The aim of this survey was to look into the clinical result of ICSI with epididymal and azoospermic patients with unequal spermatogenesis advancement in order to understand the possible factors which might impact ICSI result. Wholly, 92 clogging azoospermic ( OA ) patients and 42 non-obstructive azoospermic ( NOA ) patients with hypospermatogenesis were included in this survey. The instances with normal spermatogenesis were diagnosed as OA patients ( OA group ) . The 92 OA patients attempted 112 ICSI rhythms and were divided into two subgroups harmonizing to the sperm retrieval methods: 1 ) OA-PESA group ( n=51 ) transdermal sperm aspiration ( PESA ) rhythms, and 2 ) OA-TEFNA group ( n=61 ) testicular all right needle sperm aspiration ( TEFNA ) rhythms. While the hypospermatogenesis instances were considered as NOA patients ( NOA group ) , which performed 42 ICSI rhythms, all NOA patients were treated with TEFNA. The consequences showed that there were no statistical differences for the fertilisation, cleavage, clinical gestation and abortion 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 % , severally ) . However, when considered NOA group with OA group, fertilisation, cleavage and clinical gestation rates showed a important difference ( 44.9 % VS 64.1 % i??P & A ; lt ; 0.001, 79.8 % VS 89.0 % , P & A ; lt ; 0.001, 21.4 % VS 40.2 % , P= 0.047, severally ) . Furthermore, the abortion rate in NOA group was exteriorly higher although there was no statistical difference ( 33.3 % VS 15.6 % , P= 0.433 ) . This survey demonstrated that spermatogenesis instead than sperm retrieval methods affect the clinical result of ICSI in azoospermic patients.
More than 14 per centums twosomes were enduring from sterility ( Thonneau et al. , 1991 ) , while 10 per centums of male sterility were azoospermia. The adventage of intracytoplasmic sperm injection ( ICIS ) technique developed by Palermo et Al were used for some sterile twosomes with male factors ( Palermo et al. 1992 ) . From so on, the male sterility intervention improved a batch and new hope were given to those who were antecedently considered incurable male sterility. ICSI technique has been extensively used all over the universe to obtain successful gestations with sperm surgically retrieved from the testicle 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 sorts of methods have been developed to recover sperm for ICSI, including transdermal sperm aspiration ( PESA ) ( Shrivastav et al. 1994 ; Tsirigotis et Al. 1995 ) , microsurgical epididymal sperm aspiration ( MESA ) ( Tournaye et al. 1994 ) , testicular unfastened biopsy ( TOB ) , testicular mulct 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 attacks were proposed to recover epididymal sperm, while the sidelong attacks were used to roll up sperm from testicle if the epididymal sperm was unaccessible. In 1996, testicular sperm retrieved from hypergonadotropic azoospermia patient by TEFNA were used for ICSI and obtained a unrecorded babe ( Lewin et al. 1996 ) . While MD-TESE was chiefly performed to recover testicular sperm for NOA patients, including hypospermatogenesis, ripening apprehension and even in Sertoli Cell Only ( SCO ) syndrome patients ( Okada et al. 2002 ; Schlegel and Li 1998 ) . However, it is non clear whether these different sperm retrieval methods affect the clinical result of ICSI or non.
Many surveies were focused on the influence of sperm beginning and the spermatogenesis advancement on the clinical results of ICSI. Several surveies found that the defect of spermatogenesis affects the clinical results of ICSI ( Ghazzawi et al. 1998 ; Monzo et Al. 2001 ; Pasqualotto et Al. 2002 ) . However, it has been reported that embryos from testicular sperm keep a superior development potency than that from epididymal sperm ( Dozortsev et al. 2006 ) .
The aim of this survey was to compare the clinical results 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 results of ICSI.
