Outcome of testicular sperm retrieval procedures in non-obstructive azoospermia: percutaneous aspiration versus open biopsy

Ramazan Mercan, Bulent Urman1, Cengiz Alatas, Senai Aksoy, Alp Nuhoglu, Aycan Isiklar and Basak Balaban

Assisted Reproduction Unit, American Hospital of Istanbul, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to evaluate whether the extraction of testicular spermatozoa with percutaneous versus open biopsy has an effect on the treatment outcome with intracytoplasmic sperm injection (ICSI) in men with non-obstructive azoospermia. Regardless of testicular size, follicle stimulating hormone concentration, and previous biopsy result, percutaneous testicular sperm aspiration (PTSA) using a 21-gauge butterfly needle was attempted first and if this failed testicular sperm extraction (TESE) was performed. In 63 men spermatozoa were found with PTSA whereas in 228 men TESE had to be undertaken. More men in the PTSA group had previously been diagnosed with hypospermatogenesis (82 versus 50%). Compared with the PTSA group, more men in the TESE group had germ cell aplasia (27 versus 10%) or maturation arrest (22 versus 8%). There was no difference between the groups regarding mean age of men and their partners, duration of stimulation, oestradiol concentration on the day of human chorionic gonadotrophin, number of oocytes retrieved, fertilization rate, and embryo quality between the two groups. The number of embryos transferred (4.38 versus 3.90) was significantly higher in the PTSA group (P < 0.05), reflecting the increased number of embryos available for transfer. Implantation rate per embryo was 20.7% in the PTSA and 13.3% in the TESE group (P < 0.05). Clinical pregnancy rates were 46 and 29% in the PTSA and TESE groups respectively (P < 0.05). Clinical abortion rates were similar (21.2 versus 24%). It is concluded that in men with non-obstructive azoospermia, easier sperm retrieval, which is most likely indicative of a more favourable histopathology, is associated with higher implantation rates per embryo.

Key words: azoospermia/pregnancy/percutaneous testicular sperm aspiration/testicular sperm extraction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The utilization for intracytoplasmic sperm injection (ICSI) of spermatozoa extracted from the testes was first reported in 1993 (Craft et al., 1993Go) in men with obstructive azoospermia. However, within a short period of time indications for testicular sperm extraction (TESE) were expanded to include subjects with azoospermia due to non-obstructive causes. In most of the early studies, comparable fertilization and pregnancy rates were achieved with testicular and ejaculated spermatozoa (Silber et al., 1995Go; Fahmy et al., 1996Go; Tarlatzis et al., 1996). It was shown that there was no difference in fertilization and pregnancy rates between ejaculated spermatozoa from oligoasthenoteratozoospermic men, and epididymal spermatozoa from men with obstructive azoospermia and testicular spermatozoa from men with non-obstructive azoospermia (Ghazzawi et al., 1998Go). However, in two recent studies fertilization and pregnancy rates were reported to be significantly higher in cases of obstructive azoospermia when compared to non-obstructive azoospermia (Kahraman et al., 1996Go; Mansour et al., 1997Go). Similarly, significantly lower fertilization and pregnancy rates were shown (Aboulghar et al., 1997Go) with ICSI of testicular spermatozoa from non-obstructive cases compared with ICSI of ejaculated spermatozoa or spermatozoa from obstructive azoospermia cases.

Percutaneous epidydymal sperm aspiration (PESA) is the widely accepted and most commonly practised method of sperm retrieval when obstruction is present. In a retrospective controlled study (Tournaye et al., 1998Go) no difference was shown in fertilization, cleavage, and implantation rates when open biopsy was compared to fine needle aspiration in azoospermic men with normal spermatogenesis. When spermatogenesis is absent or only focal, the yield of percutaneous sperm aspiration or fine neeedle aspiration is presumed to be low and open testicular biopsy is usually preferred. However, there have been no studies evaluating the effectiveness of percutaneous testicular sperm aspiration (PTSA) in a large series of patients with non-obstructive azoospermia. In this study we report the results of 291 ICSI cycles undertaken in men with non-obstructive azoospermia where spermatozoa were searched for initially using a percutaneous approach. A TESE procedure was performed when the percutaneous approach failed to yield spermatozoa. The outcome of the two procedures was compared.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
This study included a total of 291 men with azoospermia of non-obstructive origin who yielded spermatozoa during PTSA or in a subsequent TESE. The total number of patients who underwent a first cycle of PTSA/TESE was 452. Of these, spermatozoa were retrieved in 291 (64.4%) and 13 of the patients with positive TESE cycles patients underwent a second, four patients a third and one patient a fourth TESE cycle. All these repeat treatment cycles were excluded from the analysis. When both PTSA and subsequent TESE were negative, a histopathological confirmation was obtained. All negative patients showed either complete maturation arrest or germ cell aplasia.

