Different sperm sources and parameters can influence intracytoplasmic sperm injection outcomes before embryo implantation

Different sperm sources and parameters can influence intracytoplasmic sperm injection outcomes before embryo implantation 1 2 first-author 1 1 1 1 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jinfan@zju.edu.cn †E-mail:jinfan@zju.edu.cn jinfan@zju.edu.cn Department of Reproductive Endocrinology, Key Laboratory of Reproductive Genetics, Ministry of Education, and Key Laboratory of Women’s Reproductive Health of Zhejiang Province, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Reproductive Endocrinology, Key Laboratory of Reproductive Genetics, Ministry of Education, and Key Laboratory of Women’s Reproductive Health of Zhejiang Province, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Reproductive Health, Shaoxing Women and Children Hospital, Shaoxing 312000, China Department of Reproductive Health, Shaoxing Women and Children Hospital, Shaoxing 312000, China To evaluate the effects of sperm with different parameters and sources on the outcomes of intracytoplasmic sperm injection (ICSI), 1972 ICSI cycles were analyzed retrospectively. Groups 1 to 5 were composed of cycles using ejaculated sperm and were grouped according to sperm quantity, quality, and morphology into normal (288 cycles), or mild (329 cycles), moderate (522 cycles), severe (332 cycles), and extremely severe (171 cycles) oligozoospermia and/or asthenozoospermia and/or teratozoospermia (OAT) groups. Group 6 was composed of 250 cycles using testicular or epididymal sperm, and Group 7 consisted of 80 cycles using frozen-thawed sperm. We found that fertilization rates were gradually reduced from Groups 1 to 6, and reached statistical difference in Groups 5 and 6 ( P < 0.05). The high-quality embryo rate was higher in Group 1 than in Groups 2, 3, 5, 6, and 7 ( P < 0.05). No statistical differences were observed in the rates of embryo cleavage, clinical pregnancy, miscarriage, live-birth, premature birth, low birth weight, weeks of premature birth, average birth weight, or sex ratio for all seven groups ( P > 0.05). A total of nine cases of malformation were observed, with a malformation rate of 1.25% (9/719). In conclusion, different sperm sources and parameters can affect ICSI outcomes before embryo implantation. A full assessment of offspring malformation will require further study using a larger sample size. Intracytoplasmic sperm injection (ICSI) Sperm Sperm source Sperm parameters Malformation Introduction 1 Intracytoplasmic sperm injection (ICSI), first introduced by Palermo et al. ( 1992 1992 ), is an assisted-fertilization micromanipulation technique for fertilizing an egg by directly injecting a selected sperm into the ooplasm. Compared with conventional in-vitro fertilization (IVF), ICSI greatly reduces the requirements for sperm quantity, motility, and fertilization ability. With the development of micromanipulation, ICSI has become an important part of assisted reproductive techniques (ARTs) for the treatment of cases with male factor infertility (Sarkar, 2007 2007 ). With ICSI, an egg can be fertilized not only with fresh sperm with mild or moderate abnormalities but also with sperm following a frozen-thawed procedure, sperm of a severely or extremely severely abnormal concentration, motility or morphology, or even sperm that have been surgically retrieved from the epididymis or testis (van Steirteghem et al., 1993 1993 ; Devroey et al., 1994 1994 ; Redgment et al., 1994 1994 ; Tournaye et al., 1994 1994 ; Chen et al., 1996 1996 ). Nevertheless, sperm retrieved from the testis, epididymis or ejaculation may have a different concentration, motility or morphology and differ in their degree of maturity, chromosomal or DNA composition, and gene imprinting conditions (Holt and North, 1991 1991 ; Holt et al., 1992 1992 ; Giraud et al., 2000 2000 ; Chatterjee and Gagnon, 2001 2001 ; Pegg, 2002 2002 ; Chohan et al., 2004 2004 ; Liu et al., 2004 2004 ; Derijck et al., 2007 2007 ; Kobayashi et al., 2007 2007 ). For instance, rates of DNA fragmentation, mitochondrial dysfunction, and chromosomal aneuploidy are significantly higher in the sperm of oligozoospermia and/or asthenozoospermia and/or teratozoospermia (OAT) males compared with unaffected controls (Liu et al., 2004 2004 ; Derijck et al., 2007 2007 ). Furthermore, aberrant DNA methylation of imprinted loci in sperm from oligospermic patients has been reported (Kobayashi et al., 2007 2007 ). Generally, testicular and epididymal sperm are considered to have a low degree of maturity and a high malformation rate. The accumulated evidence also shows that the process of freezing and thawing sperm could cause damage to the sperm (Holt and North, 1991 1991 ; Holt et al., 1992 1992 ; Giraud et al., 2000 2000 ; Chatterjee and Gagnon, 2001 2001 ; Pegg, 2002 2002 ; Chohan et al., 2004 2004 ). Therefore, the consequences of using these different sources of sperm have attracted a great deal of attention since the first introduction of ICSI. There is an ongoing debate about this question (Bukulmez et al., 2001 2001 ; Miller and Smith, 2001 2001 ; Göker et al., 2002 2002 ; Aoki et al., 2004 2004 ; Dozortsev et al., 2006 2006 ; Loutradi et al., 2006 2006 ; Tepla et al., 2006 2006 ; Nilsson et al., 2007 2007 ; Naru et al., 2008 2008 ; Verza and Esteves, 2008 2008 ). Here, we report an analysis of 1972 ICSI cycles to explore the possible relationships between sperm quantity, quality, morphology, source, and the outcomes of ICSI. Materials and methods 1 Subjects 2 A total of 1 925 couples who underwent 1 972 ICSI cycles from January 2004 to June 2008 in the Center for Reproductive Medicine of the Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China were included in this retrospective study. Institutional review board approval and written consents from patients were obtained prior to data collection. In all cycles studied, the tubal factor was identified as the sole cause of female factor infertility, thereby excluding other female factors such as myoma, endometrial polypus, endometriosis, and uterus deformation. Based on the sperm quality and source on the day of ICSI, the subjects were divided into seven groups. Group 1 was composed of 288 ICSI cycles with normal semen