embryologist and any sperm obtained was
either frozen or used for injection for a
concurrent cycle of ICSI.
Percutaneous epididymal sperm aspiration
(PESA) or microsurgical epididymal sperm
aspiration (MESA) were performed as
previously described under a local or general
anaesthesia [12]. For MESA, spermatozoa
were obtained from the microsurgically
opened epididymal tubule with a micropipette
or a 24-gauge cannula using a binocular
microscope (magnification × 25). Each
specimen was examined for the presence of
motile spermatozoa using a phase contrast
microscope (magnification × 400), starting at
the cauda epididymis and continuing to a
tubule 0.5 cm above the first, until motile
spermatozoa were aspirated. For PESA, a
needle was placed into the epididymis
through the skin blindly to aspirate sperm. The
aspirated sample was then assessed for the
presence of sperm using a phase contrast
microscope (magnification × 400).
TREATMENT PROTOCOL
Ovarian stimulation was carried out with
either recombinant FSH, human menopausal
gonadotrophin or urinary FSH. A transvaginal scan was performed before ovarian
©
FREEZING AND THAWING OF PESA/MESA
OR TESE SPERM
For freezing, an equal volume of spermfreezing medium was added slowly and drop-
wise to the semen, agitating the sample
gently throughout. Samples of 1–1.5 mL were
aliquoted into pre-labelled cryotubes. The
aliquots were placed in the vapour phase of a
liquid N2 (−147 °C) bank for 15 min and then
transferred into the liquid phase for storage
(−190 °C). For thawing, a single cryotube was
removed from the sperm storage vessel into a
holding flask containing liquid N2 (−190 °C).
The sample was then removed from holding
flask and kept at room temperature for
15 min or until the sample had thawed.
STATISTICAL ANALYSIS
Data were collected from Medical System for
IVF (MedicalSys, London, UK) and analysed
with the Statistics Package for Social Sciences
(SPSS, Surrey, UK). Descriptive statistical
analysis was performed initially to examine
the normality of distribution of all continuous
variances for parametric statistical tests.
Associations between fresh or cryopreserved
sperm groups and PR, miscarriage and live
birth rates were examined with a chi-squared
cross-tabulation test. Analysis of variance was
then used to assess the relationships between
fresh or cryopreserved sperm groups and
women’s mean age, ratio of MII/total oocytes
collected, number of oocytes injected, mean
number of normal fertilized embryos and
mean number of embryos transferred.
Statistical significance was set at P < 0.05.
RESULTS
The mean (range) maternal age was 33.7
(21–47) years. There was no difference in
maternal age between the four groups.
There were no differences between the FES
and FTES groups with respect to maternal age,
number of oocytes retrieved, oocyte maturity
and number of embryos transferred (Table 1).
A statistically higher number of oocytes were
injected in the frozen group than in the fresh
group (P = 0.018) but the two groups did not
differ with respect to FR, PR, live birth rate or
miscarriage rate (P > 0.05).
There were no differences between the FTS
and FTTS groups with respect to maternal age,
number of oocytes retrieved and oocyte
maturity (Table 1). However, there were
differences between the frozen and fresh
groups with respect to number of embryos
transferred (P = 0.001), FR (P = 0.021) and PR
(P = 0.047). Despite this, there was no
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K A L S I ET AL.
TABLE 1 Outcome of first-attempt ICSI–ET with respect to source of sperm (FES, FTES, FTS, FTTS)
Number of patients
Mean (±SD) age of women, years
Mean (±SD) number of oocytes collected
MII/total oocytes collected ratio (%)
Number (±SD) of oocytes injected
Mean (±SD) number of normal fertilized embryos
Mean (±SD) number of embryos transferred
PR (%)
Live birth rate (%)
Miscarriage rate (%)
Epididymal sperm
FES
279
33.61 ± 5.1
11.86 ± 6.6
78.7 ± 17.6
9.3 ± 5.7
5.07 ± 3.5
2.15 ± 0.78
44.4 (124/279)
35.4 (99/279)
20.3 (25/124)
FTES
59
33.92 ± 5.4
13.39 ± 6.3
83.2 ± 14.2
11.2 ± 5.6
6.07 ± 3.8
2.26 ± 0.86
40.7 (24/59)
30.5 (18/59)
25.0 (6/24)
P-value
NA
NS 0.673
NS 0.098
NS 0.058
0.018
NS 0.051
NS 0.329
NS 0.351
NS 0.295
NS 0.392
Testicular sperm
FTS
112
33.96 ± 5.4
11.82 ± 6.7
81.3 ± 17.4
9.42 ± 5.1
4.62 ± 3.1
2.18 ± 0.75
39.3 (44/112)
29.5 (33/112)
25.0 (11/44)
FTTS
43
33.57 ± 4.6
12.59 ± 7.6
75.5 ± 19.7
9.78 ± 6.6
6.04 ± 4.4
1.74 ± 0.71
55.8 (24/43)
39.5 (17/43)
29.2 (7/24)
P-value
NA
NS 0.184
NS 0.528
NS 0.731
NS 0.709
0.021
0.001
0.047
NS 0.320
NS 0.461
Testicular sperm
FTS
28
36.48 ± 5.7
10.39 ± 6.3
84.6 ± 16.6
8.77 ± 5.5
5.5 ± 3.5
2.10 ± 0.87
32.1* (9/28)
28.6* (8/28)
11.1* (1/9)
FTTS
15
35.75 ± 3.1
12.69 ± 8.2
84.2 ± 14.8
10.94 ± 7.6
6.3 ± 4.7
1.81 ± 0.65
60.0* (9/15)
60.0* (9/15)
0.0* (0/9)
NS, difference not statistically significant ( P > 0.05); NA, not applicable.
