Outcomes in couples undergoing ICSI: comparison between fresh and frozen–thawed surgically...

Outcomes in couples undergoing ICSI: comparison between

fresh and frozen–thawed surgically retrieved spermatozoa

ULUN ULUG, FARUK BENER, LEVENT KARAGENC, NADIR CIRAY and

MUSTAFA BAHCECI

Bahceci Women Health Care Center and German Hospital, Istanbul, Turkey

SummaryWe retrospectively evaluated the impact of cryopreservation on spermatozoa obtained

from patients with azoospermia and used for intracytoplasmic sperm injection (ICSI).

Frozen–thawed epididymal spermatozoa (FTEPS) was used in 34 couples, whereas

frozen–thawed testicular spermatozoa (FTTS) was used in 50 couples for ICSI during

assisted conception, and these results were compared with results using fresh spermatozoa

for ICSI in the same individuals. The fertilization rate (FR) was significantly lower for

FTTS (65.8%) but not for FTEPS (73.1%) compared with the FR using fresh

spermatozoa (72.3% and 73.2% respectively). In contrast, neither the implantation nor

the pregnancy rate was altered when FTEPS or FTTS was used. In conclusion, our

results indicate that surgically retrieved spermatozoa can be efficiently used for ICSI after

freezing and thawing without compromising the outcome.

Keywords: azoospermia, epididymis, intracytoplasmic sperm injection, frozen–thawed,

spermatozoa, testis

IntroductionIntracytoplasmic sperm injection (ICSI) has been utilized

worldwide in recent years, particularly for the alleviation of

male factor infertility. Azoospermia patients, who account

for 10% of the incidence male infertility (Chandley, 1979),

can become fertile by ICSI subsequent to microsurgical

epididymal aspiration (MESA) or testicular sperm extraction

(TESE). The success rates of ICSI with fresh testicular or

epididymal spermatozoa are equivalent to those achieved by

in vitro fertilization (IVF) using ejaculated spermatozoa

(Aboulghar et al., 1997; Ghazzawi et al., 1998; Van

Steirteghem et al., 1998).

In patients with male factor infertility, cryopreservation of

male gametes can increase treatment options. Frozen–

thawed spermatozoa have therefore been used in artificial

intrauterine insemination cycles, as well as in IVF pro-

grammes. Testicular or epididymal spermatozoa can be

extracted from azoospermic males, frozen, and used in

subsequent cycles of assisted conception without the need for

repeat surgical sperm retrieval procedures. In addition,

cryopreservation of spermatozoa leads to a reduction in the

logistic complications of arranging concurrent spermatozoa

and oocyte retrievals (Oates et al., 1996). Moreover, the use

of cryopreserved epididymal and testicular spermatozoa for

ICSI has been shown to yield acceptable fertilization and

pregnancy rates (PR) (Palermo et al., 1999; Kupker et al.,

2000).

The impact of cryopreservation of spermatozoa on

assisted reproduction treatment (ART) outcome has been

evaluated by comparing it with outcomes obtained by using

fresh spermatozoa from patients with obstructive or non-

obstructive azoospermia in ICSI cycles (Tournaye et al.,

1999; Nicopoullos et al., 2004; Wood et al. 2002). The

retrieval site of frozen–thawed spermatozoa may also affect

ART outcome when used in subsequent cycles (Wood et al.,

2002). Spermatozoa at different maturational stages are likely

to have different cryobiological characteristics. Little is

known regarding ICSI outcome using frozen–thawed

epididymal and testicular spermatozoa, although there is

Correspondence: Dr Mustafa Bahceci, Azer Is Merkezi, 44/17, Kat

5, Abdi Ipekci Cad, Nisantasi 80200, Istanbul, Turkey. E-mail:

[email protected]

international journal of andrology, 28:343–349 (2005) doi: 10.1111/j.1365-2605.2005.00559.x

� 2005 Blackwell Publishing Ltd.

evidence showing that testicular and epididymal spermatozoa

are more sensitive to cooling than ejaculated spermatozoa

(Gilmore et al., 1998). A comprehensive assessment of ART

outcomes using cryopreserved spermatozoa retrieved from

the epididymis or testis may enhance our understanding of

the efficiency of using surgically retrieved, cryopreserved

spermatozoa for assisted conception. We have therefore

retrospectively documented our results in a group of patients

with azoospermia undergoing ICSI cycles with fresh and

frozen–thawed epididymal and testicular spermatozoa.

