The spermatogenesis rather than sperm retrieval method affects the outcome of ICSI for azoospermic patients
Sperm retrieval method affects the outcome of ICSI
The objective of this study was to investigate the clinical outcome of ICSI with epididymal and azoospermic patients with unequal spermatogenesis progress in order to understand the possible factors which might affect ICSI outcome. Totally, 92 obstructive azoospermic (OA) patients and 42 non-obstructive azoospermic(NOA) patients with hypospermatogenesis were included in this study. The cases with normal spermatogenesis were diagnosed as OA patients (OA group). The 92 OA patients attempted 112 ICSI cycles and were divided into two subgroups according to the sperm retrieval methods: 1) OA-PESA group (n=51) percutaneous sperm aspiration (PESA) cycles, and 2) OA-TEFNA group (n=61) testicular fine needle sperm aspiration (TEFNA) cycles. While the hypospermatogenesis cases were considered as NOA patients (NOA group), which performed 42 ICSI cycles, all NOA patients were treated with TEFNA. The results showed that there were no statistical differences for the fertilization, cleavage, clinical pregnancy and miscarriage rates between OA-PESA and TEFNA groups (65.4% VS 63.1%, 91.0% VS 87.4%, 43.1% VS 37.7%, 13.6% VS 17.4%, respectively). However, when considered NOA group with OA group, fertilization, cleavage and clinical pregnancy rates showed a significant difference (44.9% VS 64.1%，P<0.001, 79.8% VS 89.0%, P<0.001, 21.4% VS 40.2%, P= 0.047, respectively). Moreover, the miscarriage rate in NOA group was exteriorly higher although there was no statistical difference (33.3% VS 15.6%, P= 0.433). This study demonstrated that spermatogenesis rather than sperm retrieval methods affect the clinical outcome of ICSI in azoospermic patients.
More than 14 percents couples were suffering from infertility (Thonneau et al., 1991), while 10 percents of male infertility were azoospermia. The adventage of intracytoplasmic sperm injection (ICIS) technique developed by Palermo et al were used for some infertile couples with male factors (Palermo et al. 1992). From then on, the male infertility treatment improved a lot and new hope were given to those who were previously considered incurable male infertility. ICSI technique has been extensively used all over the world to obtain successful pregnancies with sperm surgically retrieved from the testis or the epididymis (Abuzeid et al. 1997; Craft et al. 1993; Lewin et al. 1996; Schlegel and Li 1998; Tournaye et al. 1994; Tsirigotis et al. 1995). The different kinds of methods have been developed to retrieve sperm for ICSI, including percutaneous sperm aspiration (PESA) (Shrivastav et al. 1994; Tsirigotis et al. 1995), microsurgical epididymal sperm aspiration (MESA) (Tournaye et al. 1994), testicular open biopsy (TOB), testicular fine needle aspiration (TEFNA) (Lewin et al. 1996), micro dissection testicular sperm extraction (MD-TESE) (Okada et al. 2002; Schlegel and Li 1998) and so on.. The former two approaches were proposed to retrieve epididymal sperm, while the lateral approaches were used to collect sperm from testis if the epididymal sperm was inaccessible. In 1996, testicular sperm retrieved from hypergonadotropic azoospermia patient by TEFNA were used for ICSI and obtained a live baby (Lewin et al. 1996). While MD-TESE was mainly performed to retrieve testicular sperm for NOA patients, including hypospermatogenesis, maturation arrest and even in Sertoli Cell Only (SCO) syndrome patients (Okada et al. 2002; Schlegel and Li 1998). However, it is not clear whether these different sperm retrieval methods affect the clinical outcome of ICSI or not.
Many studies were focused on the influence of sperm source and the spermatogenesis progress on the clinical outcomes of ICSI. Several studies found that the defect of spermatogenesis affects the clinical outcomes of ICSI (Ghazzawi et al. 1998; Monzo et al. 2001; Pasqualotto et al. 2002). However, it has been reported that embryos from testicular sperm maintain a superior development potential than that from epididymal sperm (Dozortsev et al. 2006).
