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Lu et al.

/ J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press


Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) ISSN (Print); ISSN (Online); E-mail:

Different sperm sources and parameters can influence ICSI outcomes before embryo implantation*
Yue-hong LU§1,2, Hui-juan GAO§1, Bai-jia LI1, Ying-ming ZHENG1, Ying-hui YE1, Yu-li QIAN1, Chen-ming XU1, He-feng HUANG1, Fan JIN†‡1

(1Center for Reproductive Medicine, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China) (2Center for Reproductive Medicine, Shaoxing Women and Children Hospital, Shaoxing 312000, China)


Received July 6, 2011; Revision accepted Sept. 27, 2011; Crosschecked Sept. 4, 2011

Key words: ICSI, Sperm, Sperm source, Sperm parameters, Outcomes, Offspring, Malformation doi:10.1631/jzus.B1100216 Document code: A CLC number:

‡ §

1 Introduction

Intracytoplasmic sperm injection (ICSI), first introduced by Palermo et al. (1992), is an assisted-fertilization micromanipulation technique for fertilizing an egg by directly injecting a selected sperm into the ooplasm. Compared with conventional IVF, ICSI greatly reduces the requirements for sperm

Corresponding author These two authors contribute equally to this work * Project supported by the National Natural Science Foundation of China (No. 81070532, the National Basic Research Program of China (No. 2007CB948104), and the Zhejiang Provincial Natural Science Foundation of China (No. Z207021) © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011

ed it

Abstract: To evaluate the effects of sperm with different parameters and sources on the outcomes of intracytoplasmic sperm injection (ICSI), 1,972 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 Group 1 to Group 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% (9/718). 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.

quantity, motility and fertilization ability. With the development of micromanipulation, ICSI has become an important part of assisted reproductive techniques (ART) for the treatment of cases with male factor infertility (Sarkar, 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; Redgment et al., 1994; Tournaye et al., 1994; Devroey et al., 1994; Chen et al., 1996). Nevertheless, sperm retrieved from the testis,


endometriosis. 1 ml was used for preparation. Derijck et al. thereby excluding other female factors such as ed it epididymis or ejaculation may have a different concentration. Institutional review board approval and written consents from patients were obtained prior to data collection.. 2000. 2001. 2010 ) (total sperm number. 2007). Group 2 was composed of 329 ICSI cycles with mild OAT (total sperm number 20 – 39 × 106 sperm/ml. 2004. testicular volume. and/or progressive motility 10 – 20%).. There is an ongoing debate about this question (Bukulmez et al. In all cycles studied.. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press un 2 Materials and methods 2.. 2007).. Group 5 was composed of 171 ICSI cycles with extremely severe OAT (total sperm number < 1 × 106 sperm/ml. Nilsson et al. or abnormal results from more than two previous semen analyses. except for the cases with extremely low counts. 1991.. Göker et al. China were included in this retrospective study. and gene imprinting conditions (Holt et al.. Pegg.. Group 3 was composed of 522 ICSI cycles with moderate OAT (total sperm number 10 – 20 × 106 sperm/ml. Here. Group 1 was composed of 288 ICSI cycles with normal semen parameters. Furthermore.2 Sperm preparation Fresh ejaculated semen was collected on the day of oocyte retrieval and left to liquefy for approximately 20 – 30 min prior to semen analysis. 2004). mitochondrial dysfunction.. Pegg..972 ICSI cycles to explore the possible relationships between sperm quantity. Generally. Derijck et al. Naru et al. 2004. Therefore. and chromosomal aneuploidy are significantly higher in the spermatozoa of OAT males compared with unaffected controls (Liu et al. 2000. the tubal factor was identified as the sole cause of female factor infertility. Group 4 was composed of 332 ICSI cycles with severe OAT (total sperm number 1 – 10 × 106 sperm/ml. 2007. 2001. source and the outcomes of ICSI. 2002. Group 7 was composed of 80 ICSI cycles using frozen-thawed sperm.. based on the 5th World Health Organization (WHO) standard (WHO. and uterus deformation. 2007). morphology. Miller et al. 1991. 1992. as previously described (Mortimer. Aoki et al.1 Patients A total of 1. and/or progressive motility 1 – 10%). and diagnostic biopsy) or difficult ejaculation on the day of IVF. motility or morphology and differ in their degree of maturity. the semen specimens were processed by density gradient centrifugation. Zhejiang University School of Medicine. Verza et al. and/or progressive motility < 1%). Loutradi et al. 2001. 2001. For instance. Chohan et al. ICSI was performed for patients with failure or low fertilization rates (< 30%) in their the last in vitro fertilization (IVF) cycle.. Based on the sperm quality and source on the day of ICSI. Liu et al. Chohan et al. Giraud et al. Afterwards. 2004. quality. 2002. and morphology ≥ 4% normal forms).. Holt et al. and/or progressive motility 20 – 32%. chromosomal or DNA composition. ≥ 39 × 106 sperm/ml.. 2008). The final solution was incubated ed . rates of DNA fragmentation.. For each semen sample. Kobayashi et al.. and/or morphology < 4% normal forms). 2008. progressive motility ≥ 32%. Hangzhou.. Tepla et al.. myoma. endometrial polypus. 2006. Chatterjee et al. the patients were divided into seven groups.. Giraud et al.. aberrant DNA methylation of imprinted loci in sperm from oligospermic patients has been reported (Kobayashi et al..925 couples who underwent 1.. we report an analysis of 1. In this group. unexplained primary infertility after more than three cycles of failed artificial insemination with the male partner’s sperm. 2004. in which the main reason for ICSI was that the progressive motility of donated semen was less than 25% after thawing. 2006. testicular and epididymal sperm are considered to have a low degree of maturity and a high malformation rate. Dozortsev et al. 1992. 2002. the consequences of using these different sources of sperm have attracted a great deal of attention since the first introduction of ICSI. Group 6 was composed of 250 ICSI cycles with obstructive azoospermia (OA) (azoospermia patients with normal serum folliclular stimulating hormone (FSH)..2 Ren et al. The accumulated evidence also shows that the process of freezing and thawing sperm could cause damage to the sperm (Holt et al.972 ICSI cycles from January 2004 to June 2008 in the Center for Reproductive Medicine of the Women’s Hospital. 2007. calculated by sperm concentration × sperm volume. Chatterjee et al. 2.. 2006. using testicular or epididymal sperm. 2000)..