TABLE 2 The outcome of first-attempt ICSI–ET with respect to aetiology
Number of patients
Mean (±SD) age of women, years
Mean (±SD) number of oocytes collected
MII/total oocytes collected ratio (%)
Number of oocytes injected ± SD
Mean (±SD) number of normal fertilized
Mean (±SD) number of embryos transferred
PR (%)
Live birth rate (%)
Miscarriage rate (%)
Non-obstructive azoospermia
Testicular sperm
FTS
FTTS
41
7
33.61 ± 5.1
34.14 ± 4.1
12.89 ± 7.8
11.86 ± 2.9
80.8 ± 16.6
86.0 ± 5.1
10.32 ± 6.8
10.14 ± 2.3
5.25 ± 3.8
6.43 ± 1.9
2.36 ± 0.89
2.29 ± 0.76
36.6* (15/41)
57.1* (4/7)
31.7* (13/41)
57.1* (4/7)
13.3* (2/15)
00.0* (0/4)
Obstructive azoospermia
Epididymal sperm
FES
FTES
173
42
34.03 ± 4.8
33.77 ± 5.2
11.57 ± 6.4
13.48 ± 6.8
82.5 ± 15.4
77.8 ± 17.2
8.9 ± 5.4
11.3 ± 6.3
5.25 ± 3.5
6.07 ± 4.3
2.24 ± 0.81
2.14 ± 0.79
46.2† (80/173)
35.7† (15/42)
33.5† (58/173)
23.8† (10/42)
27.5† (22/80)
33.3† (5/10)
*Significant statistical comparison using chi-squared cross-tabulation test with P < 0.05 between fresh and frozen sperm within one particular group.
†
Not statistically significant between fresh and frozen sperm within one particular group.
difference between the groups in the live birth
and miscarriage rates.
When all groups were compared, there were
no significant differences between them with
respect to FR, PR and live birth rate. Sub-set
analysis was then performed to assess if
there were any differences within the
aetiological groups (Table 2). In the
obstructive azoospermia (OA) group, there
was a statistically higher PR and live birth
rate for FTTS than for FTS, but this was not
the case for FTES vs FES. In the nonobstructive azoospermia group (NOA), there
was a difference between FTTS and FTS with
respect to PR, live birth rate and miscarriage
rate.
11 2 6
DISCUSSION
Previous studies have suggested that using
FTTS could be associated with a lower
fertilizing potential than is found when using
FTES [13]. There is also evidence to suggest
that testicular and epididymal spermatozoa
are more sensitive to cooling than ejaculated
spermatozoa [14]. One study evaluated
the impact of cryopreservation on sperm
(FTES, FTTS) obtained from patients with
azoospermia and used for ICSI, and compared
the results with fresh spermatozoa (FES, FTS)
for ICSI in the same individuals [13]. The
results suggested no difference between the
two groups with respect to FRs or clinical PRs.
However, only 16% of the NOA and 39% of
the OA patients had their sperm frozen.
Moreover, the impact of cycle optimization
with second and subsequent cycles cannot be
excluded from the results of the present
study.
The effects of cryopreservation on sperm have
been reviewed previously. Extensive cryoinjury to spermatozoa can occur during a
number of steps of the freeze–thaw process,
including cooling, thawing and addition or
removal of the cryoprotectant (e.g. glycerol)
[15]. It has previously been shown that
freezing of sperm can cause swelling and
rupture of the inner and outer acrosomal and
plasma membranes, therefore making them
unusable [16]. Moreover, the production of
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OUTCOME OF INTRACYTOPLASMIC SPERM INJECTION
oxygen free radicals has been found to
increase during both the cooling and freeze–
thaw processes, leading to free radical injury
secondary to plasma membrane lipid
peroxidation [17]. Some studies suggest that
cryopreservation of ejaculated spermatozoa
results in an increase in the proportion of
sperm with broken necks after thawing [18],
and this might, in turn, be associated with a
lower fertilization capacity. Conversely, other
studies have shown that the FR in ICSI cycles
using frozen-thawed, surgically retrieved
spermatozoa did not differ significantly from
that obtained using fresh, surgically retrieved
spermatozoa [19–22] . Moreover, similar
outcomes have been reported after FTES and
FTTS [23]. Reports have shown that the use of
FTTS results in lower FR and PR than the use
of FTS [9,10].