The objective of this study was to compare the clinical outcomes of ICSI with testicular and epididymal sperm from azoospermic patients with normal and defect spermatogenesis in order to understand the possible factors involved in the outcomes of ICSI.
Table 1 shows the clinical outcome of ICSI from OA group and NOA group. Mean female and male ages, mean number of the injected oocytes per cycle and the mean number of transferred embryos were similar between OA group and NOA group, while the fertilization rate in OA group was significantly higher than that in NOA group (64.1% VS 44.9%, P <0.001). Moreover, the same trend of cleavage rate and clinical pregnancy rate was observed between the two groups (89.0% VS 79.8%, P <0.001, 40.2% VS 21.4%, P= 0.047, respectively). A higher miscarriage rate was observed in NOA group than that in OA group (33.3% VS 15.6%), but the trend was not statically significant.
Table 2 shows the results of the two subgroups (OA-PESA group (n=51) and OA-TEFNA group (n=61)). Basic parameters in the two subgroups, such as mean ages of the couples, number of the injected oocytes and the number of transferred embryos per cycle were not statistically different. There were no differences in fertilization, cleavage and clinical pregnancy rates between OA-PESA and OA-TEFNA groups (65.4% VS 63.1%， 91.0% VS 87.4%， 43.1% VS 37.7%，P>0.05, respectively). Moreover, miscarriage rate also did not reach a statistical difference between OA-PESA and OA-TEFNA groups (13.6% VS 17.4%, P>0.05).
It is well know that the use of ICSI was the milestone for the treatment of oligoasthenoteratozoospermia (OAT) (Palermo et al. 1992) and OA patients (Abuzeid et al. 1997; Tsirigotis et al. 1995), and even some NOA patients took some advantages from this novel micromanipulation (Mansour et al. 1997; Schlegel and Li 1998). However, for those azoospermic patients, the paternal gametes were obtained by various operations from epididymis, such as PESA, MESE. Those protocols were usually used for OA patients and always presented with abundant sperm for ICSI, sometimes with progressive motile sperm. The procedure of PESA was simple and with fewer complications. Moreover, the epididymal sperm were much easier to process before ICSI.
In addition to epididymal sperm aspiration, there was testicular sperm retrieval, such as TESE, TEFNA, MD-TESE. TESE was rarely used in recent years due to its more damages to testis compared with TEFNA. TEFNA was an effective and necessary procedure for azoospermic patients to evaluate the condition of spermatogenesis before ICSI attempts (Bettella et al. 2005; Zukerman et al. 2000). TEFNA was always performed when epididymal sperm were unavailable in some OA patients or for the sperm retrieval attempt of some NOA patients, such as hypospermatogenesis patients (Lewin et al. 1999). MD-TESE was the last attempt for NOA patients. It has been reported that sperm retrieval rate could be up to 81%, 44% for hypospermatogenesis and maturation arrest patients, respectively (Ramasamy et al. 2005). Encouragingly, in this retrospective study, 92 of the 237 SCO patients recover some sperm by MD-TESE. However, both the MESE and MD-TESE were expensive and time-consuming, so PESA and TEFNA were the suggested operations for azoospermic patients in our reproductive medicine center.
Palermo et al.(Palermo et al. 2002) analysis the chromosome 18, 21, X and Y of sperm from OA and NOA patients by fluorescent in-situ hybridization (FISH), found that the chromosome abnormalities rate was significantly higher in NOA patients compared with that in OA patients, and the sex chromosome aneuploidy was the predominant abnormalities. This finding was similar to the report of Rodrigo et al (Rodrigo et al. 2004). But whether the fertilization ability of testicular sperm reduced compared with that from epididymis was conflicting according to the previous studies.
