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Elena Sereni received her Biological Sciences degree in 1995 from the University of Bologna, Italy. Her experimental thesis entitled ‘Spermatozoa and granulosa cells in co-culture’ was carried out at the Laboratory of Reproductive Physiopathology, S Orsola Hospital, Bologna. After graduation, she completed a 1-year training period at the hospital’s Assisted Reproduction Centre. Since October 1996 she has been working as an embryologist at the assisted reproduction centre Tecnobios Procreazione in Bologna. Her areas of interest include gamete studies such as sperm and oocyte cryopreservation.

Dr Elena Sereni

E Sereni, MA Bonu, L Fava, R Sciajno, L Serrao, S Preti, V Distratis, A Borini1 Tecnobios Procreazione, Via Dante 15, 40125 Bologna, Italy1Correspondence: Tel: +39 051 2867511; e-mail: borini@tecnobiosprocreazione.it

Abstract

Testicular fine needle aspiration (TEFNA) of spermatozoa in azoospermic patients in advance of intracytoplasmic sperm injection could be useful to avoid the possibility of no recovery of spermatozoa on the day of oocyte retrieval. The conventional freezing procedure for these spermatozoa is not appropriate because of their very low number and poor in-situ motility. This article presents a new procedure for the freezing of TEFNA-recovered spermatozoa. A total of 1063 spermatozoa (10–340 cells/sample) were frozen by this method for research purposes. Before freezing, 13.7% were motile. The recovery rate after thawing was 100%. After thawing, 3.6% motility was observed. In a separate study group, the total number of frozen spermatozoa was 431 (2–300 cells/sample). Before freezing, the sperm motility rate was 3.5%. After thawing, 100% of the spermatozoa were retrieved with a motility rate of 2.3%. One biochemical pregnancy was obtained. The procedure yielded excellent recovery and good motility rates after thawing. However, because of the low number of cases, any conclusion about the efficiency of the technique is premature.

Keywords: azoospermia, cryopreservation, freezing, testicular spermatozoa

Cryopreservation of human spermatozoa is considered a routine procedure. Since the first attempts (Polge et al., 1949; Bunge and Sherman, 1953), many studies have been conducted to develop the optimal freezing technique (Royere et al., 1996). Recovery rate and cell functionality after thawing are two important markers of the efficiency of the procedure. Intracytoplasmic sperm injection (ICSI) (Palermo et al., 1992) can compensate for the loss of spermatozoa after thawing in normospermic, but not in azoospermic patients, where a limited number of cells are available, particularly in non-obstructive azoospermia where very few spermatozoa are often retrieved.

Patients with obstructive azoospermia could not be treated until microsurgical epididymal sperm aspiration (Temple-Smith et al., 1985) and percutaneous epididymal sperm aspiration were introduced. When spermatozoa cannot be collected from the epididymis and in non-obstructive azoospermia, the only possibilities are testicular sperm biopsy or testicular sperm aspiration. Repeated testicular collections may negatively affect

testicular function (Schlegel and Su, 1997; Ron-El et al., 1998) and freezing the few collected spermatozoa becomes important.

Some authors have reported pregnancies using thawed spermatozoa obtained by testicular biopsy that were stored in traditional containers according to conventional freezing protocols (Fisher et al., 1996; Gil-Salom et al., 1996; Podsiadly et al., 1996; Allan and Cotman, 1997; Khalifeh et al., 1997; Perraguin-Jayot et al., 1997; Chia et al., 2000). One further technique was introduced to freeze a few spermatozoa using an empty oocyte zona pellucida (Cohen et al., 1997; Walmsley et al., 1998; Montag et al., 1999; Borini et al., 2000; Hsieh et al., 2000; Liu et al., 2000; Levi-Setti et al., 2003). However, the latter procedure generated many ethical problems and is no longer used.

