1 Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy, 2 Laboratoire d'Eylau, Paris, France, 3 Urology department, University `La sapienza', Rome and 4 Andrology department, University of Pisa, Pisa, Italy
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Abstract |
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Key words: azoospermia/fertilization/ICSI/implantation/spermatozoa
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Introduction |
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Epididymal spermatozoa in conjunction with standard in-vitro fertilization (IVF) have been used for more than 10 years (Temple-Smith, 1985). However, the introduction of ICSI was able to improve fertilization and pregnancy rates in couples with microsurgically retrieved epididymal spermatozoa (Tournaye et al., 1994; Silber et al., 1994
), as well as to treat successfully most cases of obstructive azoospermia in which microsurgical epididymal sperm aspiration (MESA) could not be performed, by using spermatozoa retrieved from the testes (Schoysman et al., 1993
). Moreover, ICSI could be considered an effective fertility treatment for patients with non-obstructive azoospermia who hitherto had been considered irreversibly sterile (Devroey et al., 1994
, 1995
).
In the last 5 years several studies have been carried out to assess fertilization and pregnancy rates in ICSI using spermatozoa retrieved from the epididymis and from the testes in patients with obstructive and non-obstructive azoospermia, and good results have been reported (Tournaye et al., 1994, 1998
; Devroey et al., 1995
, 1996
; Nagy et al., 1995
; Craft et al., 1995
; Silber et al., 1995
, 1996
; Tsirigotis et al., 1996
; Holden et al., 1997
; De Croo et al., 1998
). Some authors, however, observed a better chance of conceiving with ICSI using spermatozoa from obstructed than non-obstructed azoospermic patients (Kahraman et al., 1996
; Aboulghar et al., 1997
; Mansour et al., 1997
; Ghazzawi et al., 1998
). Moreover, it was not clearly stated whether ejaculated spermatozoa even from patients with severe male factor infertility perform better than surgically retrieved spermatozoa when used in conjunction with ICSI. Because of the contradictions in the published reports, which may be partly due to sperm-unrelated factors, such as the female age and ovarian pathology, the actual impact of the source of spermatozoa and the underlying testicular pathology on sperm reproductive capacity is not known.
This is the first controlled study in which fertilization, pregnancy, implantation and abortion rates after ICSI using testicular spermatozoa from obstructed and non-obstructed men were compared with those using ejaculated spermatozoa from patients with severe male factor infertility.
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Materials and methods |
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Complete history, physical examinations and hormonal evaluations were performed. All patients were evaluated with karyotype examination of peripheral leukocytes and 19 patients with congenital absence of vas deferens (CAVD) were screened for the carrier status for cystic fibrosis (CF) by typing the most common CF gene mutation, F508 and other less common mutations in our population (W1282X, G542X, R117H, R553X, N1303K, R1162X, R334W, G551D, 17171G > A, 349 + 10 < kbC > T). All patients were informed about the current concerns (Cummins, 1997
; Tournaye and Van Steirteghem, 1997a
) regarding ICSI technique with testicular spermatozoa and the possible complications of the surgical procedures such as infection, testicular haematoma and impaired testicular blood flow (Schlegel and Su, 1997
).
Each testicular sperm ICSI cycle was matched with an ejaculated sperm ICSI cycle with semen parameters 5x106/ml and
10% progressive motility. The control cases were matched for female age (within 3 years), rank of trial, basal FSH serum concentrations and close proximity to the study group's procedure.