parameters, based on the 5th World Health Organization standard (WHO, 2010 2010 ) (total sperm number, calculated by sperm concentration×sperm volume, ≥39×10 6 sperm/ml, progressive motility ≥32%, and morphology ≥4% normal forms). In this group, ICSI was performed for patients with failure or low fertilization rates ( < 30%) in their last IVF cycle, unexplained primary infertility after more than three cycles of failed artificial insemination with the male partner’s sperm, or abnormal results from more than two previous semen analyses. Group 2 was composed of 329 ICSI cycles with mild OAT (total sperm number 20×10 6 –39×10 6 sperm/ml, and/or progressive motility 20%–32%, and/or morphology < 4% normal forms). Group 3 was composed of 522 ICSI cycles with moderate OAT (total sperm number 10×10 6 –20×10 6 sperm/ml, and/or progressive motility 10%–20%). Group 4 was composed of 332 ICSI cycles with severe OAT (total sperm number 1×10 6 –10×10 6 sperm/ml, and/or progressive motility 1%–10%). Group 5 was composed of 171 ICSI cycles with extremely severe OAT (total sperm number < 1×10 6 sperm/ml, and/or progressive motility < 1%). Group 6 was composed of 250 ICSI cycles with obstructive azoospermia (OA) (azoospermia patients with normal serum follicle-stimulating hormone (FSH), testicular volume, and diagnostic biopsy) or difficult ejaculation on the day of IVF, using testicular or epididymal sperm. Group 7 was composed of 80 ICSI cycles using frozen-thawed sperm, in which the main reason for ICSI was that the progressive motility of donated semen was less than 25% after thawing. Sperm preparation 2 Fresh ejaculated semen was collected on the day of oocyte retrieval and left to liquefy for approximately 20–30 min prior to semen analysis. Afterwards, the semen specimens were processed by density gradient centrifugation, as previously described (Mortimer, 2000 2000 ). For each semen sample, 1 ml was used for preparation, except for the cases with extremely low counts. The final solution was incubated at 37 °C, 5% CO 2 for 0.5–1.0 h prior to the ICSI procedure. Sperm retrieval from the testis was performed percutaneously by testicular sperm aspiration (TESA) (Watkins et al., 1997 1997 ) under local anesthesia. Seminiferous tubules were dissected mechanically, and the presence of sperm was examined using an inverted microscope (Olympus, Tokyo, Japan) at 200× magnification. The supernatant was processed by density gradient centrifugation (Mortimer, 2000 2000 ). Sperm retrieval from the epididymis was performed by percutaneous epididymal sperm aspiration (PESA) (Lin et al., 2000 2000 ). The aspirate was washed into a Petri dish with a small volume of modified human tubal fluid (M-HTF; Irvine Scientific, CA, USA), and the presence of sperm was confirmed using an inverted microscope at 400× magnification. ICSI was performed immediately. The frozen sperm was thawed (Royere et al., 1996 1996 ) on the day of ICSI. The thawed sperm was processed using the same method as used for fresh ejaculated sperm. The final solution was incubated at 37 °C, 5% CO 2 for 0.5–1.0 h prior to the ICSI procedure. Ovarian stimulation and oocyte retrieval 2 All female patients were treated with a long gonadotrophin-releasing hormone agonist (GnRH-a; Decapeptyl, Ferring, Kiel, Germany)/gonadotrophin protocol (Tarlatzis et al., 1993 1993 ). Ovulation was induced by injection of human chorionic gonadotrophin (HCG; Ovidrel®, Serono, Geneva, Switzerland) when at least two follicles reached a diameter of ≥18 mm. Oocytes were retrieved 36 h after HCG administration using follicular aspiration with guidance from transvaginal sonography. After follicular aspiration, the tubes with the follicular fluid were transferred to the laboratory and examined using stereomicroscopy to identify the cumulus-oocyte complexes (COCs). The COCs were transferred to pre-balanced G-IVF (Vitrolife, Goteborg, Sweden) culture medium and incubated at 37 °C, 5% CO 2 for 2 h prior to oocyte handling. Oocyte handling 2 The oocytes were stripped of the surrounding cumulus cells (2 h after ovum collection) using hyaluronidase (80 U/ml; Vitrolife) in M-HTF for 10–15 s. Then, using a 135 μm diameter pipette (Flexipet, Cook, Bloomington, IN, USA), the oocytes were transferred to M-HTF for complete removal of the corona cells. The denuded oocytes were then rinsed and incubated in M-HTF until the time of injection (2–3 h after collection). The ICSI procedure was performed only for metaphase II (MII) oocytes that had extruded their first polar bodies. ICSI procedure 2 The ICSI procedure (Miller and Smith, 2001 2001 ; Göker et al., 2002 2002 ) was performed 38–42 h post-HCG injection. The microinjection procedure was performed on a heated stage at 37 °C under an inverted microscope at 400× magnification. Sperm were selected at the periphery of the polyvinylpyrrolidone (PVP; Vitrolife) micro-droplet. A selected spermatozoon was immobilized by repeatedly touching the tail and was then aspirated, tail first, into the injecting pipette. The oocyte was held by a holding pipette with the polar body at the twelve or six o’clock position. The injecting pipette containing the spermatozoon was introduced at the three o’clock position through the zona pellucida and oolemma, deep into the cytoplasm. A small part of the cytoplasm was aspirated. The spermatozoon and the cytoplasm were injected, and the injecting pipette was withdrawn gently. After injection, the oocytes were rinsed and incubated in G-IVF medium. Embryo culture and pregnancy establishment 2 Fertilization was confirmed 16–20 h post-ICSI procedure by the presence of two pronuclei. Cleavage and embryo quality were assessed 48–72 h post-ICSI procedure. The embryos were classified as high-quality when they exhibited 3–4 symmetric blastomeres on the second day of culture and 7–8 symmetric blastomeres on the third day of culture, in the absence of multinucleation and zona pellucida alterations, Grade I (absence of fragmentation) or II (up to 20% of cytoplasmic fragments) of cytoplasmic fragmentation (Veek, 1990 1990 ). A total of 1–3 embryos were chosen for transfer to the female partners 2 or 3 d after oocyte retrieval. Females older than 35 years or on their second round of ART received at most three embryos, in accordance with China’s ART Regulations. The