Females older than thirty-five years or on their second round of ART received at most three embryos. After injection. Japan) at 200× magnification. The spermatozoon and the cytoplasm were injected. Seminiferous tubules were dissected mechanically. A small part of the cytoplasm was aspirated. Decapeptyl.. and the presence of sperm was confirmed using an inverted microscope at 400× magnification. 2000). 1996) on the day of ICSI. 2000). USA). the tubes with the follicular fluid were transferred to the laboratory and examined using stereomicroscopy to identify the cumulus-oocyte complexes (COCs). 5% (v/v) CO2 for 0. The denuded oocytes were then rinsed and incubated in M-HTF until the time of injection (2 – 3 h after collection). The COCs were transferred to pre-balanced G-IVF (Vitrolife. Ovidrel®. 1990). 2. The final solution was incubated at 37°C. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press 3 2. Goteborg. Vitrolife) micro-droplet. The injecting pipette containing the spermatozoon was introduced at the 3 o’clock position through the zona pellucida and oolemma. Vitrolife) in M-HTF for 10 – 15 s. 2. Serum ed .6 Embryo culture and establishment of pregnancy Fertilization was confirmed 16 – 20 h post ICSI procedure by the presence of two pronuclei. 2. tail first. 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. Oocytes were retrieved 36 h after HCG administration using follicular aspiration with guidance from transvaginal sonography. Bloomington. and the injecting pipette was withdrawn gently.. A total of 1 – 3 embryos were chosen for transfer to the female partners 2 or 3 d after oocyte retrieval. Irvine Scientific.5 – 1 h prior to the ICSI procedure. Sweden) culture medium and incubated at 37°C. grades Ⅰ (absence of fragmentation) or Ⅱ (up to 20% of vitelineous space occupied by fragments) of cytoplasmic fragmentation (Veek. ICSI was performed immediately. Serono. Ovulation was induced by injection of human chorionic gonadotrophin (HCG. Tokyo. 5% (v/v) CO2 for 2 h prior to oocyte handling. Then. Spermatozoa were selected at the periphery of the polyvinylpyrrolidone (PVP. deep into the cytoplasm. DebioPharm.. the oocytes were rinsed and incubated in G-IVF medium. A selected spermatozoon was immobilized by repeatedly touching the tail and was then aspirated. 1993). Sperm retrieval from the epididymis was performed by percutaneous epididymal sperm aspiration (PESA) (Lin et al. USA). using a 135 μm diameter pipette (Flexipet. The aspirate was washed into a petri dish with a small volume of modified human tubal fluid (M-HTF. un ed it at 37°C. The microinjection procedure was performed on a heated stage at 37°C under an inverted microscope at 400× magnification. Switzerland)/gonadotrophin protocol (Tarlatzis et al. The luteal phase was supported by progesterone ampules. into the injecting pipette. 5% (v/v) CO2 for 0.4 Oocyte handling The oocytes were stripped of the surrounding cumulus cells (2 h after ovum collection) using hyaluronidase (80 IU/ml. Geneva. in accordance with China’s ART Regulations. The frozen sperm was thawed (Royere et al. 2001) was performed 38 – 42 h post-HCG injection. the oocytes were transferred to M-HTF for complete removal of the corona cells. Cook. Geneva.. The supernatant was processed by density gradient centrifugation (Mortimer.. Cleavage and embryo quality was assessed 48 – 72 h post-ICSI procedure.5 – 1 h prior to the ICSI procedure.5 ICSI procedure The ICSI procedure (Miller et al. and the presence of sperm was examined using an inverted microscope (Olympus. The oocyte was held by a holding pipette with the polar body at the 12 or 6 o’clock position. Sperm retrieval from the testis was performed percutaneously by testicular sperm aspiration (TESA) (Watkins et al. The thawed sperm was processed using the same method as used for fresh ejaculated sperm. IN. The ICSI procedure was performed only for oocytes that had extruded their first polar bodies.Lu et al. in the absence of multinucleation and zona pellucida alterations. 1997) under local anesthesia. Switzerland) when at least two follicles reached a diameter of ≥ 18 mm. CA.3 Ovarian stimulation and oocyte retrieval All female patients were treated with a long gonadotrophin-releasing hormone agonist (GnRH-a. After follicular aspiration.