Ghazzawi et al reported that sperm of the ejaculates, epididymis and testis from OAT patients, OA patients and NOA patients, respectively, were able to fertilize equally by using ICSI. However, pregnancy rate was significantly reduced in NOA patients compared to the other two groups (Ghazzawi et al. 1998). Another two studies also found that NOA patients had lower fertilization, embryo quality, and clinical pregnancy but higher miscarriage rate than those of OA patients (Monzo et al. 2001; Pasqualotto et al. 2002). Recently, a dissimilar result about the paternal impacts on embryos potential development ability of OA patients was reported by Dozortsev et al. (Dozortsev et al. 2006). The fertilization ability of testicular sperm, performed ICSI was lower than that of epididymal sperm, however, the following embryos demonstrated an superior implantation rate to the epididymal sperm.
In the present study, we took into consideration of the influence of spermatogenesis on the outcome of ICSI by comparing OA group and NOA group. A significant higher fertilization , cleavage and clinical pregnancy rates were observed in OA group compared with that in NOA group. Moreover, the miscarriage rate in NOA group was 33.3% compared with 15.6% in OA group but did not reach a statistical difference. These findings were generally concordant to most of the previous reports, which referred the defect spermatogenesis affected the outcome of ICSI (Balaban et al. 2001; Ghazzawi et al. 1998; Monzo et al. 2001; Pasqualotto et al. 2002).
When only cases of OA patients were analyzed, there was no difference of fertilization rate, cleavage rate, clinical pregnancy rate and miscarriage rate between OA-PESA group and NOA-TEFNA group, which was in agreement to some of the previous studies (Balaban et al. 2001; Nicopoullos et al. 2004). But the date was not in concordance with Dozortsev's report (Dozortsev et al. 2006). Some studies may explain these controversial findings. O'Connel et al. detected mtDNA and determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from OA patients, and investigated that mtDNA and nDNA of testicular sperm have fewer mutation and fragmentation than epididymal sperm (O'Connell et al. 2002). It was universally acknowledged that epididymis was the essential organ for sperm maturation in a normal physiological condition. However, one clinical study suggested that once the sperm enter the vas deference, those fresh sperm less than 7 days maintained the best fertilization ability and developmental potential(Pellestor et al. 1994). Levitas et al. analyzed 9,489 semen samples found that the peak mean sperm motility was observed after 1 day abstinence(Levitas et al. 2005). Both of the studies suggested that long period staying in spermatic duct was not good for sperm motility and function. Nicopoullos et al. analysed their own date of ICSI outcome from 154 OA patients and demonstrated that fertilization and live-birth rates were highest in men with a previous vasectomy and no infective cause (vasectomy 51% and 23%; not infective 53% and 29%, respectively) and lowest in men with infective or inflammatory causes (Nicopoullos et al. 2004). We often found that epididymal sperm from those infective caused OA patients were less motile, sometimes entirely immotile. This may due to hydrolyzing enzymes, hyaluromidase enzymes from inflammatory cells and acrosin from dead and dying sperm acrosome (Cummins et al. 1994).
In conclusion, the clinical outcome of ICSI was affected by the spermatogenesis process of the patients rather than the sperm retrieval methods. Moreover, in OA patients, PESA was the dominant choice for sperm retrieval because of its simple procedure, less complication and reasonable pregnancy rate. However, if no motile sperm presented or abundant inflammatory cells mixed together, testicular sperm would attribute to a stable clinical ICSI outcome.