At the study centre, testicular fine needle aspiration (TEFNA) is routinely performed before ICSI in azoospermic patients in case of suspected reduced testicular spermatogenesis. As far as is known, no reports have yet been published about freezing a

Article

Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique

Introduction

Article - Freezing a low number of spermatozoa: a new technique - E Sereni et al.

very low number of spermatozoa obtained by TEFNA. This article presents a new freezing procedure for the few spermatozoa obtained by diagnostic TEFNA from azoospermic men.

Materials and methods

Test samples

Between January 2000 and February 2002 azoospermic patients undergoing diagnostic TEFNA at the study centre consented to donate their spermatozoa for research purposes. The samples were frozen and then thawed to test the efficiency of the new testicular sperm freezing procedure (detailed below). Approval for the use of the donated spermatozoa was obtained from the internal review board.

Study samples

Subsequently, 15 patients underwent diagnostic TEFNA which revealed 14 with obstructive and one with non-obstructive azoospermia. The spermatozoa retrieved during the procedure were frozen (as detailed below) and stored until the day of oocyte retrieval.

Between September 2002 and January 2004, partners of the patients underwent an ICSI treatment. A second TEFNA was performed on the day of oocyte retrieval. Fresh spermatozoa were recovered from 10 out of 15 patients, but in one of these patients only immotile spermatozoa were present. In five patients, no spermatozoa were retrieved. Thus, in the six cases where no motile fresh spermatozoa were recovered the previously frozen TEFNA samples were used. These cases comprised the study samples.

TEFNA procedure

Testicular sperm aspiration was performed using a 23-gauge butterfly needle connected to a 60 ml syringe. After disinfecting the scrotal skin with iodine solution, the needle was inserted into the testicle. A negative pressure was created using a 60 ml syringe. An occlusive artery forceps was applied to the plastic tube to maintain suction and the needle was moved gently back and forward repeatedly and then withdrawn.

The 60 ml syringe was then replaced by a 2.5 ml syringe with 1 ml of HEPES-buffered human tubal fluid medium (Irvine Scientific, Santa Ana, California, USA) supplemented with 0.5% human albumin (IMMUNO, Italy). After removing the forceps, the medium was flushed through the butterfly needle into a 15 ml conical tube (Falcon, Becton Dickinson, USA).

Freezing procedure

After recovery, the testicular sperm suspension was immediately centrifuged for 5 min at 1800 g in the 15 ml conical tube (Figure 1a). The supernatant was then discarded and the pellet resuspended in 50 µl of sperm freezing medium (Medicult, Denmark) at room temperature. The sperm suspension was divided into 5 µl drops on a 60 mm diameter dish (Falcon, Becton Dickinson, USA), (Figure 1b). Drops (5 µl) of Sidney IVF oocyte wash buffer medium (Cook, Australia) were added to the dish and immediately removed, leaving a ‘footprint’ on

the dish, (Figure 1c,d). Next, all the microdrops were coated with 5 ml of mineral oil (Sidney IVF Culture Oil, Cook), thus obtaining a dish similar to those prepared for ICSI (Figure 1e).

Each microdrop was carefully checked to evaluate the number and motility of the cells (typically, testicular spermatozoa show poor in-situ movements) by means of an inverted microscope at ×200 magnification (Nikon Diaphot 200, Japan). The dish was then covered and wrapped in transparent plastic film (Domopack, Italy), (Figure 1f). After a brief incubation time at room temperature, the dish was first stored at 4°C for 15 min and then exposed to nitrogen vapour for 15 min and subsequently immersed in liquid nitrogen in a special tank fitted with a system of six racks, each with five shelves for boxes (LS 750, Taylor-Wharton, MidAtlantic Diagnostic) (Figure 1g).

Thawing procedure

The dish was kept at room temperature until completely thawed (Figure 2a,b) and then checked for the presence, number and motility of the spermatozoa.

A 5 µl drop of 3.6 mmol/l pentoxifylline (PF; Sigma, Germany) was added to each testicular sperm microdrop containing immotile spermatozoa. Sperm motility was evaluated after a 20 min incubation at 37°C. After pipetting the ‘footprints’ with 5 µl drops of fresh Sidney IVF oocyte wash buffer medium and Povidone K-90 (PVP Clinical Grade, Medicult, Denmark) (Figure 2c), the dish was ready to be used for ICSI (Figure 2d).