P < 0. 2.5 13111.05: Significantly higher than Groups 1.8±8483 . / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press HCG levels were measured on day 14 after embryo transfer. 4.5%) (P<0.1 Characteristics of patients A total of 1. 2. premature birth rates.1±7.6±3.1±3. ed it Group 1 288 Group 2 329 31.1±6.05) (Rank Transform method).0 11.2‡ 6.2±3.1±6.9±7.2±3.7** 4. one-way ANOVA. There were no statistical differences in the cleavage rates among the seven groups (P>0.05).05 was accepted as significant.8 6.7 12.4±92 00. 2.0±3.7 11. # p<0.9±8.3 31. p<0.05: Significantly higher than Groups 1. The fertilization rates decreased as semen quality decreased from Group 1 to Group 6 and reached statistical significance in Groups 5 (67.2 5. 5. and 3 (P < 0.7±2.0±7771 .2±7. Miscarriage was defined as pregnancy loss before 20 weeks of gestational age. 3.8±7.2 11.6±2.6±4.4±6. 3.1±3. The infertility period was the shortest in Group 6 and the longest in Group 7 (P<0.05) (Rank Transform method) ed Group 7 80 31.7%) was significantly higher than in other groups (P<0. such as the gestational age. Δp<0.0±8. 2. 2. pregnancy.9 11. and 7 (P < 0.9 6.4 31. some perinatal parameters.3 31.2±3. Major malformations were defined as malformations requiring surgical treatment or causing essential reduced functionality. cleavage.4±6. and 3 (P < 0.5 The rates of fertilization.7±7.4 un a Values are means ± SD.9±7585 . miscarriage.6§ 13222.5%) and 6 (65.4§ 13860. and 4 (P < 0. cleavage and high-quality embryos in each group are listed in Table 2.2 12.3 12840.4±2. high-quality embryos. ectopic pregnancy.8±2. Endometria were significantly thicker in Groups 6 and 7 (P<0.0±2.05) (Rank Transform method).0±4.05) (Table 2).3 13308.4±2.4 Ren et al.6±4. the females were significantly younger (P<0.1±2. live-birth rates.8±3.0±96 08.7% ± 24.9% ± 21. Gestational age was calculated as the time from an estimated first menstruation day (14 d before follicular aspiration) to the time of birth.7±1.3±6.1±7.0 6. Cycles (n) Women’s agea (y) Infertility perioda (y) Basal FSHa (IU/L) Endometrial thicknessa (mm) Peak E2a (nmol/L) Retrieved oocytesa (n) Injected MII oocytea (n) Table 1 Characteristics of the patients in all groups Group 3 Group 4 Group 5 Group 6 522 332 171 250 30.2±2.1±3. In Groups 4.972 ICSI cycles were analyzed. 3. Clinical pregnancies were confirmed by a vaginal ultrasound 30 d after embryo transfer that showed the presence of an intrauterine gestational sac with detectable fetal cardiac activity.5±1. 2. 3.2 30.1±2.5±8623 .1 6. 3. birth-weight.9 14. and 4 (P < 0.9±3.05: Significantly higher than Groups 1.05) (Rank Transform method).4±6.05: Significantly higher than Groups 1.7Δ 15. and 6 (P < 0.7±1.05: Significantly lower than Groups 1. and 7 (P < 0.2 Outcomes of ICSI All data were analyzed using SPSS 15. and major malformations were assessed.05). 4. * p<0.5±2.2 15. and 6.2 12722. the Rank Transform method.1±82 71.05) (Rank ‡ Transform method). Ⅲ.05: Significantly higher than Groups 1.0 11. † p<0.9 11.3% ± 27.4 12. 3.05: Significantly lower than Groups 1. Sex ratio was defined as male offspring / (males + females).4 11.8 Statistical analysis 3 Results 3. The peak (estradiol) E2 before HCG injection and the number of retrieved oocytes were significantly higher in Group 6 (Table 1).5§ 12.6 13. and logistic regression.05).3 14. § p<0. 2.5 .05).7 Assessments In addition to the rates of fertilization.9 11. 5.8 11. sex ratio. The high-quality embryo rate in Group 1 (70.6 12614.0 software (SPSS.6 14.1 13.6# 6.2 6.0 5. Chicago.9±3.05) (Rank Transform method). and multiple pregnancy. ** p<0. USA) and evaluated by the Chi-square test.05) (Rank Transform method).6±1.05). 2.9 5.3 6.0 * 4.6 11.9† 4.