MATERIALS AND METHODS
A total of 138 azoospermic patients who performed 154 ICSI cycles from January 2006 to January 2009, were involved in this study. All the patients performed at least 3 times semen analysis in our laboratory, using centrifugation at 1800g for 10 min failed to recover sperm. 92 men with normal hormone level were diagnosed as OA patients (OA group) whom were confirmed by one of the following evidences, abundant sperm were discovered in the epididymal fluid during PESA or normal spermatogenesis histopathologically diagnosed by TEFNA before ICSI attempt. 62 men had an acquired epididymal obstruction due to a history of epididymitis, all these patients present a turgescent epididymis or an epididymal cyst during physical examination, and some of them were confirmed by ultrasound. 21 men have a history of vasectomy and failed vasovasotomy. Another 9 congenital bilateral absence of vas deferens (CBAVD) were also involved in our study. They were lack of vas deferens with obvious laboratorial characteristics: low semen volume, semen PH<7 and very low or absence fructose in the seminal plasma, confirmed with the absence of seminal vesicle by transrectal ultrasound. The OA group was subdivided into two groups according to the sperm retrieval methods, 1) OA-PESA group: cycles with epididymal sperm (n=51) and 2) OA-TEFNA group: cycles with testicular sperm (n=61). For all the OA patients, PESA were performed firstly, if no sperm was found or epididymal fluid was mixed with too many inflammatory cells, TEFNA will be performed to retrieve testicular sperm for ICSI. There were totally 46 NOA patients attempted ICSI during the investigating period, a diagnostic TEFNA was done before ICSI attempt and confirmed as hypospermatogenesis by histophathology evaluation. Unfortunately, four of them failed to retrieve sperm from their testes on the oocytes retrieval day and changed to use the donative sperm. Finally, 42 NOA patients were involved in present study as NOA-group. All these NOA patients were treated with TEFNA.
Ovarian stimulation and oocytes preparation
Either a long or a short protocol of controlled ovarian stimulation was used for their partners. After the administration of GnRHa (Enantone, Takeda, Japan) during luteal phase or follicular phase, purified follicle-stimulating hormone (Gonal-F; Serono, Rockland, MA) and human menopausal gonadotropin (HMG, Lizhu, China) were injected for ovarian stimulation. Human chorionic gonadotrophin (hCG; Lizhu, China) was used when two or more dominant follicles in diameter were observed. 36 hours later, oocytes were retrieved by transvaginal ultrasound guided puncture.
After retrieval, oocytes were removed into microdroplets of culture media (IVF, Vitrolife, Sweden) and covered with mineral oil (IVF, Vitrolife, Sweden) and stored in an incubator for 2-4 hours. Before micromanipulation, the oocytes were briefly exposed to hyaluronidase (Type VIII; Sigma, St. Louis, MO) for 30 seconds, and the cumulus-corona complex was removed by pippetting with a plastic pipette with an inner diameter of 135μm. Each oocyte was rinsed in a series of droplets of medium to remove any remaining hyaluronidase. Finally the oocytes were kept in Gamate 100 medium (IVF, Vitrolife, Sweden) under mineral oil (IVF, Vitrolife, Sweden). Then, each oocyte was examined under an inverted microscope at ×200 magnification to assess the maturation stage. Only metaphase Ⅱ (M Ⅱ) stage oocytes were selected to perform intracytoplasmic sperm injection.
PESA and TEFNA procedures
Both PESA and TEFNA were performed under local anesthesia and spermatic cord blocking. For the PESA procedures, the caput of epididymis was held between the thumb and forefinger. A 23-gauge needle connected with a 20 ml syringe filled with culture medium was inserted into the turgescent caput, continuous suction was applied as epididymal fluid was aspirated. The epididymal fluid was examined under an inverted microscope directly. For the TEFNA procedures, the testis was held in the left hand, an avascular site was chosen to perform the puncture. A 23-gauge needle connected with a 20 ml syringe was inserted into the selected site. Puncture the testis with the fine needle back and forth, synchronized with an alternately negative pressure. Pull out the needle connected with seminiferous tubules and pull out as much tubules as possible with two micro forceps. The retrieved tubules were washed one to two times to remove the red blood cell, then minced into cell suspension with two 1 ml syringes, and checked under an inverted microscope. If no sperm was found, at most four sites on each testis were punctured.