Ovarian stimulation and oocyte retrieval
Ovarian stimulation was conducted in all patients as follows: gonadotrophin releasing hormone agonist (GnRHa) (subcutaneous buserelin acetate 0.2 mg twice daily; Suprefact®; Hoechst, Marion Roussel, Milan, Italy) was started on day 21 of the menstrual cycle. When serum oestradiol concentrations were <40 pg/ml and no ovarian cystic structures were observed on ultrasound examination, ovarian stimulation was usually started with 150225 IU/day of FSH (Metrodin®; HP 75 IU; Serono, Rome, Italy) for 4 days. The FSH dose was adapted if no oestradiol rise was observed after 4 days by adding 75 IU FSH daily. Thereafter FSH was adapted individually according to the daily serum oestradiol increment. Ovulation was induced with 10 000 IU human chorionic gonadotrophin (HCG) (Profasi®; Serono) when serum oestradiol concentration exceeded 1000 pg/ml and when at least three follicles >18 mm in diameter were recorded by ultrasound. Oocyte retrieval was performed 36 h after HCG administration under transvaginal ultrasound-guided puncture of the follicles. Oocyte and ejaculated semen preparation as well as the ICSI procedure have been extensively described elsewhere (Rienzi et al., 1998).
Fertilization was considered normal when two clearly distinct pronuclei were present. Further embryonic development was assessed 24 h later. The embryos were classified according to the following morphological criteria. Type A or excellent embryos are defined as embryos in which all blastomeres have an equal or non-equal size without fragmentation. Type B or good embryos have blastomeres of equal or non equal size, with less than 20% of the volume of the embryo with anucleate fragments. Type C or fair embryos have anucleate fragments between 20% and 50% of the volume of the embryo (Rienzi et al., 1998). According to the number of good morphological quality embryos and the age of the patient, up to four embryos (in most instances only two) were replaced into the uterine cavity approximately 70 h after the micro-injection procedures.
Surgical techniques and sperm preparation
In obstructed azoospermic patients spermatozoa were recovered either by a percutaneous fine needle aspiration (FNA) technique or by an open excisional technique (TESE). In non-obstructed azoospermic patients single or multiple testicular biopsies were obtained by TESE techniques. In most cases the surgical procedures were performed a few hours after oocyte retrieval. No surgical complications were observed. All patients received a scrotal Doppler ultrasound examination 56 months after surgery and no cases of impaired testicular blood flow were seen.
Open testicular biopsy procedure
After scrotal disinfection with clorexidine digluconate the spermatic cord was infiltrated with 3 ml of 7.5% Norepine (ASTA, Milan, Italy). The testis was then positioned with the epididymis and vas deferens directed posteriorly and the scrotal contents were extruded through a small incision in the scrotal skin and in the tunica vaginalis. The albuginea was then opened through a small 0.5 cm incision and a small piece of extruding testicular tissue was excised. The testicular specimen was transferred into a small Petri dish with Gamete 100 medium (Scandinavian IVF Science AB, Gothenburg, Sweden). This biopsy was fragmented into small pieces by means of fine needles or sterile glass microscope slides on the heated stage of a stereomicroscope. This manipulation usually lasted 25 min depending on the number of the specimens. The presence of spermatozoa in the wet preparation of the shredded specimens was assessed under the inverted microscope at x200 or x400 magnification. If spermatozoa were observed, no further testicular incision was made. If no spermatozoa had been found repeated sampling was needed in order to observe spermatozoa and biopsies were taken from the contralateral testis whenever necessary. However, spermatozoa were not always observed even after repeated sampling. One single testicular specimen per testis was sent for histological analysis. The testicle was closed by layers using 40 catgut suture.
The pieces of biopsy tissue were then removed and the medium was centrifuged at 300 g for 5 min. The pellet was gently resuspended in medium. The sperm suspension was then kept at 37°C in the incubator in Gamete 100 medium until the ICSI procedure. An individual spermatozoon was picked up from the droplet of testicular concentrate and then placed in a droplet of 10% polyvinylpyrrolidone (PVP; Scandinavian IVF Science AB, Gothenburg, Sweden), which often results in complete immotility (Nagy et al., 1995). All possible efforts were made to select morphologically normal-appearing spermatozoa showing some signs of motility for injection. However, only immotile and morphologically abnormal spermatozoa were found and injected in some cases of non-obstructive azoospermia.