Materials and Methods

PatientsPatient records of the Bahceci Women’s Health Care

Center, in which frozen–thawed spermatozoa were used

between May 2001 and May 2004, were retrospectively

evaluated. Among 1124 couples with non-obstructive

azoospermia who underwent controlled ovarian hyperstim-

ulation (COH) and TESE, 182 (16.1%) had their sperm

frozen. Among 253 couples with obstructive azoospermia

who underwent COH and MESA, 101 (39.9%) had their

sperm frozen. Eighty-four of these couples elected to

undergo a consecutive cycle of ART using their previously

frozen spermatozoa, 34 using frozen–thawed epididymal

spermatozoa (FTEPS) and 50 using frozen–thawed testicular

spermatozoa (FTTS). Results using FTEPS and FTTS were

compared with those from previous cycles using fresh

spermatozoa.

Of the 50 males, 37 (74%) had previously had a testicular

biopsy. The underlying histopathology was determined to be

hypospermatogenesis in 18, Sertoli cell only in 12 and

complete maturation arrest in seven. The remaining 13 men

had not had a previous testicular biopsy, but they showed

evidence of childhood orchiopexy because of an improperly

descended testicle.

The follow-up for sperm cryopreservation in our centre is

shown in Fig. 1. Briefly, all males with a history of

azoospermia were re-evaluated at our andrology laboratory

to confirm their diagnosis prior to commencement of ART.

In addition, all males, regardless of the underlying reason for

azoospermia (i.e. obstructive or non-obstructive), under-

went a semen analysis on the day of oocyte retrieval. If

spermatozoa were detected, ICSI was performed using fresh

ejaculated spermatozoa. The remaining individuals under-

went MESA or TESE procedures according to the diagnosis

of azoospermia.

Surgically retrieved testicular and epididymal tissue

specimens were minced thoroughly using sterile glass slides

and needles and examined. Samples were subjected to mini-

density gradient centrifugation, depending on the concen-

tration of spermatozoa and the presence of tissue debris at

initial examination. In addition, testicular samples were

washed with erythrocyte lysis solution. If <2 sperm were

found on initial examination, the samples were concentrated

without application of a density-gradient centrifugation. All

preparations were pipetted onto a dish, covered with mineral

oil and incubated for 30 min to allow the cells to settle. This

material was again examined by two laboratory personnel

using an inverted microscope equipped with Hoffman optics

and a micromanipulator at ·400 magnification. Motile

spermatozoa were recovered individually with the aid of an

in-house built micropipette, with an opening of approxi-

mately 8 lm.

If the number of detected spermatozoa exceeded the

number of metaphase II (MII) oocytes and the count of

motile spermatozoa exceeded 50, ICSI was performed using

fresh spermatozoa, and the remaining specimens were

frozen. All males diagnosed with azoospermia had under-

gone a urological examination prior to surgical sperm

retrieval. No chromosomal aberrations were detected in

peripheral lymphocyte cultures from any of the men from

whom we used frozen testicular spermatozoa. However, we

do not routinely assay for Y microdeletions in men

diagnosed with non-obstructive azoospermia. None of the

men diagnosed with obstructive azoospermia was found to

have congenital absence of the vas deferens.

Figure 1. Algorithm for sperm collection from men with previous diagnosisof azoospermia for ICSI.