Of the 291 procedures, spermatozoa were found in 63 with PTSA (group 1), whereas a TESE had to be performed in 228 (group 2). The study period was from July 1996 to June 1999. All patients first underwent PTSA, and TESE was performed if spermatozoa were not found. Fertilization, cleavage and pregnancy rates were compared between the group who yielded spermatozoa to PTSA (group 1) and the group that proceeded to TESE (group 2).

Ovarian stimulation, oocyte retrieval and embryo transfer procedures
In the majority of treatment cycles ovarian stimulation was undertaken using subcutaneous buserelin acetate (Suprefact proinjection; Hoechst AG, Frankfurt am Main, Germany) in a long protocol combined with pure FSH (Metrodin 75; Serono, Rome, Italy). Buserelin acetate (0.3 mg/day) was commenced on day 20 or 21 of the preceding cycle and continued until the day of human chorionic gonadotrophin (HCG). In cycles where the woman was predicted to respond poorly to ovarian stimulation a flare gonadotrophin releasing hormone agonist (GnRHa) protocol was used. FSH was initiated on the third day of the menstrual cycle with 2–6 ampoules depending on the patient's previous or anticipated response. The treatment was then individualized in a step-down fashion. When the leading follicle reached 20 mm in mean diameter with a serum oestradiol concentration of 200–300 pg/ml per mature follicle 10 000 U HCG (Profasi HP 5000; Serono) was administered. Oocyte retrieval was performed 36 h after the injection of HCG. ICSI was performed according to conventional protocols (Van Steirteghem et al., 1993Go) and only on metaphase II oocytes. Two to five embryos were replaced 2 or 3 days after retrieval according to number of embryos available, embryo quality and age of the woman. A serum pregnancy test was performed 10–12 days after embryo transfer. Clinical pregnancy was defined as the presence of gestational sac/s with a viable embryo shown on vaginal ultrasonography performed ~24 days after embryo transfer.

Testicular sperm aspiration and extraction procedures
Both PTSA and TESE specimens were processed in the same manner. Procedures were performed under general anaesthesia or local anaesthesia with i.v. sedation. All biopsies were performed bilaterally if spermatozoa were not retrieved from the first side. All procedures were performed 24–48 h prior to oocyte retrieval. This eased the workload on laboratory and operating room personnel. Results from a previous study showed that this had no deleterious effect on sperm viability (Urman et al., 1998Go). Scheduling PTSA/TESE 48 h prior to oocyte retrieval also obviated the need to administer HCG to the woman at risk of developing ovarian hyperstimulation syndrome in case spermatozoa could not be retrieved. PTSA was performed with a 21-gauge Butterfly needle that was inserted into the testes and moved up and down to sample a wide area. An artery forceps was secured across the attached microtubing set before the needle was withdrawn. The aspirate located in the tubing was washed into a Falcon tube (Becton Dickinson, Franklin Lakes, NJ, USA) with a small volume of media. The presence of spermatozoa was sought under x200 magnification. Percutaneous aspiration was attempted from three different areas of the testis and if spermatozoa were not observed a TESE procedure was performed.

Tissue samples measuring in size from 0.5x0.5x0.5 to 1x1x1 cm were removed until spermatozoa were identified or 4–5 biopsy pieces were extracted from each testis. Testicular tissue samples thus obtained were placed into Falcon tubes containing 2 ml EBSS medium (Earle's balanced salt solution; Sigma, Aldrich Co. Ltd, Irvine, UK) supplemented with HSA (human serum albumin; Irvine Scientific, Santa Ana, CA, USA) penicillin and pyruvate. Tissue samples were then divided into small segments, and gently crushed with micro-needles in a Petri dish containing the same medium. The sperm suspension thus obtained was then centrifuged for 5 min at 300 g in a Falcon tube. If sperm cells were identified, the suspension was centrifuged on a two-layer Percoll gradient (50% and 90%) to free the sperm from debris and red blood cells. Both gradients were washed with HSA-supplemented EBSS. Following centrifugation, the supernatant was removed and the final pellet in the HAS-supplemented EBSS solution was prepared as a swim-out droplet covered with mineral oil (Sigma). The droplet was incubated at 37°C in 5% CO2 until the ICSI procedure. As part of a concurrent study, in-vitro culture of testicular tissue samples with recombinant FSH was undertaken in 143 cycles (Balaban et al., 1999Go).

Statistical analysis
The age of patients, duration of stimulation, number of ampoules used, number of embryos transferred, oestradiol on the day of HCG, number of eggs retrieved and endometrial thickness were compared using t-test; pregnancy rates were compared using {chi}2-test. P < 0.05 was accepted as significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous testicular biopsy results were available for 50 patients in the PTSA group and 129 patients in the TESE group. In the PTSA group, 41 had hypospermatogenesis, five had germ cell aplasia, and four had incomplete maturation arrest. In the TESE group, 65 had hypospermatogenesis, 35 had germ cell aplasia, 19 had incomplete maturation arrest, and 10 had complete maturation arrest (Table IGo).