Table 1 shows the clinical result of ICSI from OA group and NOA group. Mean female and male ages, average figure of the injected oocytes per rhythm and the average figure of transferred embryos were similar between OA group and NOA group, while the fertilisation rate in OA group was significantly higher than that in NOA group ( 64.1 % VS 44.9 % , P & A ; lt ; 0.001 ) . Furthermore, the same tendency of cleavage rate and clinical gestation rate was observed between the two groups ( 89.0 % VS 79.8 % , P & A ; lt ; 0.001, 40.2 % VS 21.4 % , P= 0.047, severally ) . A higher abortion rate was observed in NOA group than that in OA group ( 33.3 % VS 15.6 % ) , but the tendency was non statically important.
Table 2 shows the consequences of the two subgroups ( OA-PESA group ( n=51 ) and OA-TEFNA group ( n=61 ) ) . Basic parametric quantities in the two subgroups, such as average ages of the twosomes, figure of the injected oocytes and the figure of transferred embryos per rhythm were non statistically different. There were no differences in fertilisation, cleavage and clinical gestation rates between OA-PESA and OA-TEFNA groups ( 65.4 % VS 63.1 % i?? 91.0 % VS 87.4 % i?? 43.1 % VS 37.7 % i??P & A ; gt ; 0.05, severally ) . Furthermore, abortion rate besides did non make a statistical difference between OA-PESA and OA-TEFNA groups ( 13.6 % VS 17.4 % , P & A ; gt ; 0.05 ) .
It is good cognize that the usage of ICSI was the milepost for the intervention 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 fresh micromanipulation ( Mansour et al. 1997 ; Schlegel and Li 1998 ) . However, for those azoospermic patients, the paternal gametes were obtained by assorted operations from epididymis, such as PESA, MESE. Those protocols were normally used for OA patients and ever presented with abundant sperm for ICSI, sometimes with progressive motile sperm. The process of PESA was simple and with fewer complications. Furthermore, the epididymal sperm were much easier to treat before ICSI.
In add-on to epididymal sperm aspiration, there was testicular sperm retrieval, such as TESE, TEFNA, MD-TESE. TESE was seldom used in recent old ages due to its more amendss to testis compared with TEFNA. TEFNA was an effectual and necessary process for azoospermic patients to measure the status of spermatogenesis before ICSI efforts ( Bettella et al. 2005 ; Zukerman et al. 2000 ) . TEFNA was ever performed when epididymal sperm were unavailable in some OA patients or for the sperm retrieval effort of some NOA patients, such as hypospermatogenesis patients ( Lewin et al. 1999 ) . MD-TESE was the last effort for NOA patients. It has been reported that sperm retrieval rate could be up to 81 % , 44 % for hypospermatogenesis and ripening apprehension patients, severally ( Ramasamy et al. 2005 ) . Encouragingly, in this retrospective survey, 92 of the 237 SCO patients retrieve 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 generative medical specialty centre.
Palermo et Al. ( Palermo et al. 2002 ) analysis the chromosome 18, 21, X and Y of sperm from OA and NOA patients by fluorescent unmoved hybridisation ( FISH ) , found that the chromosome abnormalcies rate was significantly higher in NOA patients compared with that in OA patients, and the sex chromosome aneuploidy was the prevailing abnormalcies. This determination was similar to the study of Rodrigo et Al ( Rodrigo et al. 2004 ) . But whether the fertilisation ability of testicular sperm reduced compared with that from epididymis was conflicting harmonizing to the old surveies.
Ghazzawi et al reported that sperm of the semens, epididymis and testicle from OAT patients, OA patients and NOA patients, severally, were able to fertilise every bit by utilizing ICSI. However, gestation rate was significantly reduced in NOA patients compared to the other two groups ( Ghazzawi et al. 1998 ) . Another two surveies besides found that NOA patients had lower fertilisation, embryo quality, and clinical gestation but higher abortion rate than those of OA patients ( Monzo et al. 2001 ; Pasqualotto et Al. 2002 ) . Recently, a dissimilar consequence about the paternal impacts on embryos possible development ability of OA patients was reported by Dozortsev et Al. ( Dozortsev et al. 2006 ) . The fertilisation ability of testicular sperm, performed ICSI was lower than that of epididymal sperm, nevertheless, the undermentioned embryos demonstrated an superior nidation rate to the epididymal sperm.