luteal phase was supported by progesterone ampules. Serum HCG levels were measured on Day 14 after embryo transfer. Clinical pregnancies were confirmed by a vaginal ultrasound 30 d after embryo transfer which showed the presence of an intrauterine gestational sac with detectable fetal cardiac activity. Assessments 2 In addition to the rates of fertilization, cleavage, high-quality embryos, pregnancy, miscarriage, ectopic pregnancy, and multiple pregnancy, some perinatal parameters, such as the gestational age, live-birth rates, premature birth rates, birth weight, sex ratio, and major malformations were assessed. Sex ratio was defined as male offspring/(males+females). Miscarriage was defined as pregnancy loss before 20 weeks of gestational age. Gestational age was calculated as the time from an estimated first menstruation day (14 d before follicular aspiration) to the time of birth. Major malformations were defined as malformations requiring surgical treatment or causing essential reduced functionality. Statistical analysis 2 All data were analyzed using SPSS 15.0 software (SPSS Inc., Chicago, Illinois, USA) and evaluated by the Chi-square test, one-way analysis of variance (ANOVA), the rank transform method, and logistic regression. P < 0.05 was accepted as significant. Results 1 Characteristics of patients 2 A total of 1 972 ICSI cycles were analyzed. In Groups 4, 5, and 6, the females were significantly younger ( P < 0.05). The infertility period was the shortest in Group 6 and the longest in Group 7 ( P < 0.05). Endometria were significantly thicker in Groups 6 and 7 ( P < 0.05). The peak (estradiol) E 2 before HCG injection and the number of retrieved oocytes were significantly higher in Group 6 (Table 1 1 ). All values (except those for cycle number) are expressed as mean±SD P < 0.05: significant difference compared with Groups 1, 2, and 3 P < 0.05: significant difference compared with Groups 1, 2, 3, and 4 P < 0.05: significant difference compared with Groups 1, 2, 3, 4, and 7 P < 0.05: significant difference compared with Groups 1, 2, 3, and 7 P < 0.05: significant difference compared with Groups 1, 3, 4, 5, and 6 (rank transform method) Group Cycle number Women’s age (year) Infertility period (year) Basal FSH (U/L) Endometrial thickness (mm) Peak E 2 (nmol/L) Retrieved oocytes Injected MII oocyte 1 288 31.6±4.2 5.1±3.4 6.8±2.2 11.1±2.4 13 111.0±9 608.1 13.9±7.6 11.4±6.5 2 329 31.9±3.9 5.2±3.3 6.7±2.1 11.1±2.5 12 614.8±8 483.3 14.1±7.6 11.4±6.7 3 522 31.6±4.0 5.1±3.3 6.7±1.9 11.0±2.3 12 840.1±8 271.6 13.8±7.0 11.1±6.0 4 332 31.0±4.0 * * 4.8±3.2 6.7±1.9 11.2±2.3 13 308.4±9 200.2 15.0±8.4 12.1±6.8 5 171 30.2±3.7 Δ Δ 4.9±3.3 6.5±1.8 11.4±2.2 12 722.0±7 771.6 14.9±8.7 12.1±7.2 6 250 30.1±3.9 † † 4.6±3.2 ‡ ‡ 6.5±2.0 11.4±2.4 * * 13 860.9±7 585.7 Δ Δ 15.2±7.5 * * 12.4±6.4 7 80 31.2±3.9 6.0±3.6 # # 6.6±1.9 11.6±2.6 * * 13 222.5±8 623.9 14.7±7.2 12.3±6.5 Laboratory outcomes of ICSI 2 The rates of fertilization, cleavage and high-quality embryos in each group are listed in Table 2 2 . The fertilization rates decreased as semen quality decreased from Groups 1 to 6 and reached statistical significance in Groups 5 [(67.7±24.5)%] and 6 [(65.9±21.5)%] ( P < 0.05). The high-quality embryo rate in Group 1 [(70.3±27.7)%] was significantly higher than that in other groups ( P < 0.05). There were no statistical differences in the cleavage rates among the seven groups ( P > 0.05) (Table 2 2 ). All values are expressed as mean±SD P < 0.05: significantly lower than Groups 1, 3, and 7 P < 0.05: significantly lower than Groups 1, 2, 3, 4, and 7) P < 0.05: significantly higher than Groups 3, 5, 6, and 7 (rank transform method) Group Fertilization rate (%) Cleavage rate (%) High-quality embryo rate (%) 1 73.6±20.2 97.7±11.1 70.3±27.7 † † 2 72.8±21.0 98.4±8.2 65.2±30.6 3 72.6±21.3 98.4±10.9 65.7±29.3 4 72.1±22.1 98.0±10.9 67.4±28.0 5 67.7±24.5 * * 98.3±9.1 62.0±30.4 6 65.9±21.5 Δ Δ 96.9±11.3 62.7±31.7 7 74.8±17.4 99.0±5.0 63.1±28.2 Clinical outcomes of ICSI 2 A total of 1 831 transfer cycles were performed, with a total of 4 196 embryos transferred. The cancellation rate for fresh embryo transfer was 7%, based mainly on a high risk of ovarian hyperstimulation syndrome (OHSS), fever, or poor conditions of endometrium preparation. In total, 700 female partners became clinically pregnant, with a total clinical pregnancy rate of 38.2% (700/1 831). The average numbers of embryos transferred in Groups 4, 5, and 6 were statistically lower than those in other groups ( P < 0.05). The rates of clinical pregnancy, chemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage showed no statistical differences among all seven groups ( P > 0.05) (Table 3 3 ). n TC : number of transfer cycles; n TE : number of transferred embryos; n avg : average number of embryos transferred of a subject Values are expressed as mean±SD Pregnancy rates and miscarriage rate were compared by logistic regression P < 0.05: significantly lower than Group 1 P < 0.05: significantly lower than Groups 1, 2, 3, and 7 (rank transform method) 2 Group 2 n TC 2 n TE 2 n avg a a 4 Pregnancy rate b b (%) 2 Miscarriage rate b b (%) Clinical Chemical Ectopic Multiple 1 269 655 2.4±0.6 37.5 (101/269) 3.0 (8/269) 2.2 (6/269) 29.7 (30/101) 15.8 (16/101) 2 305 712 2.3±0.5 * * 33.1 (101/305) 1.6 (5/305) 1.6 (5/305) 26.7 (27/101) 15.8 (16/101) 3 490 1 137 2.3±0.5 * * 37.1 (182/490) 1.2 (6/490) 1.4 (7/490) 28.0 (51/182) 15.9 (29/182) 4 307 678 2.2±0.5 Δ Δ 41.0 (126/307) 2.6 (8/307) 2.3 (7/307) 31.7 (40/126) 12.7 (16/126) 5 155 334 2.2±0.5 Δ Δ 40.6 (63/155) 1.9 (3/155) 1.9 (3/155) 20.6 (13/63) 17.5 (11/63) 6 234 507 2.2±0.4 Δ Δ 40.2 (94/234) 1.7 (4/234) 2.1 (5/234) 37.2 (35/94) 12.8 (12/94) 7 71 173 2.4±0.6 46.5 (33/71) 2.8 (2/71) 0.0 (0/71) 36.4 (12/33) 15.2 (5/33) Outcomes of ICSI children 2 Thirteen couples were lost to follow up and one female patient died by suicide in the second trimester. A total of 719 living newborns were delivered. No statistical differences were observed for the live-birth rate, premature birth rate, low birth weight rate, weeks of premature birth, birth weight, or sex ratio for all groups ( P > 0.05). There were two cases of stillbirth due to a circular umbilical cord in Groups 2 and 7; two cases of perinatal death, both of which were premature births, in