ICSI, fertilization and embryo development

After thawing, ICSI was performed as previously described by Borini et al. (1996). Motile spermatozoa were used when available. Each cell was transferred to a 10% PVP medium droplet and both motile and immotile cells were immobilized by puncturing their tails before ICSI. The oocytes were placed in droplets of Sidney IVF oocyte wash buffer medium and kept there by a holding pipette. Each sperm cell was aspirated into a micropipette and injected into the oocyte positioned with the polar body at 12 o’clock position. After the injection, the oocytes were incubated in cleavage medium (Cook) at 37°C in 6% CO2 atmosphere. About 16–18 h after ICSI, normal fertilization was observed. After an additional 24 hours in culture, embryo cleavage was assessed and transfer performed. The embryos were evaluated and scored according to blastomere size and rate of fragmentation (Puissant et al., 1987; Steer et al., 1992).

Results

Test samples

A total of 1063 spermatozoa were frozen for research purposes (Table 1). The number varied in the different samples from a minimum of ten to a maximum of 340. The recovery rate of the spermatozoa was 100% (1063/1063). A total of 146 spermatozoa were motile before freezing (146/1063; 13.7%). After thawing, 38 motile spermatozoa were observed (38/1063; 3.6%) (Table 1).90

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PF was added to each microdrop containing immotile spermatozoa. After incubation, 48 spermatozoa exhibited the weak motility that is a sign of viability (48/1063; 4.5%). At the end of the thawing procedure, a total of 86 viable cells was obtained (86/1063; 8.1%) (Table 1).

Study samples

The number of frozen spermatozoa varied from a minimum of two to a maximum of 300 in the six study samples (Table 2). The total number of frozen spermatozoa was 431. Before freezing,

the sperm motility rate was 3.5% (15/431). After thawing, the sperm cell recovery rate was 100% and ten motile spermatozoa were found (10/431, 2.3% motility rate). PF had no effect on spermatozoa that were immotile before freezing, and the total number of viable cells available for ICSI after the addition of PF was 15/431 (3.5%). Fifty-one oocytes were treated by ICSI (Table 3). In four out of six cases, only immotile spermatozoa were injected (Table 3). The fertilization, cleavage and good quality embryo rates were 17.6% (9/51), 77.8% (7/9) and 71.4% (5/7) respectively. An average of two embryos were transferred. One biochemical pregnancy was obtained.

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Figure 1. Description of the freezing procedure: (a) the testicular sperm suspension was centrifuged for 5 min at 1800 g in a 15 ml conical tube; (b) the sperm suspension was divided into 5 µl drops in a 60 mm diameter dish; (c,d) 5 µl drops of Sidney IVF Oocyte Wash Buffer medium were added and immediately removed to leave a ‘footprint’ on the dish; (e) all the microdrops were coated with 5 ml of mineral oil; (f) the dish was covered and wrapped in transparent plastic film; (g) the dish was immersed into liquid nitrogen and stored until thawing.

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Figure 2. Description of the thawing procedure: (a,b) the dish was kept at room temperature until completely thawed; (c) the ‘footprints’ were pipetted with oocyte wash buffer medium and 5 µl microdrops of povidone K-90 before injecting; (d) fully prepared dish, ready to use.

Table 1. Recovery and motility of frozen–thawed of spermatozoa from the test samples. Parameter Number (%)

Frozen spermatozoa 1063 (100)Motile frozen spermatozoa 146 (13.7)Recovered spermatozoa after thawing 1063 (100)Motile spermatozoa after thawing 38 (3.6)Motile spermatozoa after PF 48 (4.5)Vital spermatozoa after thawing 86 (8.1)

PF = pentoxifylline.

Table 2. Recovery and motility of frozen–thawed spermatozoa from the study samples (Patients 1–6).