In the present survey, we took into consideration of the influence of spermatogenesis on the result of ICSI by comparing OA group and NOA group. A important higher fertilisation, cleavage and clinical gestation rates were observed in OA group compared with that in NOA group. Furthermore, the abortion rate in NOA group was 33.3 % compared with 15.6 % in OA group but did non make a statistical difference. These findings were by and large concordant to most of the old studies, which referred the defect spermatogenesis affected the result of ICSI ( Balaban et al. 2001 ; Ghazzawi et Al. 1998 ; Monzo et Al. 2001 ; Pasqualotto et Al. 2002 ) .
When lone instances of OA patients were analyzed, there was no difference of fertilisation rate, cleavage rate, clinical gestation rate and abortion rate between OA-PESA group and NOA-TEFNA group, which was in understanding to some of the old surveies ( Balaban et al. 2001 ; Nicopoullos et Al. 2004 ) . But the day of the month was non in harmony with Dozortsev ‘s study ( Dozortsev et al. 2006 ) . Some surveies may explicate these controversial findings. O’Connel et Al. detected mtDNA and determine atomic DNA ( nDNA ) atomization in testicular and epididymal sperm from OA patients, and investigated that mtDNA and nDNA of testicular sperm have fewer mutant and atomization than epididymal sperm ( O’Connell et al. 2002 ) . It was universally acknowledged that epididymis was the indispensable organ for sperm ripening in a normal physiological status. However, one clinical survey suggested that one time the sperm enter the vessel respect, those fresh sperm less than 7 yearss maintained the best fertilisation ability and developmental potency ( Pellestor et al. 1994 ) . Levitas et Al. analyzed 9,489 seeds samples found that the peak average sperm motility was observed after 1 twenty-four hours abstention ( Levitas et al. 2005 ) . Both of the surveies suggested that long period remaining in spermous canal was non good for sperm motility and map. Nicopoullos et Al. analysed their ain day of the month of ICSI result from 154 OA patients and demonstrated that fertilisation and live-birth rates were highest in work forces with a old vasectomy and no morbific cause ( vasectomy 51 % and 23 % ; non infective 53 % and 29 % , severally ) and lowest in work forces with morbific or inflammatory causes ( Nicopoullos et al. 2004 ) . We frequently found that epididymal sperm from those morbific caused OA patients were less motile, sometimes wholly nonmotile. This may due to hydrolysing enzymes, hyaluromidase enzymes from inflammatory cells and acrosin from dead and deceasing sperm acrosome ( Cummins et Al. 1994 ) .
In decision, the clinical result of ICSI was affected by the spermatogenesis procedure of the patients instead than the sperm retrieval methods. Furthermore, in OA patients, PESA was the dominant pick for sperm retrieval because of its simple process, less complication and sensible gestation rate. However, if no motile sperm presented or abundant inflammatory cells assorted together, testicular sperm would impute to a stable clinical ICSI result.