344 U. Ulug et al.

� 2005 Blackwell Publishing Ltd, International Journal of Andrology, 28, 343–349

Protocol for controlled ovarian hyperstimulationOvulation induction was initiated by pituitary desensiti-

zation with daily doses of 0.5 mg leuprolide acetate (LA)

(Lucrin, Abbot, France) during the mid-luteal phase of the

preceding menstrual cycle. Beginning on the third day of the

preceding menstrual period, the daily dose of LA was

decreased to 0.25 mg, and patients were administered daily

doses of 2–4 ampoules of gonadotropins (purified follicle-

stimulating hormone, Metrodin HP 75 IU and HMG,

Pergonal 75 IU; Serono, Aubonne, Switzerland, in equal

doses). Pituitary desensitization was confirmed by serum E2

assay on the third day (<50 pg/mL). The starting regimen

was fixed for the first 4 days, after which the gonadotropin

dose was adjusted according to the individual patient’s

response. When at least two follicles reached 18 mm in

diameter, 10 000 IU human chorionic gonadotropin (hCG;

Pregnyl; Organon, Oss, The Netherlands) was administered

i.m. Oocytes were retrieved under general anaesthesia 32–

38 h later and were subjected to ICSI. Fertilization was

assessed 16–18 h after ICSI. Embryos were cultured at

37 �C, in an atmosphere of 6.0% CO2 in air in individual

30 lL drops of human tubal fluid (HTF)-based medium

(Vitrolife, Gothemburg, Sweden) covered with mineral oil.

Transfers were selected according to day 3 embryo quality,

based on the criteria introduced by Steer et al. (1992). Three

days after oocyte retrieval, embryos were transferred trans-

cervically under ultrasonographic guidance. The luteal phase

was supported by 100 mg/day progesterone in oil i.m.

Clinical pregnancy was defined as a demonstrable gestational

sac on a transvaginal ultrasonogram, concomitant with a rise

in b-hCG values.

Retrieval of testicular spermatozoaMicrodissection TESE was performed as described

(Schlegel, 1999). Briefly, a wide opening was made in the

tunica albuginea near its mid-portion to optimize visualiza-

tion of the testicular parenchyma without affecting the

testicular blood supply. Direct examination of the testicular

parenchyma was carried out at 20–25· magnification under

the operating microscope to identify larger individual

seminiferous tubules. Small tissue samples were excised from

larger and more opaque tubules and were cut into smaller

pieces to release spermatozoa. Additional incisions in the

same or contralateral testis were made to find spermatozoa.

Retrieval of epididymal spermatozoaThe MESA was performed as previously described

(Schroeder-Printzen et al., 2000) under general anaesthesia.

After scrotal exploration, spermatozoa were sampled from

the microsurgically opened epididymal tubule with a

micropipette or a 24-gauge cannula using a binocular

microscope (magnification ·15). Each specimen was exam-

ined 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. In some

cases, it was necessary to open the ductuli efferentes.

Freezing and thawing of MESA or TESE spermEach preparation was washed, concentrated to approxi-

mately 0.5 mL and transferred to a cryovial (Nunc, Cryo-

Tube Vials, Denmark). An equal volume of sperm freezing

medium (Vitrolife) was slowly added at room temperature to

allow the cells to adapt to the changing osmolarity. After

incubation at room temperature for 5 min, the cryovial was

incubated in overnight in liquid nitrogen vapour and in liquid

nitrogen until used. On the day of use, the vial was removed

from the tank, kept in air for 30 sec, and immersed in a 30 �Cwater bath until thawed. The freezing solution was removed

from the sample by mini-gradient centrifugation.

Statistical analysisStatistical analysis consisted of McNemar test and Fisher’s

exact test for paired and unpaired non-continuous variables

and Student’s t-test for paired continuous variables as

applicable. A probability <0.05 was considered statistically

significant.

ResultsIn all cycles, a sufficient number of motile spermatozoa to

inject oocytes was detected after the thawing process. Two

cycles using FTEPS and six cycles using FTTS were

cancelled because of inadequate embryo development, and

ET was not performed. The mean duration between ICSI

cycles using fresh and frozen spermatozoa was 13.9 ±

6.4 months (median, 10 months; range, 3–27 months).