Groups 2 and 6. A total of nine cases of malformation were observed, including one case of multiple structural abnormality and one case of fetal edema, both terminated in the second trimester; four boys with hypospadias (two boys), congenital cardiopathy, or hexadactylism, and one girl with megacolon diagnosed in the newborn period; one boy with cryptorchidism and another with epilepsy diagnosed in infancy (Table 4 4 ). n n : number of newborns; n tw : number of twins; n tr : number of triplets; r lb : live-birth rate/transfer cycle; r pb : premature birth rate; t pb : weeks of premature birth; m B : birth weight; r lbw : low birth weight rate; r s : sex ratio; n m : number of malformations. Values are expressed as mean±SD Values are males/(males+females) No statistical differences were observed for any group ( P > 0.05). Live-birth rate, premature birth rate, low birth weight rate, and sex ratio were compared by the Chi-square test, weeks of premature birth were compared by the K-W test, and weight of newborns was compared by one-way ANOVA Group n n n tw n tr r lb (%) r pb (%) t pb a a (week) m B a a (g) r lbw (%) r s b b n m 1 104 23 1 29.4 (79/269) 30.4 (24/79) 35.1±1.2 2 981.9±702.3 20.2 (21/104) 0.53 (55/104) 0 2 99 21 0 25.6 (78/305) 25.6 (20/78) 33.7±3.1 2 965.1±671.1 18.2 (18/99) 0.45 (45/99) 3 3 183 43 0 28.6 (140/490) 26.4 (37/140) 33.9±2.2 2 891.7±652.0 25.7 (47/183) 0.48 (87/183) 4 4 130 30 1 31.9 (98/307) 29.6 (29/98) 34.5±1.8 2 799.0±715.8 32.4 (42/130) 0.53 (69/130) 0 5 58 10 0 31.0 (48/155) 20.8 (10/48) 34.8±1.6 3 042.2±623.1 15.5 (9/58) 0.45 (26/58) 1 6 107 31 0 32.9 (77/234) 32.5 (25/77) 34.4±1.8 2 757.5±596.1 29.9 (32/107) 0.49 (52/107) 1 7 38 9 0 40.8 (29/71) 20.7 (6/29) 35.0±2.0 2 898.7±639.1 18.4 (7/38) 0.53 (20/38) 0 Discussion 1 The question of whether male factor infertility would affect the outcome of ICSI and the health of the offspring has gained great attention since the introduction of ICSI for the treatment of patients with extremely poor-quality sperm (Bonduelle et al., 1999 1999 ). Currently, ICSI can be successfully used for even the most severe cases of OATs or azoospermia. The wider application of ICSI and the improvements in our understanding of sperm biology have encouraged more detailed investigations into sperm-derived effects on ICSI outcomes and children’s health. We compared the outcomes of ICSI based on the sperm source and the quality of the ejaculates. A tendency towards reduced fertilization rates was observed in Groups 1 to 5 as the sperm quality decreased, indicating that sperm quality can affect the fertilization process. Analyses of unfertilized oocytes after ICSI have revealed that the main causes of failed fertilization are failure to complete oocyte activation and sperm head decondensation (Flaherty et al., 1995 1995 ; Dubey et al., 1997 1997 ; 1998 1998 ). Although ICSI can help sperm penetrate the zona pellucida and reach the cytoplasm of the oocytes, the completion of oocyte activation and the formation of the male and female pronuclei are determined by the intrinsic nature of the sperm and oocytes (Perreault et al., 1988 1988 ; Perreault, 1992 1992 ; Schatten, 1994 1994 ; Yanagimachi, 1994 1994 ). High rates of DNA fragmentation (Tavalaee et al., 2009 2009 ), acrosomal defects (Cummins et al., 1991 1991 ; Fenichel et al., 1991 1991 ), and epigenetic factors such as sperm-specific phospholipase C zeta (PLC ζ ) (Swann et al., 2004 2004 ; Heytens et al., 2009 2009 ) in poor-quality sperm are all considered to be possible reasons for fertilization failure. Rates of DNA fragmentation, mitochondrial dysfunction, and chromosomal aneuploidy are significantly higher in the sperm of OAT males (Liu et al., 2004 2004 ; Derijck et al., 2007 2007 ). In our view, sperm morphology is a crucial factor for sperm quality. We selected sperm under 400× magnification, at the periphery of the PVP micro-droplet, to guarantee that the sperm selected had a relatively normal form and good motility. However, a normal-looking sperm could still contain serious nuclear malformations and chromosomal aneuploidies (Huszar et al., 2007 2007 ; Oliveira et al., 2010 2010 ). There are published reports that when intracytoplasmic morphologically selected sperm injection (IMSI) was performed with sperm selected under a magnification level above 6 000× (Antinori et al., 2008 2008 ; Wilding et al., 2011 2011 ), the rate of sperm nucleus normality was significantly higher. In addition, Huszar et al. ( 2007 2007 ) have reported that in sperm selected with hyaluronic acid, the frequencies of chromosomal disomy and diploidy were reduced 4- to 6-fold. More importantly, with these two methods, ICSI outcomes were significantly improved (Antinori et al., 2008 2008 ; Parmegiani et al., 2010 2010 ; Wilding et al., 2011 2011 ). Therefore, more detailed sperm selection may help to further improve the ICSI outcomes. Significantly lower fertilization rates were observed when testicular or epididymal sperm from men with OA or ejaculation failure were used for ICSI, when compared with all of the groups that used ejaculated sperm. This is in contrast to prior studies that reported that sperm from OA had the same fertilization rates after ICSI as normal ejaculated sperm (Bukulmez et al., 2001 2001 ; Verza and Esteves, 2008 2008 ). Our data seem to support the notion that sperm from the testis or epididymis have decreased fertility potential after ICSI (Loutradi et al., 2006 2006 ). Possible explanations for this result may be that testicular and epididymal sperm are less mature and subsequently less competent at fertilization than ejaculated sperm. Furthermore, epididymal obstruction also can cause sperm damage (Shapiro et al., 1998 1998 ; Kolettis et al., 1999 1999 ); it has been reported that high percentages of cells with DNA damage were found in sperm samples from men with OA or anejaculation (Ramos et al., 2002 2002 ). Although we did not observe statistically significant differences in cleavage rates among different groups, the high-quality embryo rate in Group 1, with normal semen parameters, was significantly higher than that in other groups. This result suggests that after completing fertilization, different quantities, qualities, morphologies, or sources of sperm have no obvious effects on pronuclei