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Table I. Testicular pathology as documented in a previous diagnostic biopsy
 
There was no difference regarding the partner's age, duration of infertility, days of gonadotrophin stimulation, number of FSH ampoules used, oestradiol concentrations on the day of HCG, endometrial thickness, number of oocytes retrieved, and two pronuclear fertilization rate, and cleavage rate between the two groups (Table IIGo). Embryo quality was graded (Staessen et al., 1992Go). The number of G1+G2 and G3+G4 embryos on day 2 or 3 was similar in the two groups. Implantation rates in the PTSA and TESE groups were 20.7 and 13.3% respectively (P < 0.05). Clinical pregnancy rate per embryo transfer was 46.0% in the PTSA and 28.9% in the TESE group (P < 0.05).


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Table II. Cycle characteristics and outcome of embryo transfer
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study show that in men with non-obstructive azoospermia, easier sperm recovery, which is most likely reflective of a lesser degree of spermatogenetic impairment, is associated with a higher implantation and pregnancy rate following ICSI when PTSA is used compared with TESE. Embryos implanted at a significantly higher rate when derived from ICSI of spermatozoa recovered by PTSA compared with TESE. A higher proportion of men in the needle aspiration group had hypospermatogenesis reported in their previous diagnostic biopsies compared with a higher incidence of germ cell aplasia and complete maturation arrest in the TESE group. It has been reported that the incidence of chromosomal abnormalities is higher in patients with oligoasthenoteratozoospermia (OATS) and azoospermia (Chandley et al., 1995) and the incidence of these anomalies is positively correlated with the severity of spermatogenetic impairment (Retief et al., 1984Go; Yoshida et al., 1995Go). Several studies showed an increased rate of deletions of DNA sequences in the long arm of the Y chromosome, Robertsonian translocations of chromosomes 13, 14 and 21 in men with OATS (Ma et al., 1992Go; Johannison et al., 1993; Koyabashi et al., 1994Go; Moosani et al., 1995Go; Pang et al., 1995Go; Rejio et al., 1995Go; Martin, 1996; In't veld et al., 1997Go; Lahdetie et al., 1997; Bernardini et al., 1998Go). The incidence of these chromosomal abnormalities is probably increased with increasing severity of the spermatogenetic defect, thus resulting in genetically abnormal embryos that have a lower implantation potential.

It has been shown that abnormal sperm decondensation is an important component of fertilization failure (Dozortsev et al., 1994Go). Alterations in sperm chromatin might result in defective decondensation and DNA activation during fertilization that may result in fertilization failure, early embryonic wastage or poor embryonic development (Hamamah et al., 1997Go). It is also known that maternal genes control the first two cell division cycles of the embryo (Braude et al., 1988Go). The paternal genetic effect is not exerted until the 4-cell stage; therefore, any potential detrimental paternal effect would only be observed after that particular stage. Evidence has been provided for a strong paternal effect on human preimplantation embryo development by studying blastocyst formation in co-culture (Janny and Ménézo, 1994Go). These workers compared blastocyst formation in IVF cycles undertaken with either normal spermatozoa, donor spermatozoa, or spermatozoa from severely oligozoospermic men. The overall number of blastocysts and the number of patients having at least one blastocyst were severely reduced when spermatozoa from severely oligozoospermic men were used to inseminate the oocytes.

Delayed fertilization and poor embryonic development were reported to be associated with impaired semen quality following IVF (Ron-El et al., 1991Go). It was later suggested that the spermatozoon is involved in the embryonic quality, even in early stages of development, and that this shows an association between abnormal sperm morphology and poor embryo morphology (Parinaud et al., 1993Go).

It is possible that the severity of spermatogenic defect is associated with ease of sperm retrieval and affects embryo implantation. There may be chromatin defects and DNA abnormalities in spermatozoa from men with non-obstructive azoospermia and the frequency of these abnormalities may be related to testicular pathology or the severity of spermatogenic impairment. It seems reasonable to conclude from our results that, in couples with non-obstructive azoospermia, implantation potential is significantly lower in patients who require TESE after failing PTSA. The difference is most probably due to the severity of testicular pathology, i.e. the more severe the spermatogenic defect, the more difficult the retrieval of spermatozoa from the testis.


    Notes
 
1 To whom correspondence should be addressed at: Bulent Urman M.D., VKV American Hospital, Guzelbahce sok. No:20, Nisantasi 80200, Istanbul, Turkey. E-mail: burman{at}superonline.com Back


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 Materials and methods
 Results
 Discussion
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Submitted on December 30, 1999; accepted on April 3, 2000.