MATERIALS AND METHODS
A sum of 138 azoospermic patients who performed 154 ICSI rhythms from January 2006 to January 2009, were involved in this survey. All the patients performed at least 3 times semen analysis in our research lab, utilizing centrifugation at 1800g for 10 min failed to retrieve sperm. 92 work forces with normal endocrine degree were diagnosed as OA patients ( OA group ) whom were confirmed by one of the undermentioned groundss, abundant sperm were discovered in the epididymal fluid during PESA or normal spermatogenesis histopathologically diagnosed by TEFNA before ICSI effort. 62 work forces had an acquired epididymal obstructor due to a history of epididymitis, all these patients present a turgescent epididymis or an epididymal cyst during physical scrutiny, and some of them were confirmed by ultrasound. 21 work forces have a history of vasectomy and failed vasovasotomy. Another 9 inborn bilateral absence of vessel deferens ( CBAVD ) were besides involved in our survey. They were deficiency of vessel deferens with obvious laboratorial features: low seeds volume, seeds PH & A ; lt ; 7 and really low or absence fruit sugar in the seminal plasma, confirmed with the absence of seminal cyst by transrectal ultrasound. The OA group was subdivided into two groups harmonizing to the sperm retrieval methods, 1 ) OA-PESA group: rhythms with epididymal sperm ( n=51 ) and 2 ) OA-TEFNA group: rhythms with testicular sperm ( n=61 ) . For all the OA patients, PESA were performed foremost, if no sperm was found or epididymal fluid was assorted with excessively many inflammatory cells, TEFNA will be performed to recover testicular sperm for ICSI. There were wholly 46 NOA patients attempted ICSI during the investigating period, a diagnostic TEFNA was done before ICSI effort and confirmed as hypospermatogenesis by histophathology rating. Unfortunately, four of them failed to recover sperm from their testicles on the oocytes retrieval twenty-four hours and changed to utilize the donative sperm. Finally, 42 NOA patients were involved in present survey as NOA-group. All these NOA patients were treated with TEFNA.
Ovarian stimulation and oocytes readying
Either a long or a short protocol of controlled ovarian stimulation was used for their spouses. After the disposal of GnRHa ( Enantone, Takeda, Japan ) during luteal stage or follicular stage, purified follicle-stimulating endocrine ( Gonal-F ; Serono, Rockland, MA ) and human menopausal gonadotrophin ( HMG, Lizhu, China ) were injected for ovarian stimulation. Human chorionic gonadotropin ( human chorionic gonadotropin ; Lizhu, China ) was used when two or more dominant follicles in diameter were observed. 36 hours subsequently, oocytes were retrieved by transvaginal ultrasound guided puncture.
After retrieval, oocytes were removed into microdroplets of civilization media ( IVF, Vitrolife, Sweden ) and covered with mineral oil ( IVF, Vitrolife, Sweden ) and stored in an brooder 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 composite was removed by pippetting with a plastic pipette with an interior diameter of 135?m. Each oocyte was rinsed in a series of droplets of medium to take any staying spreading factor. 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 upside-down microscope at -200 magnification to measure the ripening phase. Merely metaphase a…? ( M a…? ) phase oocytes were selected to execute intracytoplasmic sperm injection.
PESA and TEFNA processs
Both PESA and TEFNA were performed under local anaesthesia and spermous cord blocking. For the PESA processs, the caput of epididymis was held between the pollex and index. A 23-gauge acerate leaf connected with a 20 milliliter syringe filled with civilization medium was inserted into the turgescent caput, uninterrupted suction was applied as epididymal fluid was aspirated. The epididymal fluid was examined under an upside-down microscope straight. For the TEFNA processs, the testicle was held in the left manus, an avascular site was chosen to execute the puncture. A 23-gauge acerate leaf connected with a 20 milliliter syringe was inserted into the selected site. Puncture the testicle with the all right acerate leaf back and Forth, synchronized with an alternately negative force per unit area. Pull out the needle connected with seminiferous tubules and draw out every bit much tubules as possible with two micro forceps. The retrieved tubules were washed one to two times to take the ruddy blood cell, so minced into cell suspension with two 1 milliliter panpipes, and checked under an upside-down microscope. If no sperm was found, at most four sites on each testicle 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 milliliter conelike tubing incorporating 5 milliliter of Fertilization medium ( Cook, Aus ) . The suspension was centrifugated at 800 g for 1 min. The pellet was kept in the tubing in instance of no motile sperm were found in the supernatant. The supernatant was removed into another clean tubing, centrifuged at 1000 g for 10 min, the pellet was washed once more with 3 milliliters Fertilization medium ( Cook, Aus ) and centrifugated at 1000 g for 5 min. The concluding pellet was resuspended with 0.3-0.5 milliliter Gamate 100 medium ( Vitrolife, Sweden ) . 10 ?l was used for sperm rating. If no sperm was found, the pellet after first centrifugation was washed with the same process to look for sperm. The concluding suspension was incubated in 5 % CO2 at 37a„? until usage.