syngamy, embryonic genome formation, or the initiation of mitotic division. However, those factors seem able to affect the formation of high-quality embryos, and a high percentage of good-quality sperm may be advantageous for generating a high percentage of high-quality embryos. Sperm DNA fragmentation is a specific paternal factor that can influence embryo development when it increases above a certain level (Sakkas et al., 1999 1999 ; Seli et al., 2004 2004 ; Virro et al., 2004 2004 ). The development of a human embryo in the early stages is controlled by maternally inherited mRNA. The embryonic genome is activated at approximately the 8-cell stage, and then the paternal influences on embryo development begin to be apparent (Braude et al., 1988 1988 ). These factors might explain the discrepancy between the effects of ICSI on cleavage and the formation of high-quality embryos. The numbers of transferred embryos were significantly lower in Groups 4, 5, and 6. Aside from this factor, the data on ICSI clinical outcomes did not show statistically significant differences in the rates of clinical pregnancy, chemical pregnancy, multiple pregnancies, ectopic pregnancy, or miscarriage; the same was true for the perinatal parameters in all seven groups. This result indicates that although different sources and different qualities of sperm could influence the fertilization process and the formation of high-quality embryos, the potential viability of ICSI embryos after transfer is nearly equal no matter what type of sperm is used. Therefore, it is thought that there is no significant effect on the clinical outcome and perinatal parameters of offspring when different qualities and sources of sperm are used for ICSI (Ludwig and Katalinic, 2003 2003 ; Tepla et al., 2006 2006 ). Furthermore, this result also demonstrates that the fertilization process and the formation of high-quality embryos might act as a mechanism for selecting the truly normal sperm. Definite male-factor infertility in Groups 4, 5, and 6 led to the couples visiting a fertility clinic at younger ages. Hence, the percentages of female patients younger than 35 years old were much higher in these groups, resulting in lower numbers of transferred embryos, in accordance with China’s ART Regulations. Decreased fertilization rates and lower percentages of high-quality embryos could also contribute to the reduced numbers of transferable embryos. Aside from the perinatal parameters, the malformation rate was another important indicator to evaluate the safety of ICSI. In the present study, two cases of stillbirth, two cases of perinatal death, and nine cases of malformation occurred. The malformation rate was 1.25% (9/719), which is consistent with previously reported malformation rates ranging from 1.1% to 9.7% for ICSI (Rimm et al., 2004 2004 ). Unfortunately, our cases were too few for a precise statistical analysis. There is an ongoing debate about whether sperm quality and source affect the malformation rate and genetic materials of ICSI children. Some investigators believe that there are some effects (Bonduelle et al., 2002 2002 ; Fedder et al., 2007 2007 ; FIVNAT et al., 2007 2007 ), while others do not (Ludwig and Katalinic, 2003 2003 ). Further multi-center studies or meta-analyses will be required to obtain a definitive answer. In conclusion, our results demonstrate that sperm quality and source affect the fertilization process and the formation of high-quality embryos after ICSI. After embryo transfer, the potential viability of ICSI embryos is nearly equal regardless of the type of sperm used. Larger databases or a meta-analysis would be necessary to find a definitive answer as to whether sperm quality and source affect the malformation rate and genetic material of the offspring. References M Antinori , E Licata , G Dani , F Cerusico , C Versaci , D d′Angelo , S Antinori . Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial . Reprod Biomed Online , 2008 . 16 ( 6 ): 835 - 841 . 18549694 DOI: 10.1016/S1472-6483(10)60150-2 http://doi.org/10.1016/S1472-6483(10)60150-2 . Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial VW Aoki , AL Wilcox , C Thorp , BD Hamilton , DT Carrell . Improved in vitro fertilization embryo quality and pregnancy rates with intracytoplasmic sperm injection of sperm from fresh testicular biopsy samples vs. frozen biopsy samples . Fertil Steril , 2004 . 82 ( 6 ): 1532 - 1535 . 15589855 DOI: 10.1016/j.fertnstert.2004.05.092 http://doi.org/10.1016/j.fertnstert.2004.05.092 . Improved in vitro fertilization embryo quality and pregnancy rates with intracytoplasmic sperm injection of sperm from fresh testicular biopsy samples vs. frozen biopsy samples M Bonduelle , M Camus , A de Vos , C Staessen , H Tournaye , E van Assche , G Verheyen , P Devroey , I Liebaers , A van Steirteghem . Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children . Hum Reprod , 1999 . 14 ( s1 ): 243 - 264 . 10573038 . Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children M Bonduelle , E van Assche , H Joris , K Keymolen , P Devroey , A van Steirteghem , I Liebaers . Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters . Hum Reprod , 2002 . 17 ( 10 ): 2600 - 2614 . 12351536 DOI: 10.1093/humrep/17.10.2600 http://doi.org/10.1093/humrep/17.10.2600 . Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters P Braude , V Bolton , S Moore . Human gene expression first occurs between the four- and eight-cell stages of preimplantation development . Nature , 1988 . 332 ( 6163 ): 459 - 461 . 3352746 DOI: 10.1038/332459a0 http://doi.org/10.1038/332459a0 . Human gene expression first occurs between the four- and eight-cell stages of preimplantation development O Bukulmez , A Yucel , H Yarali , I Bildirici , T Gurgan . The origin of spermatozoa does not affect intracytoplasmic sperm injection outcome . Eur J Obstet Gynecol Reprod Biol , 2001 . 94 ( 2 ): 250 - 255 . 11165734 DOI: 10.1016/S0301-2115(00)00347-X http://doi.org/10.1016/S0301-2115(00)00347-X . The origin of spermatozoa does not affect intracytoplasmic sperm injection outcome S Chatterjee , C Gagnon . Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing . Mol Reprod Dev , 2001 . 59 ( 4 ): 451 - 458 . 11468782 DOI: 10.1002/mrd.1052 http://doi.org/10.1002/mrd.1052 . Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing SU Chen , HN Ho , HF Chen , SC Huang , TY Lee , YS Yang . Pregnancy achieved by intracytoplasmic sperm injection using cryopreserved semen from a man with testicular cancer . Hum Reprod , 1996 . 11 ( 12 ): 2645 - 2647 . 9021367 . Pregnancy achieved by intracytoplasmic sperm injection using cryopreserved semen from a man with testicular cancer KR Chohan , JT Griffin , DT Carrell . Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation . Andrologia , 2004 . 36 ( 5 ): 321 - 326 . 15458552 DOI: 10.1111/j.1439-0272.2004.00626.x http://doi.org/10.1111/j.1439-0272.2004.00626.x . Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation JM Cummins , SM Pember , AM Jequier , JL Yovich , PE Hartmann . A test of the human sperm acrosome reaction following ionophore challenge: relationship to fertility and other seminal parameters . J Androl , 1991 . 12 ( 2 ): 98 - 103 . 2050585 . A test of the human sperm acrosome reaction following ionophore challenge: relationship to fertility and other seminal parameters AA Derijck , GW van der Heijden , L Ramos , M Giele , JA Kremer , P de Boer . Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence . Hum Reprod , 2007 . 22 ( 9 ): 2368 - 2376 . 17580297 DOI: 10.1093/humrep/dem166 http://doi.org/10.1093/humrep/dem166 . Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence P Devroey , J Liu , A Nagy , H Tournaye , SJ Silber , AC van Steirteghem . Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection . Fertil Steril , 1994 . 62 ( 3 ): 639 - 641 . 8062963 . Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection D Dozortsev , R Neme , MP Diamond , S Abdelmassih , V Abdelmassih , F Oliveira , R Abdelmassih . Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia . Fertil Steril , 2006 . 86 ( 3 ): 606 - 611 . 16952508 DOI: 10.1016/j.fertnstert.2006.01.036 http://doi.org/10.1016/j.fertnstert.2006.01.036 . Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia AK Dubey , AS Penzias , AE Emmi , LC Layman , RH Reindollar , T Ducibella . Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation . Fertil Steril , 1997 . 68 ( 4 ): 714 - 717 . 9341616 DOI: 10.1016/S0015-0282(97)00313-0 http://doi.org/10.1016/S0015-0282(97)00313-0 . Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation AK Dubey , AS Penzias , RH Reindollar , T Ducibella . Technical and physiological aspects associated with the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human . Theriogenology , 1998 . 49 ( 1 ): 33 - 41 . 10732119 DOI: 10.1016/S0093-691X(97)00400-7 http://doi.org/10.1016/S0093-691X(97)00400-7 . Technical and physiological aspects associated with the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human J Fedder , A Gabrielsen , P Humaidan , K Erb , E Ernst , A Loft . Malformation rate and sex ratio in 412 children conceived with epididymal or testicular sperm . Hum Reprod , 2007 . 22 ( 4 ): 1080 - 1085 . 17224411 DOI: 10.1093/humrep/del488 http://doi.org/10.1093/humrep/del488 . Malformation rate and sex ratio in 412 children conceived with epididymal or testicular sperm P Fenichel , M Donzeau , D Farahifar , B Basteris , N Ayraud , BL His . Dynamics of human sperm acrosome reaction: relation with in vitro fertilization . Fertil Steril , 1991 . 55 ( 5 ): 994 - 999 . 2022277 . Dynamics of human sperm acrosome reaction: relation with in vitro fertilization FIVNAT , J de Mouzon , R Levy , Z Mourouvin , J Belaisch-Allart , A Bachelot , D Royère . Semen characteristics and quality of the conceptus in fertilization in vitro . Gynecol Obstet Fertil , 2007 . 35 ( 3 ): 216 - 223 . (in French) 17321779 DOI: 10.1016/j.gyobfe.2007.01.017 http://doi.org/10.1016/j.gyobfe.2007.01.017 . Semen characteristics and quality of the conceptus in fertilization in vitro SP Flaherty , D Payne , NJ Swann , CD Mattews . Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection . Hum Reprod , 1995 . 10 ( 10 ): 2623 - 2629 . 8567782 . Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection MN Giraud , C Motta , D Boucher , G Grizard . Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa . Hum Reprod , 2000 . 15 ( 10 ): 2160 - 2164 . 11006192 DOI: 10.1093/humrep/15.10.2160 http://doi.org/10.1093/humrep/15.10.2160 . Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa EN Göker , F Sendag , R Levi , H Sendag , E Tavmergen . Comparison of the ICSI outcome of ejaculated sperm with normal, abnormal parameters and testicular sperm . Eur J Obstet Gynecol Reprod Biol , 2002 . 104 ( 2 ): 129 - 136 . DOI: 10.1016/S0301-2115(02)00067-2 http://doi.org/10.1016/S0301-2115(02)00067-2 . Comparison of the ICSI outcome of ejaculated sperm with normal, abnormal parameters and testicular sperm E Heytens , J Parrington , K Coward , C Young , S Lambrecht , SY Yoon , RA Fissore , R Hamer , CM Deane , M Ruas , 等 . Reduced amounts and abnormal forms of phospholipase C zeta (PLC ζ ) in spermatozoa from infertile men . Hum Reprod , 2009 . 24 ( 10 ): 2417 - 2428 . 19584136 DOI: 10.1093/humrep/dep207 http://doi.org/10.1093/humrep/dep207 . Reduced amounts and abnormal forms of phospholipase C zeta (PLCζ) in spermatozoa from infertile men WV Holt , RD North . Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa . J Reprod Fertil , 1991 . 91 ( 2 ): 451 - 461 . 2013873 DOI: 10.1530/jrf.0.0910451 http://doi.org/10.1530/jrf.0.0910451 . Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa WV Holt , MF Head , RD North . Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy . Biol Reprod , 1992 . 46 ( 6 ): 1086 - 1094 . 1391306 DOI: 10.1095/biolreprod46.6.1086 http://doi.org/10.1095/biolreprod46.6.1086 . Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy G Huszar , A Jakab , D Sakkas , CC Ozenci , S Cayli , E Delpiano , S Ozkavukcu . Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects . Reprod Biomed Online , 2007 . 14 ( 5 ): 650 - 663 . 17509211 DOI: 10.1016/S1472-6483(10)61060-7 http://doi.org/10.1016/S1472-6483(10)61060-7 . Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects H Kobayashi , A Sato , E Otsu , H Hiura , C Tomatsu , T Utsunomiya , H Sasaki , N Yaegashi , T Arima . Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients . Hum Mol Genet , 2007 . 16 ( 21 ): 2542 - 2551 . 17636251 DOI: 10.1093/hmg/ddm187 http://doi.org/10.1093/hmg/ddm187 . Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients PN Kolettis , RK Sharma , FF Pasqualotto , D Nelson , AJ Thomas , A Agarwal . Effect of seminal oxidative stress on fertility after vasectomy reversal . Fertil Steril , 1999 . 71 ( 2 ): 249 - 255 . 9988393 DOI: 10.1016/S0015-0282(98)00467-1 http://doi.org/10.1016/S0015-0282(98)00467-1 . Effect of seminal oxidative stress on fertility after vasectomy reversal YM Lin , CC Hsu , TC Kuo , JS Lin , ST Wang , KE Huang . Percutaneous epididymal sperm aspiration versus microsurgical epididymal sperm aspiration for irreparable obstructive azoospermia—experience with 100 cases . J Formos Med Assoc , 2000 . 99 ( 6 ): 459 - 465 . 10925551 . Percutaneous epididymal sperm aspiration versus microsurgical epididymal sperm aspiration for irreparable obstructive azoospermia—experience with 100 cases CH Liu , HM Tsao , TC Cheng , HM Wu , CC Huang , CI Chen , DP Lin , MS Lee . DNA fragmentation, mitochondrial dysfunction and chromosomal aneuploidy in the spermatozoa of oligoasthenoteratozoospermic males . J Assist Reprod Genet , 2004 . 21 ( 4 ): 119 - 126 . 15270210 DOI: 10.1023/B:JARG.0000029495.22787.83 http://doi.org/10.1023/B:JARG.0000029495.22787.83 . DNA fragmentation, mitochondrial dysfunction and chromosomal aneuploidy in the spermatozoa of oligoasthenoteratozoospermic males KE Loutradi , BC Tarlatzis , DG Goulis , L Zepiridis , T Pagou , E Chatziioannou , GF Grimbizis , I Papadimas , I Bontis . The effects of sperm quality on embryo development after intracytoplasmic sperm injection . J Assist Reprod Genet , 2006 . 23 ( 2 ): 69 - 74 . 16575547 DOI: 10.1007/s10815-006-9022-8 http://doi.org/10.1007/s10815-006-9022-8 . The effects of sperm quality on embryo development after intracytoplasmic sperm injection M Ludwig , A Katalinic . Pregnancy course and health of children born after ICSI depending on parameters of male factor infertility . Hum Reprod , 2003 . 18 ( 2 ): 351 - 357 . 12571173 DOI: 10.1093/humrep/deg048 http://doi.org/10.1093/humrep/deg048 . Pregnancy course and health of children born after ICSI depending on parameters of male factor infertility JE Miller , TT Smith . The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro . Hum Reprod , 2001 . 16 ( 5 ): 918 - 924 . 11331638 DOI: 10.1093/humrep/16.5.918 http://doi.org/10.1093/humrep/16.5.918 . The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro D Mortimer . Sperm preparation methods . J Androl , 2000 . 21 ( 3 ): 357 - 366 . 10819443 . Sperm preparation methods T Naru , MN Sulaiman , A Kidwai , MH Ather , S Waqar , S Virk , JH Rizvi . Intracytoplasmic sperm injection outcome using ejaculated sperm and retrieved sperm in azoospermic men . Urol J , 2008 . 5 ( 2 ): 106 - 110 . 18592463 . Intracytoplasmic sperm injection outcome using ejaculated sperm and retrieved sperm in azoospermic men S Nilsson , U Waldenström , AB Engström , D Hellberg . Single blastocyst transfer after ICSI from ejaculate spermatozoa, percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) . J Assist Reprod Genet , 2007 . 24 ( 5 ): 167 - 171 . 17318393 DOI: 10.1007/s10815-006-9091-8 http://doi.org/10.1007/s10815-006-9091-8 . Single blastocyst transfer after ICSI from ejaculate spermatozoa, percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) JB Oliveira , CG Petersen , FC Massaro , RL Baruffi , AL Mauri , LF Silva , J Ricci , JG Franco . Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles . Reprod Biol Endocrinol , 2010 . 8 ( 1 ): 56 20529256 DOI: 10.1186/1477-7827-8-56 http://doi.org/10.1186/1477-7827-8-56 . Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles G Palermo , H Joris , P Devroey , AC van Steirteghem . Pregnancies after intracytoplasmic injection of a single permatozoon into an oocyte . Lancet , 1992 . 340 ( 8810 ): 17 - 18 . 1351601 DOI: 10.1016/0140-6736(92)92425-F http://doi.org/10.1016/0140-6736(92)92425-F . Pregnancies after intracytoplasmic injection of a single permatozoon into an oocyte L Parmegiani , GE Cognigni , S Bernardi , E Troilo , W Ciampaglia , M Filicori . “Physiologic ICSI”: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality . Fertil Steril , 2010 . 93 ( 2 ): 598 - 604 . 19393999 DOI: 10.1016/j.fertnstert.2009.03.033 http://doi.org/10.1016/j.fertnstert.2009.03.033 . “Physiologic ICSI”: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality DE Pegg . The history and principles of cryopreservation . Semin Reprod Med , 2002 . 20 ( 1 ): 5 - 13 . 11941530 DOI: 10.1055/s-2002-23515 http://doi.org/10.1055/s-2002-23515 . The history and principles of cryopreservation SD Perreault . Chromatin remodeling in mammalian zygotes . Mutat Res , 1992 . 296 ( 1-2 ): 43 - 55 . 1279407 DOI: 10.1016/0165-1110(92)90031-4 http://doi.org/10.1016/0165-1110(92)90031-4 . Chromatin remodeling in mammalian zygotes SD Perreault , RR Barbee , VL Slott . Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes . Dev Biol , 1988 . 125 ( 1 ): 181 - 186 . 3334716 DOI: 10.1016/0012-1606(88)90070-X http://doi.org/10.1016/0012-1606(88)90070-X . Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes L Ramos , P Kleingeld , E Meuleman , R van Kooy , J Kremer , D Braat , A Wetzels . Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia . Fertil Steril , 2002 . 77 ( 2 ): 233 - 237 . 11821077 DOI: 10.1016/S0015-0282(01)02962-4 http://doi.org/10.1016/S0015-0282(01)02962-4 . Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia CJ Redgment , D Yang , M Tsirigotis , N Yazdani , T al Shawaf , IL Craft . Experience with assisted fertilization in severe male factor infertility and unexplained failed fertilization in vitro . Hum Reprod , 1994 . 9 ( 4 ): 680 - 683 . 8046023 . Experience with assisted fertilization in severe male factor infertility and unexplained failed fertilization in vitro AA Rimm , AC Katayama , M Diaz , KP Katayama . A meta-analysis of controlled studies comparing major malformation rates in IVF and ICSI infants with naturally conceived children . J Assist Reprod Genet , 2004 . 21 ( 12 ): 437 - 443 . 15704519 DOI: 10.1007/s10815-004-8760-8 http://doi.org/10.1007/s10815-004-8760-8 . A meta-analysis of controlled studies comparing major malformation rates in IVF and ICSI infants with naturally conceived children D Royere , C Barthelemy , S Hamamah , J Lansac . Cryopreservation of spermatozoa: a 1996 review . Hum Reprod Update , 1996 . 2 ( 6 ): 553 - 559 . 