Parameter (n) Patient Total (%) 1 2 3 4 5 6

Frozen spermatozoa 40 23 21 45 2 300 431 (100)Motile frozen spermatozoa 0 0 0 3 0 12 15 (3.5)Recovered spermatozoa after thawing 40 23 21 45 2 300 431 (100)Motile spermatozoa after thawing 0 0 0 2 0 8 10 (2.3)Motile spermatozoa after PF 0 0 0 3 0 2 5 (1.2)Vital spermatozoa after thawing 0 0 0 5 0 10 15 (3.5)

PF = pentoxifylline.

DiscussionTEFNA can be considered as a quick, simple and easy method to recover spermatozoa in azoospermic patients. One of the advantages is that it does not require anaesthesia or sedation and can therefore be performed at an IVF centre without difficulty.

Despite the simplicity of the technique, it has not been adopted routinely. Data in the literature are contradictory. Some authors (Foresta et al., 1992; Gottschalk-Sabag et al., 1993; Mallidis and Baker, 1994; Craft et al., 1997; Dajani et al., 1998; Lewin et al., 1999; Aridogan et al., 2003; Bettella et al., 2005; Amin et al., 2006) agree on the efficiency of the technique, while others (Friedler et al., 1997a; Ezeh et al., 1998; Shufaro et al., 2002) prefer open biopsy. However, many consider testicular fine needle aspiration as a reliable and simple method for the evaluation of testicular pathology in male-factor infertility (Srivastava et al., 2004; Bettella et al., 2005; Hussein et al., 2005; Viswaroop et al., 2005; Amin et al., 2006; Kumar et al., 2006).

Obviously, the chances of aspirating testicular spermatozoa are highest when spermatogenesis is active in the seminiferous tubule. If this is not the case, open testicular biopsy would still seem to be necessary. In azoospermic patients, not retrieving spermatozoa on the day of oocyte retrieval compromises the results of the treatment. Despite the high predictive value of diagnostic TEFNA in patients with non-obstructive azoospermia (Fasouliotis et al., 2002), freezing their spermatozoa might be very useful to avoid the risk of no recovery of spermatozoa on the day of oocyte retrieval. The development of this new freezing technique, that allows a very low number of cells to be frozen, might be especially useful in the treatment of non-obstructive azoospermia. The need to cryopreserve testicular spermatozoa obtained by fine needle aspiration should encourage the adoption of this new freezing procedure because the established freezing procedures cause cell loss after thawing (Borini et al., 2000).

Glycerol is the penetrating cryoprotectant of choice, because of its high buffering effect on human spermatozoa at low temperatures (Hammerstedt et al., 1990). On the other hand,

the use of yolk added to the cryoprotectant is still controversial in the case of testicular spermatozoa. Some authors use glycerol as cryoprotectant with yolk (Friedler et al., 1997b; Khalifeh et al., 1997; Liu et al., 1997; Rives et al., 1998) and others without (Allan and Cotman, 1997; De Croo et al., 1998; Walmsley et al., 1998). With testicular spermatozoa, the authors prefer to use a freezing medium without yolk, as the microdrops can be contaminated by yolk particles, thus reducing the rate of sperm recovery.

The classical ‘vehicles’ (cryovials or straws) are inadequate when the number of cells is very low. Also, the stress caused by centrifugation to wash and concentrate the sample after thawing makes classical storage systems unsuitable for spermatozoa obtained by TEFNA. It has been found that freezing these spermatozoa in straws using the conventional freezing procedure results in only 1% recovery after thawing (Borini et al., 2000).

Apart from the conventional system, several techniques exist for cryopreservation of a limited number of sperm cells. The use of the zona pellucida as a vehicle for freezing a few spermatozoa was introduced by Cohen et al. in 1997 (Walmsley et al., 1998; Montag et al., 1999; Borini et al., 2000; Hsieh et al., 2000; Liu et al., 2000; Levi-Setti et al., 2003). The zona pellucida seems to be a good vehicle for freezing spermatozoa obtained by TEFNA because of the good recovery rate (Borini et al., 2000), but it requires oocytes and a homologous system to be available, which cannot be obtained from the partner unless she decides to undergo oocyte retrieval specifically for this.