Fine needle aspiration procedure
Under local anaesthesia fine needle aspiration (FNA) was performed by using 21-gauge butterfly needles attached to a 20 ml syringe. With a single puncture the butterfly needle was percutaneously placed into the testis. Suction was then applied (1520 ml) and a small artery forceps was used to clamp the butterfly needle's microtubing. The needle was then moved in and out keeping the tip within the substance of the testis. Ten to twenty needle excursions were performed before withdrawing the needle from the testis. The 20 ml syringe was disconnected and an insulin syringe loaded with Gamete 100 medium was attached to the butterfly needle. After releasing the artery forceps the contents of the needle and the tubing were flushed into a 2003 Falcon tube. The aspirate was then checked at x400 magnification under an inverted microscope for the presence of spermatozoa.
For ICSI, the sperm suspension was put into separated droplets containing Hepes buffered medium. Spermatozoa with some signs of motility were collected from these droplets and transferred to the PVP drop. Selected spermatozoa were then used for the ICSI procedure.
Luteal-phase assessment and establishment of pregnancy
The luteal phase was supported by means of natural progesterone in oil, 50 mg/day i.m. (Prontogest® 100 mg; Amsa, Barberino del Mugello, Italy) starting on the day of the oocyte retrieval. Pregnancy was confirmed by serial rise in serum HCG concentrations on two consecutive occasions 12 days after embryo replacement. Clinical pregnancy was determined by ultrasound demonstration of cardiac activity at 7 weeks. An abortion was considered preclinical when HCG levels did not reach 1000 mIU/ml and no gestational sac was detected at the ultrasound examination.
Statistical analysis
Whenever indicated, the 2 test and the Student's t-test were applied at the 5% level of significance.
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Results |
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Male serum FSH levels were significantly higher in group IIa compared to group Ia and to groups Ib and IIb. However, in the non-obstructive group serum FSH levels were comparable between the group of patients with successful and failed sperm recovery (Table II). In groups Ib and IIb the mean ± SD sperm concentration and progressive motility were 2132x103 ± 1702x103/ml and 3.2% ± 4.9% respectively.
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Discussion |
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Silber et al. showed that diagnostic biopsy is predictive for the finding of spermatozoa in subsequent TESE, claiming that testicular histology in non-obstructive azoospermic men is homogeneous (Silber et al., 1997). Other authors (Devroey et al., 1995
; Gil-Shalom et al., 1995
; Tournaye et al., 1997b
) found different histological patterns. According to these different assumptions, attitudes vary concerning the number of testicular samples that should be taken when performing TESE procedures in non-obstructive azoospermic men: some perform multiple biopsies, while others perform a single testicular biopsy (Silber et al., 1997
). In the present study we took up to five testicular samples per testis in group IIa. If we had performed a single biopsy, we would have found no spermatozoa in 11 patients of group IIa. Similar results have been recently reported (Ezeh et al., 1998
; Hauser et al., 1998
). Performing multiple biopsies, however, lengthens operation time and may increase the risk of complications. Attention has been drawn to the injuries that can be inflicted on the testis due to devascularization and to inflammatory changes following testicular biopsy (Schlegel and Su, 1997
). These risks can be significantly reduced by careful observation of the distribution of blood vessels in the tunica albuginea to avoid cutting them and by the use of meticulous haemostasis. However, all patients should undergo colour Doppler and power Doppler sonography 36 months after TESE procedure to assess testicular function (Foresta et al., 1998
).
In obstructive azoospermic patients with normal spermatogenesis we used in all cases testicular spermatozoa recovered either by FNA or TESE. Similar results between epididymal and testicular spermatozoa have been reported (Mansur et al., 1996; Abuzeid et al., 1997) and TESE and FNA procedures are relatively simple, can be done under local anaesthesia, require less training, shorter operative time, no special equipment and show reduced costs compared to the microsurgical epididymal sperm aspiration procedure.