Comparison of fresh epididymal spermatozoa and FTEPS forICSIThere were no differences between the fresh epididymal

spermatozoa and FTEPS groups in maternal age, paternal

age, number of oocytes retrieved, oocyte maturity and

number of embryos transferred (Table 1). These groups did

not differ in either fertilization rate (FR) or implantation rate

(IR). Although there were trends towards higher PR and

lower early pregnancy loss in the FTEPS group, the

differences did not reach statistical difference. Of the 15

pregnancies in the fresh epididymal spermatozoa group, four

(26.6%) aborted prior to 12 weeks of gestation. In compar-

ison, two of the 21 pregnancies (9.0%) in the FTEPS group

aborted prior to 12 weeks (p > 0.05).

Comparison of fresh testicular spermatozoa and FTTS forICSIThere were no differences between the fresh testicular

spermatozoa and FTTS groups in maternal age, paternal age,

number of oocytes retrieved, oocyte maturity and number

of embryos transferred (Table 2). Although FR was

significantly decreased in the FTTS group compared with

Outcomes in couples undergoing ICSI 345


the fresh testicular spermatozoa group (p ¼ 0.04, OR: 1.3,

95% CI: 1.0–1.8), IR and PR did not differ between these

two groups. None of the pregnancies in the fresh testicular

spermatozoa group aborted prior to 12 weeks of gestation,

compared with two of the 20 pregnancies (10.0%) in the

FTEPS group (p > 0.05).

Comparison of FTEPS and FTTS for ICSIWhen the FTEPS and FTTS groups were compared, we

found that FR was significantly lower in the FTTS group

(p ¼ 0.003, OR: 1.4, 95%CI: 1.0–1.9). Although there was

a trend towards increased PR in the FTEPS group compared

with the FTTS group, the difference was not significant

(p ¼ 0.13, OR: 0.4, 95% CI: 0.1–1.1). The cumulative PR

per oocyte retrieval, however, was significantly higher in the

FTEPS group than in the FTTS group (52.9% vs. 36.0%,

respectively; p ¼ 0.03, OR: 0.5, 95% CI: 0.2–0.9).

DiscussionThe effects of cryopreservation on male gametes have

been intensively examined. Extensive cryoinjury to sperma-

Table 1. Cycle characteristics of patientsundergoing consecutive assisted concep-tion treatment with fresh and frozen–thawed epididymal spermatozoa

Fresh epididymalspermatozoa (n ¼ 34) FTEPS (n ¼ 34) p-value

Female age (mean ± SD) 31.4 ± 4.7 32.11 ± 4.7 NSMale age (mean ± SD) 35.52 ± 5.5 36.26 ± 5.8 NSTotal oocytes retrieved (mean ± SD) 15.46 ± 7.4 14.03 ± 7.6 NSMII/total oocyte ratio (%) 79.3 81.2 NSInjected oocytes 393 3652 PN 288 267Fertilization rate (%) 73.2 73.1 NSCleavage rate (%) 94.7 95.1 NSCancelled cycle – 2ET (mean ± SD) 3.53 ± 1.2 3.46 ± 1.0 NSImplantation rate (%) 26/116 (22.4) 30/111 (32.4) NSPregnancies 15 21Pregnancy rate/cycle 44.1 61.7 NSPregnancy rate/ET (%) 44.1 65.6 NSMultiple pregnancies 8 7Early pregnancy loss 4 2

Table 2. Cycle characteristics of patientsundergoing consecutive assisted concep-tion treatment with fresh and frozen–thawed testicular spermatozoa

Fresh testicularspermatozoa (n ¼ 50) FTTS (n ¼ 50) p-value

Female age (mean ± SD) 33. 47 ± 5.3 34.16 ± 5.5 NSMale age (mean ± SD) 37.97 ± 6.8 38.92 ± 6.0 NSTotal oocytes retrieved (mean ±SD)

11.43 ± 7.2 11.88 ± 7.5 NS

MII/total oocyte ratio (%) 77.5 80.6 NSInjected oocytes 390 4222 PN 282 278Fertilization rate (%) 72.3 65.8 0.04Cleavage rate (%) 94.6 94.9 NSCancelled cycle – 6ET (mean ± SD) 3.52 ± 1.3 3.2 ± 1.5 NSImplantation rate (%) 19/169 (11.2) 24/140 (19.2) NSPregnancies 16 20Pregnancy rate/cycle 32.0 40.0Pregnancy rate/ET (%) 32.0 45.4 NSMultiple pregnancies 4 4Early pregnancy loss 0 2