Other techniques have recently been developed as an alternative to conventional sperm cryopreservation procedures. Some authors have reported on sperm cryopreservation in ‘pills’ of testicular tissue obtained by testicular biopsy (Gil-Salom et al., 1996; Romero et al., 1996). More recently, Schuster et al. (2003) have reported on cryopreservation in ‘cryoloops’ of normospermic semen samples and others (Desai et al., 2004) have shown their efficiency with samples of poor semen. Herrler et al. (2006) have developed a method for freezing few spermatozoa in polymerized alginic acid drops.

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Table 3. Fertilization, cleavage and number of good-quality embryos following ICSI using the study samples (Patients 1–6). Parameter (n) Patient Total (%) 1 2 3 4 5 6

Injected oocytes 10 18 11 5 1 6 51 (100)Vital spermatozoa No No No Yes No Yes –Fertilized oocytes 4 3 0 1 0 1 9 (17.6)Cleaved embryos 3 2 0 1 0 1 7 (77.8)Good-quality embryos 2 2 0 1 0 0 5 (71.4)Embryos transferred 3a 2 0 0 0 1 6 (85.7)Embryos frozen 0 0 0 1 0 0 1 (14.3)

aBiochemical pregnancies.

Article - Freezing a low number of spermatozoa: a new technique - E Sereni et al.

Spermatozoa recovered from the testicles are often immotile or show a very poor in-situ motility. Despite their immotility, it is unreasonable to consider them not viable. Using PF after thawing, a considerable number of viable spermatozoa for ICSI were obtained. PF is a 3′,5′-nucleotidase phosphodiesterase inhibitor that enhances sperm motility by increasing intracellular cAMP. In this study, some spermatozoa were immotile before and after freezing, and also after adding PF. The stimulating effect of PF on sperm motility is well documented in the literature (Yovich et al., 1990; Tesarik et al., 1992), also in the case of testicular or epididymal spermatozoa (Terriou et al., 2000). Previous experience showed (Bonu et al., 2000) the efficacy of PF on immotile testicular spermatozoa and the authors favour the use of this motility activator over other systems (e.g. hypo-osmotic swelling test, HOST) (Casper et al., 1996; Barros et al., 1997; Angelopoulos et al., 1999). The incidence of false positives and the technical problems due to the low number of cells available make it difficult to use HOST for immotile spermatozoa obtained by TEFNA. These results show how PF enhances motility of immotile spermatozoa when motility is present before freezing, but cannot stimulate sperm motility if it is absent before freezing.

Many authors recommend the incubation of testicular spermatozoa for the selection of viable cells for ICSI (Liu et al., 1997; Morris et al., 2006), although others have evidence that incubation of fresh and frozen–thawed testicular spermatozoa may cause damage to DNA resulting in a significant increase in sperm DNA fragmentation (Dalzell et al., 2004). The cryopreservation of testicular spermatozoa has a negative effect on viability and on cell morphology too. In fact, intracellular ice formation may cause morphological alterations to plasma membranes that make sperm nuclei more sensitive to reactive oxygen species, so affecting DNA integrity (Dalzell et al., 2004). This could explain the low fertilization rate that has been obtained; a higher number of cases must be investigated to understand its causes.

In conclusion, it is possible to freeze very few spermatozoa collected by diagnostic TEFNA before ICSI treatment. Because of the small number of study samples, any final conclusions about the efficiency of the technique are premature. The procedure yields excellent recovery and good motility rates after thawing. Further investigations are necessary to test the fertilizing capability and efficiency of the thawed cells.

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Declaration: The authors report no financial or commercial conflicts of interest.

Received 31 July 2007; refereed 17 August 2007; accepted 3 October 2007.

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