Epididymal and Testicular sperm processing
After PESA, the epididymal fluid was placed in a petri dish and examined for the presence of sperm. If sperm were found, they were transferred into a 15 ml conical tube containing 5 mL of Fertilization medium (Cook, Aus). The suspension was centrifugated at 800 g for 1 min. The pellet was kept in the tube in case of no motile sperm were found in the supernatant. The supernatant was removed into another clean tube, centrifuged at 1000 g for 10 min, the pellet was washed again with 3 ml Fertilization medium (Cook, Aus) and centrifugated at 1000 g for 5 min. The final pellet was resuspended with 0.3-0.5 ml Gamate 100 medium (Vitrolife, Sweden). 10 μl was used for sperm evaluation. If no sperm was found, the pellet after first centrifugation was washed with the same procedure to look for sperm. The final suspension was incubated in 5% CO2 at 37℃ until use.
For testicular sperm processing, the seminiferous tubules were placed in a petri dish and minced into cell suspension with two 1 ml syringes. Removed all the cell suspension and tissues into a new 15 ml conical tube, contain 5 ml Fertilization medium (Cook, Aus). The suspension was centrifugated at 800 g for 30 seconds. The pellet was kept in the tube in case of no motile sperm were found in the supernatant. The supernatant was removed into another clean tube, washed twice as the same procedure mentioned before. The final pellet was resuspended with 0.3- 0.5 ml Gamate 100 medium (Vitrolife, Sweden). 10 μl sample was used for sperm evaluation. If no sperm was found, the resuspended pellet after first centrifugation was washed with the same procedure to look for available sperm. Testicular sperm was cultured in a incubator in 5% CO2 at 37℃ for 2- 4 hours before ICSI. In most cases, viable sperm began to twitch after several hours culture. These slight motile sperm was selected for ICSI. When no motile sperm was detected, sperm was chosen on the basis of normal morphology.
Intracytoplasmic sperm injection
Intracytoplasmic sperm injection (ICSI) was performed at ×400 magnification stage phase-contrast inverted microscope (Olympus Japan). M Ⅱ stage oocytes were placed in Gamete medium drops (IVF, Vitrolife, Sweden). Epididymal sperm was placed in PVP c(Polyvinilpirrolidone, Vitrolife, Sweden) for immobilization, while oligodynamic testicular sperm was placed in a Gamete medium micro droplet (IVF, Vitrolife, Sweden) directly for ICSI. The tail of the sperm was mechanically immobilized by the tip of the microinjection needle (Cook, USA) and aspirated tail-first inside the needle. The oocyte was held with a holding micropipette (Cook, USA) with polar body at the 6 o'clock position. The sperm was injected into the oocyte at 3 o'clock position by using electrohydraulic micromanipulators (Olympus, Japan). Each injected oocyte was transferred into one micro droplet with Gamate medium (IVF, Vitrolife, Sweden) covered with mineral oila for single embryo culture.
Fertilization and embryo culture
The injected oocytes were incubated for 16-18 hours in 6.5% CO2 at 37℃ before fertilization evaluation. The fertilization was confirmed by the presence of two pronuclei and two polar bodied at ×200 magnification under stage phase-contrast inverted microscope (Olympus, Japan). Embryos cleavage were assessed 24 hours later, and two to three embryos were retransferred into a new balanced micro droplet for another 24 hours culture in 6.5% CO2 at 37℃. 48 hours after fertilization, two to three morphologically good quality embryos were selected by two experienced embryologists for embryo transfer. The left morphologically good quality embryos were frozen by vitrification.
Embryo transfer and clinical result
Embryo transfer was performed 48 hours after fertilization assessment. The selected morphologically good quality embryos were loaded into an Edwards-Wallace catheter (SIMS Portex Ltd, Kent, UK). The catheter was inserted into uterine cavity under ultrasound guiding. After embryos transfer, the catheter was checked under dissecting microscope to confirm all the embryos were transferred into the uterine. Luteal phase support started on the day of oocyte retrieval with 60 mg progesterone intramuscular injection everyday. The beta hCG serum level was assessed 14 days after embryos transfer for chemical pregnancy. A clinical pregnancy was defined as the visualization of a heartbeat by transvaginal ultrasound at 5 weeks after embryos transfer.
Numerical variables between two groups were compared using t test. Categorical variables were compared using chi-square test. Results were considered to be significant when P<0.05.
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