346 U. Ulug et al.


tozoa can occur during several steps of the freeze–thaw

process, including the addition of cryoprotectant to the

tissue, cooling, thawing and the removal of the cryoprotec-

tant (Agca & Critser, 2002). Freezing of sperm can cause

swelling and rupture of the inner and outer acrosomal and

plasma membranes (Nogueira et al., 1999). The production

of oxygen radicals has been found to increase during both the

cooling and freeze–thaw processes (Chatterjee & Gagnon,

2001). Furthermore, cryopreservation of ejaculated sperma-

tozoa results in an increase in the proportion of sperm with

broken necks after thawing (Verheyen et al., 1997), and this

has been associated with a lower fertilization capacity.

Numerous studies, however, have demonstrated that FR

in ICSI cycles using frozen–thawed, surgically retrieved

spermatozoa did not differ significantly compared with FR

using fresh, surgically retrieved spermatozoa (Gil-Salom

et al., 1996; Friedler et al., 1998; Habermann et al., 2000;

Fukunaga et al., 2001; Makhseed et al., 2002; Ben Rhouma

et al., 2003; Park et al., 2003). Moreover, comparable

clinical outcomes, including PR and live birth rates, have

been obtained following FTEPS and FTTS (De Croo et al.,

1998; Habermann et al., 2000; Fukunaga et al., 2001).

However, several reports have indicated that the use of

FTTS results in lower FR and PR compared with fresh

testicular spermatozoa (Holden et al., 1997; Thompson-Cree

et al., 2003).

The impact of cryopreservation on clinical outcome

would be most clearly determined by comparing patients

undergoing consecutive cycles of ICSI with fresh and

frozen–thawed spermatozoa. It is therefore important that

most of the above-mentioned studies used retrospective or

historical data from unmatched control populations to

compare outcomes. Ideally, paired case–control studies can

reliably demonstrate the impact of cryopreservation of

surgically retrieved spermatozoa on clinical outcome. In this

regard, few studies have compared the outcomes of FTEPS

or FTTS with those obtained during previous ICSI matched

cycles using fresh epididymal or testicular spermatozoa

(Table 3). In one study (Wood et al., 2002), 13 couples

where the male had azoospermia underwent consecutive

ICSI cycles using fresh and FTEPS, and no difference was

observed in ICSI outcome. In a study of 19 paired patients,

both FR and PR did not differ between fresh and freeze–

thawed epididymal spermatozoa (Cayan et al., 2001).

Tournaye et al. (1999) observed no difference in ICSI

outcome in 67 couples who underwent ICSI cycles using

both fresh and FTEPS. Similarly, Friedler et al. (1998)

compared 13 cycles using FTEPS with 13 using fresh

epididymal spermatozoa and observed no differences in FR,

IR and PR. Wood et al. (2002) also found that FR, but not

PR, was decreased when FTTS was compared with fresh

testicular spermatozoa in 18 paired couples. Our results are in

substantial agreement with the earlier findings, in that we

found that FR was not affected in FTEPS but was reduced in

FTTS compared with their appropriate controls.

In this study, we have compared the clinical outcomes in

a large series of couples undergoing consecutive cycles of

ICSI using fresh and frozen–thawed surgically retrieved

spermatozoa. It should be considered, however, that

evaluating subsequent ICSI cycles makes the interpretation

of these results difficult, inasmuch as the frequency of a

second ICSI cycle can be determined to a large extent by the

failure of the first ICSI cycle. Thus, a significant proportion

of couples undergoing ICSI with frozen–thawed spermato-

zoa could have had negative results during their first ICSI

cycle using fresh spermatozoa, thus making comparisons

difficult. Thus, our finding of reduced IR and PR in patients

undergoing injections with fresh testicular spermatozoa

compared with subsequent injections with FTTS was not

unexpected. Moreover, previous paired case controlled

studies have shown a tendency towards increased PR when

using frozen–thawed spermatozoa after fresh spermatozoa

(Table 3). The higher success rates observed in couples

undergoing a second ICSI cycle may also result from

optimizing the stimulation of the female partner, not just

from sperm factors. However, in the current study, it should

also be noted that the total number of oocytes retrieved and

oocyte maturity, as assessed by metaphase II, did not differ

between cycles using fresh and frozen spermatozoa.