The effects of cryopreservation on clinical
outcome can be best determined by
comparing patients undergoing cycles of
ICSI with both fresh and frozen-thawed
spermatozoa in their first cycle. The present
study is the first one to have specifically
examined this. Previous studies have used
data either from consecutive cycles or from
unmatched control populations to compare
the outcomes [24]. Using data from
consecutive cycles can be misleading as
higher success rates observed in couples
undergoing a second or subsequent ICSI cycle
could result from optimizing the stimulation
of the female partner and not just from male
variables [13]. Furthermore, these studies
have involved only a few of patients, which
can make statistical analysis difficult to
interpret (n = 24 [19]; n = 19 [25]). In the
present study, we compared the clinical
outcomes in a large series of couples
undergoing a first cycle of ICSI using fresh
and frozen-thawed surgically retrieved
spermatozoa. Our results suggest that
cryopreservation does not have a significant
impact on outcome of first-attempt ICSI
regardless of the source of the sperm. Indeed,
the results suggest that FTS has a higher FR
and PR than fresh samples. This contrasts
with other studies which have reported lower
FR in FTTS than in FTS [26,27]. The results of
the present study can be partly explained by
an inherent bias to freeze only sperm with
good characteristics that might survive the
freeze–thaw cycle.
Cryopreservation has previously been shown
to have a detrimental effect on the
morphology of both testicular and ejaculated
©
spermatozoa because of the formation of
intracellular ice, which results in the plasma
membrane rupturing [18,28,29]. This, in turn,
might allow free radical oxygen species to
access sperm nuclei, adversely affecting DNA
integrity [30]. The cytoplasm of testicular
spermatozoa is not usually sufficient for
antioxidant protection and might thus permit
oxidative damage [31]. Compared with
epididymal sperm, testicular sperm are more
vulnerable because their chromatin packaging
is not completed until the Sulphur-Hydrogen
(SH) bonds are oxidized during transit
through the epididymis. Moreover, prolonged
incubation after thawing of cryopreserved
testicular spermatozoa might damage nuclear
DNA, thus reducing the quality of sperm used
for ICSI [32].
In the present study, when all aetiological
groups were assessed, there was no
significant difference in outcome with respect
to the underlying aetiology. This is in contrast
to previous studies which have shown a lower
FR in the FTTS group [33]. Moreover, sub-set
analysis suggests a higher success rate for
FTTS in both the OA and NOA groups than for
their respective fresh counterparts. However,
these results must be interpreted with caution
as they probably represent an inherent bias to
cryopreserve good-quality sperm only and as
the numbers in the frozen groups are small.
Our interpretation of the results would
suggest that frozen sperm is as effective as
fresh sperm, irrespective of the underlying
aetiology.
In conclusion, we have shown in the present
study that surgically retrieved spermatozoa
can be efficiently used for ICSI after freezing
and thawing, without significantly
compromising outcome, independent of the
site of retrieval or the underlying aetiology.
Freezing of surgically retrieved spermatozoa
allows ICSI cycles to be more appropriately
planned and could therefore increase the
probability of conception in couples with
infertility secondary to azoospermia.
Using frozen sperm has the advantage of
avoiding repeated surgical sperm retrieval
with each cycle and ensures the availability of
sperm before beginning the IVF cycle, which
could reduce the cost and avoid unnecessary
cycles.
CONFLICT OF INTEREST
None declared.
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Correspondence: Jas Kalsi, Department of
Andrology, University College London
Hospitals NHS Foundation Trust, 250 Euston
Road, London, Greater London, NW1 2PG, UK.
e-mail: [email protected]
Abbreviations: ET, embryo transfer; FES, fresh
epididymal sperm; FR, fertilization rate;
FTES, frozen-thawed epididymal sperm;
FTS, fresh testicular sperm; FTTS, frozenthawed testicular sperm; ICSI,
intracytoplasmic sperm injection; IVF, in vitro
fertilization; MESA, microsurgical epididymal
sperm aspiration; MII, Metaphase II; NOA,
non-obstructive azoospermia; OA,
obstructive azoospermia; PESA, percutaneous
epididymal sperm aspiration; PR, pregnancy
rate; SH, Sulphur-Hydrogen; TESE, testicular
sperm extraction.
©
BJU INTERNATIONAL
©
2010 THE AUTHORS
2010 BJU INTERNATIONAL