9111188 DOI: 10.1093/humupd/2.6.553 http://doi.org/10.1093/humupd/2.6.553 . Cryopreservation of spermatozoa: a 1996 review D Sakkas , E Mariethoz , G Manicardi , D Bizzaro , PG Bianchi , U Bianchi . Origin of DNA damage in ejaculated human spermatozoa . Rev Reprod , 1999 . 4 ( 1 ): 31 - 37 . 10051100 DOI: 10.1530/ror.0.0040031 http://doi.org/10.1530/ror.0.0040031 . Origin of DNA damage in ejaculated human spermatozoa NN Sarkar . Intracytoplasmic sperm injection: an assisted reproductive technique and its outcome to overcome infertility . J Obstet Gynaecol , 2007 . 27 ( 4 ): 347 - 353 . 17654183 DOI: 10.1080/01443610701327339 http://doi.org/10.1080/01443610701327339 . Intracytoplasmic sperm injection: an assisted reproductive technique and its outcome to overcome infertility G Schatten . The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization . Dev Biol , 1994 . 165 ( 2 ): 299 - 335 . 7958403 DOI: 10.1006/dbio.1994.1256 http://doi.org/10.1006/dbio.1994.1256 . The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization E Seli , DK Gardner , WB Schoolcraft , O Moffatt , D Sakkas . Extend of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization . Fertil Steril , 2004 . 82 ( 2 ): 378 - 383 . 15302287 DOI: 10.1016/j.fertnstert.2003.12.039 http://doi.org/10.1016/j.fertnstert.2003.12.039 . Extend of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization RH Shapiro , CH Muller , G Chen , RE Berger . Vasectomy reversal associated with increased reactive oxygen species production by seminal fluid leukocytes and sperm . J Urol , 1998 . 160 ( 4 ): 1341 - 1346 . 9751351 DOI: 10.1016/S0022-5347(01)62532-1 http://doi.org/10.1016/S0022-5347(01)62532-1 . Vasectomy reversal associated with increased reactive oxygen species production by seminal fluid leukocytes and sperm L Swann , MG Larman , CM Saunders , FA Lai . The cytosolic sperm factor that triggers Ca 2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCζ . Reproduction , 2004 . 127 ( 4 ): 431 - 439 . 15047934 DOI: 10.1530/rep.1.00169 http://doi.org/10.1530/rep.1.00169 . The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCζ BC Tarlatzis , G Pados , J Bontis , S Lagos , G Gribizis , E Spanos , S Mantalenakis . Ovarian stimulation with buserilin/HMG/HCG: prospective randomized study of short versus long protocol . Hum Reprod , 1993 . 8 ( 6 ): 807 - 812 . 8345067 . Ovarian stimulation with buserilin/HMG/HCG: prospective randomized study of short versus long protocol M Tavalaee , S Razavi , MH Nasr-Esfahani . Influence of sperm chromatin anomalies on assisted reproductive technology outcome . Fertil Steril , 2009 . 91 ( 4 ): 1119 - 1126 . 18504041 DOI: 10.1016/j.fertnstert.2008.01.063 http://doi.org/10.1016/j.fertnstert.2008.01.063 . Influence of sperm chromatin anomalies on assisted reproductive technology outcome O Tepla , J Peknicova , K Koci , J Mika , M Mrazek , F Elzeinova . Evaluation of reproductive potential after intracytoplasmic sperm injection of varied human semen tested by antiacrosomal antibodies . Fertil Steril , 2006 . 86 ( 1 ): 113 - 120 . 16750209 DOI: 10.1016/j.fertnstert.2005.12.019 http://doi.org/10.1016/j.fertnstert.2005.12.019 . Evaluation of reproductive potential after intracytoplasmic sperm injection of varied human semen tested by antiacrosomal antibodies H Tournaye , P Devroey , J Liu , Z Nagy , W Lissens , A van Steirteghem . Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens . Fertil Steril , 1994 . 61 ( 6 ): 1045 - 1051 . 8194615 . Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens AC van Steirteghem , Z Nagy , H Joris , J Liu , C Staessen , J Smitz , A Wisanto , P Devroey . High fertilization and implantation rates after intracytoplasmic sperm injection . Hum Reprod , 1993 . 8 ( 7 ): 1061 - 1066 . 8408487 . High fertilization and implantation rates after intracytoplasmic sperm injection LL Veek . BA Keel , BW Webster . The Morphological Assessment of Human Oocytes and Early Conception . Hand-Book of the Laboratory Diagnosis and Treatment of Infertility , 1990 . : Voca Raton CRC Press , 353 - 369 . . The Morphological Assessment of Human Oocytes and Early Conception SJr Verza , SC Esteves . Sperm defect severity rather than sperm source is associated with lower fertilization rates after intracytoplasmic sperm injection . Int Braz J Urol , 2008 . 34 ( 1 ): 49 - 56 . 18341721 DOI: 10.1590/S1677-55382008000100008 http://doi.org/10.1590/S1677-55382008000100008 . Sperm defect severity rather than sperm source is associated with lower fertilization rates after intracytoplasmic sperm injection MR Virro , KL Larson-Cook , DP Evenson . Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles . Fertil Steril , 2004 . 81 ( 5 ): 1289 - 1295 . 15136092 DOI: 10.1016/j.fertnstert.2003.09.063 http://doi.org/10.1016/j.fertnstert.2003.09.063 . Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles W Watkins , F Nieto , H Bourne , B Wutthiphan , A Speirs , HW Baker . Testicular and epididymal sperm in a microinjection program; methods of retrieval and results . Fertil Steril , 1997 . 67 ( 3 ): 527 - 535 . 9091342 DOI: 10.1016/S0015-0282(97)80081-7 http://doi.org/10.1016/S0015-0282(97)80081-7 . Testicular and epididymal sperm in a microinjection program; methods of retrieval and results M Wilding , G Coppola , L di Matteo , A Palagiano , E Fusco , B Dale . Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa . J Assist Reprod Genet , 2011 . 28 ( 3 ): 253 - 262 . 21072684 DOI: 10.1007/s10815-010-9505-5 http://doi.org/10.1007/s10815-010-9505-5 . Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa WHO WHO Laboratory Manual for the Examination and Processing of Human Semen , 2010 . 5th Ed : Geneva World Health Organization Press , 7 - 114 . . WHO Laboratory Manual for the Examination and Processing of Human Semen R Yanagimachi . E Knobil , JD Neil . Mammalian Fertilization . Physiology of Reproduction , 1994 . : New York Raven Press , 189 - 317 . . Mammalian Fertilization 10.1631/jzus.B1100216 Project supported by the National Natural Science Foundation of China (No. 81070532), the National Basic Research Program (973) of China (Nos. 2012CB944901 and 2007CB948104), and the Zhejiang Provincial Natural Science Foundation of China (No. Z207021) 2011-07-06 2011-12-08 2011-09-27 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 13