When we compared the outcome relative to the retrieval

site of frozen–thawed spermatozoa, we found that FR was

significantly lower in FTTS than in FTEPS, suggesting that

Table 3. Outcomes in paired ICSI cycles using fresh and frozen–thawed epididymal or testicular spermatozoa

Author Cycles (n) Retrieval site

Fresh spermatozoa Frozen–thawed spermatozoa

Fertilization rate (%) Pregnancy rate (%) Fertilization rate (%) Pregnancy rate (%)

Cayan et al. (2001) 19 Epididymis 58.4 31.6 62 36.8Wood et al. (2002) 13 Epididymis 58 15.3 57 23.7Tournaye et al. (1999) 67 Epididymis 60.1 32.1 53 35.2Friedler et al. (1998) 21 Epididymis 56 32 53 37Wood et al. (2002) 18 Testicle 71 11.1 53 27.7



testicular spermatozoa are more susceptible to the cryopre-

servation/thawing process than are epididymal spermatozoa.

The lower FR in the FTTS group is compatible with

previous results showing that FR in cases of non-obstructive

azoospermia is lower than that in cases with obstructive

azoospermia (Vernaeve et al., 2003; Wood et al., 2004).

The retrieval site of spermatozoa may also contribute to

the extent of cryoinjury and thus the clinical outcome. Our

results have shown that FTTS have a reduced fertilization

capacity relative to the identical fresh preparations. Similarly,

the overall FR of FTTS has been found to be lower than that

obtained using fresh testicular spermatozoa (De Croo et al.,

1998; Park et al., 2003). This decline in FR is possibly the

result of a dysfunction of the essential paternal centrosome

located in the neck region (Verheyen et al., 1997). Although

DNA damage was shown not to differ in fresh and FTTS

(Steele et al., 1999), when testicular sperm DNA was assessed

by the alkaline Comet assay, significant damage was observed

after slow freezing and fast thawing (Thompson-Cree et al.,

2003). In addition, the freezing and thawing processes may

affect the decondensation of the sperm nucleus after its

insertion into the oocyte (Sakkas et al., 1996). Moreover,

histone–protamine exchange during DNA condensation and

decondensation may be impaired more drastically in testi-

cular than in epididymal spermatozoa.

Cryopreservation has been found to have a detrimental

effect on the morphology of both testicular and ejaculated

spermatozoa because of the formation of intracellular ice,

which causes the plasma membrane to rupture (Mossad et al.,

1994; Verheyen et al., 1997; O’Connell et al., 2002). This,

in turn, may allow radical oxygen species to access sperm

nuclei, adversely affecting DNA integrity (Dalzell et al.,

2004). The cytoplasm of testicular spermatozoa is not

sufficient for antioxidant protection, thus permitting oxida-

tive damage (Aitken et al., 1998). Compared with epididy-

mal sperm, testicular sperm are more vulnerable because

their chromatin packaging is not completed until the SH

bonds are oxidized during transit through the epididymis.

Moreover, prolonged incubation after thawing of cryopre-

served testicular spermatozoa may damage nuclear DNA,

thus reducing the quality of sperm used for ICSI (Dalzell

et al., 2003).

In conclusion, we have shown here that surgically

retrieved spermatozoa can be efficiently used for ICSI after

freezing and thawing, without significantly compromising

outcome. Freezing of surgically retrieved spermatozoa

provides repeatable ART cycles, which can increase the

probability of conception for couples with male factor due to

azoospermia.

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Receipt 23 February 2005; revised 12 May 2005; accepted 16

May 2005



Outcomes in couples undergoing ICSI: comparison between fresh and frozen–thawed surgically...

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