For testicular sperm processing, the seminiferous tubules were placed in a petri dish and minced into cell suspension with two 1 milliliter panpipes. Removed all the cell suspension and tissues into a new 15 ml conelike tubing, contain 5 ml Fertilization medium ( Cook, Aus ) . The suspension was centrifugated at 800 g for 30 seconds. The pellet was kept in the tubing in instance of no motile sperm were found in the supernatant. The supernatant was removed into another clean tubing, washed twice as the same process mentioned before. The concluding pellet was resuspended with 0.3- 0.5 milliliter Gamate 100 medium ( Vitrolife, Sweden ) . 10 ?l sample was used for sperm rating. If no sperm was found, the resuspended pellet after first centrifugation was washed with the same process to look for available sperm. Testicular sperm was cultured in a brooder in 5 % CO2 at 37a„? for 2- 4 hours before ICSI. In most instances, feasible sperm began to jerk after several hours civilization. These little motile sperm was selected for ICSI. When no motile sperm was detected, sperm was chosen on the footing of normal morphology.
Intracytoplasmic sperm injection
Intracytoplasmic sperm injection ( ICSI ) was performed at -400 magnification phase phase-contrast inverted microscope ( Olympus Japan ) . M a…? phase oocytes were placed in Gamete medium beads ( IVF, Vitrolife, Sweden ) . Epididymal sperm was placed in PVP degree Celsius ( Polyvinilpirrolidone, Vitrolife, Sweden ) for immobilisation, while oligodynamic testicular sperm was placed in a Gamete medium micro droplet ( IVF, Vitrolife, Sweden ) straight for ICSI. The tail of the sperm was automatically immobilized by the tip of the microinjection acerate leaf ( Cook, USA ) and aspirated tail-first inside the acerate leaf. The oocyte was held with a keeping micropipette ( Cook, USA ) with polar organic structure at the 6 o’clock place. The sperm was injected into the oocyte at 3 o’clock place by utilizing electrohydraulic micromanipulators ( Olympus, Japan ) . Each injected oocyte was transferred into one micro droplet with Gamate medium ( IVF, Vitrolife, Sweden ) covered with mineral oila for individual embryo civilization.
Fertilization and embryo civilization
The injected oocytes were incubated for 16-18 hours in 6.5 % CO2 at 37a„? before fertilisation rating. The fertilisation was confirmed by the presence of two pronuclei and two polar bodied at -200 magnification under phase phase-contrast inverted microscope ( Olympus, Japan ) . Embryos cleavage were assessed 24 hours subsequently, and two to three embryos were retransferred into a new balanced micro droplet for another 24 hours civilization in 6.5 % CO2 at 37a„? . 48 hours after fertilisation, two to three morphologically good quality embryos were selected by two experient embryologists for embryo transportation. The left morphologically good quality embryos were frozen by vitrification.
Embryo transportation and clinical consequence
Embryo transportation was performed 48 hours after fertilisation appraisal. The selected morphologically good quality embryos were loaded into an Edwards-Wallace catheter ( SIMS Portex Ltd, Kent, UK ) . The catheter was inserted into uterine pit under ultrasound guiding. After embryos transportation, the catheter was checked under dissecting microscope to corroborate all the embryos were transferred into the uterine. Luteal stage support started on the twenty-four hours of oocyte retrieval with 60 milligrams progesterone intramuscular injection everyday. The beta human chorionic gonadotropin serum degree was assessed 14 yearss after embryos transportation for chemical gestation. A clinical gestation was defined as the visual image of a pulse by transvaginal ultrasound at 5 hebdomads after embryos transportation.
Numeric variables between two groups were compared utilizing t trial. Categorical variables were compared utilizing chi-square trial. Consequences were considered to be important when P & A ; lt ; 0.05.