In the present study the mean number of cumulus-complexes oocytes retrieved/cycle and the fertilization rate were comparable between the four groups (Table III). Similarly the mean number of embryos transferred/cycle and the mean number of type A, type B and type C embryos transferred/cycle were comparable between the study groups and between the study and the control groups (Table II
). These data show that the severity of oligozoospermia or oligoasthenoteratozoospermia even with 100% abnormal morphology in the ejaculated control groups has no adverse effect on fertilization and cleavage rates (Nagy et al., 1995
) and that testicular spermatozoa either from obstructive or non-obstructive azoospermic patients perform as well as pathological ejaculated spermatozoa with regard to fertilization and cleavage rates. Similar results have been reported recently (Ghazzawi et al., 1998
).
We achieved 51.5 and 55.1% total pregnancy rates in obstructive and non-obstructive azoospermic patients respectively which compares favourably with their respective control groups (42.4 and 58.6%, groups Ib and IIb respectively). These figures could not be influenced by confounding factors of a technical nature because all work was performed only by two experienced biologists (Z.P.N., L.R) and one clinician (F.U.). The clinical pregnancy rates and the delivery/ongoing pregnancy rate did not differ statistically between the four groups, although in the non-obstructive group the delivery/ongoing pregnancy rate was 34.4% compared to 42.4% (obstructive group) and to 44.8% (control group). These results are in agreement with those reported by some (Devroey et al., 1995, 1996
; Tournaye et al., 1995
) but not all authors (Kahraman et al., 1996
; Aboulgar et al., 1997; Mansur et al., 1997; Ghazzawi et al., 1998
). However, the implantation rate was significantly lower in the non-obstructive group compared to the matched control group. This may be due to the fact that the severe impairment of the spermatogenesis in patients with non-obstructive azoospermia might have a genetic origin (Martin-du-Pan and Campana, 1993; Chandley, 1995
; Vogt, 1995
). Therefore, despite succeeding in extracting live spermatozoa in such patients, doubts have arisen about the developmental competence of the embryos obtained. Irrespective of the sperm genetic contribution, embryo viability can also be influenced by the cytoplasmic maturity of the spermatozoa used for ICSI. It should be emphasized that insufficiency of the reproducing element of the centrosome, the oocyte activating factor, nuclear proteins and other sperm cytoplasmic components (Sofikitis et al., 1998a
) may be responsible for the pre-implantation or post-implantation developmental failure after ICSI techniques using spermatozoa extracted from men with testicular defects. Furthermore, it is pertinent to note that an unambiguous distinction between a dysmorphic spermatozoon and an elongated spermatid is impossible with the means of observation that is currently used in ICSI, and this is particularly true in cases of disturbed spermatogenesis when spermatids are released prematurely from the Sertoli cells (Tesarik, 1997
). Embryos obtained by fertilizing oocytes with elongated spermatids have been reported to have a relatively low implantation potential compared to those obtained by ICSI with mature spermatozoa (Araki et al., 1997
; Vanderzwalmen et al., 1997
; Sofikitis et al., 1998b
). Cytoplasmic immaturity of the male germ cells used for assisted reproduction can result in fertilization failure, but it can also cause developmental abnormalities in embryos after apparently normal fertilization (Tesarik et al., 1998a
). Hence, the relatively low implantation rates in our group of patients with non-obstructive azoospermia may be due to inadvertent injection of elongated spermatids with incomplete cytoplasmic maturation in some of these cases. Because spermatids can complete the final phase of cytoplasmic maturation in vitro (Tesarik et al., 1998b
) previous in-vitro culture of testicular biopsy samples before ICSI may improve the implantation potential of the resulting embryos.
In conclusion, ICSI with surgically retrieved spermatozoa achieved fertilization and cleavage rates comparable to those obtained when pathological ejaculated spermatozoa were used. However, the implantation rate resulting from the use of testicular spermatozoa in non-obstructive cases was significantly lower then in obstructive and in ejaculated cases.
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Notes |
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References |
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Submitted on March 4, 1999; accepted on August 20, 1999.