05) (Rank Transform method).5 ** Group 7 74.9±11.6 Table 2 Outcomes of ICSI Group 3 Group 4 Group 5 72. 6.2 73. A total of 718 living newborns were delivered.5* 101/305 (33.4±8. Transfer cycles (n) Transferred embryos (n) Average no. in Groups 2 and 6.3) 40/126 (31.3±0.2 97. 3. 3. No statistical differences were observed for the live-birth rate.7±11.7) 5/234 (2.05) (Rank Transform method) ed Group 6 234 507 2. one girl with megacolon diagnosed in the newborn period.6) 11/63 (17. or hexadactylism.3±27.05: Significantly lower than Groups 1.5) 3. The cancellation rate for fresh embryo transfer was 7%.7±29.05: Significantly lower than Groups 1. † p<0. c Pregnancy rates and miscarriage rate were compared by logistic regression.0) 29/182 (15.2±0.05) (Rank Transform method).9 67.Lu et al.0±5. and 7 (P < 0.4±10.9±21.1) 35/94 (37.2±0.1±28. 700 female partners became clinically pregnant.2) 12/94 (12. ** p<0.05: Significantly lower than Group 1 (P < 0.196 embryos transferred.2 65.9) 2.9 65. low birth weight. In total. 2.05) (Rank Transform method). congenital cardiopathy.8±21. ** p<0.05: Significantly lower than Groups 1. two cases of perinatal death.7) 16/101 (15.05: Significantly higher than Groups 3.831 transfer cycles were performed.6) 27/101 (26. and 7 (P < 0.0 67. There were two cases of stillbirth due to a circular umbilical cord in Groups 2 and 7.4) 5/33 (15.1) 5/305 (1.6 2.1±22. diagnosed in infancy (Table 4).05). of embryos transferreda (n) Clinical pregnancy ratebc (%) Chemical pregnancy ratebc (%) Ectopic pregnancy ratebc (%) Multiple pregnancy ratebc (%) Miscarriage ratebc (%) a Table 3 Clinical outcomes of ICSI Group 3 Group 4 Group 5 490 307 155 un Values are means ± SD. fever.7) 101/269 (37. 2. weeks of premature birth.4 * Group 6 65.05) (Rank Transform method) 3.2) 30/101 (29.8±17.4±28.0) 8/307 (2.05).4±0.6 33/71 (46. and 6 were statistically lower than in other groups (P<0. 3. A total of nine cases of malformation were observed.4 99.4) 51/182 (28.2) .0 63.5* 182/490 (37.4 ** 94/234 (40. both of which were premature births. and 7 (P < 0.7±24. * p<0. chemical pregnancy. multiple pregnancy.0) 6/269 (2.8) 173 2.4±0. or sex ratio for all groups (P>0. birth weight.8) 2. b Values in parentheses are percentages.4 Outcomes of ICSI children ed it Group 1 269 Group 2 305 655 712 1137 678 334 2.8) 63/155 (40.0±10.5) 2/71 (2.0 98. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press 5 Group 1 Fertilization rate a (%) Cleavage rate a (%) High-qualit y embryo rate a (%) a Group 2 72.8) 12/33 (36.5** 0/71 Group 7 71 2.7† 96.2% (700/1.6) 3/155 (1. and miscarriage showed no statistical differences among all 7 groups (P>0.1) 6/490 (1.5 98.0±30.7±31.2±30. * p<0. or poor conditions of endometrium preparation. and 7 (P < 0.3 72. with a total clinical pregnancy rate of 38. The average numbers of embryos transferred in Groups 4.9) 3/155 (1. four boys with hypospadias (two boys).7) 16/101 (15.7) 16/126 (12.3 98. including one case of multiple structural abnormality and one case of fetal edema. premature birth rate.6±20.1 98.5** 126/307 (40. 4.7 Values are means ± SD.2±0.3 62. based mainly on a high risk of ovarian hyperstimulation syndrome (OHSS).1 62. with a total of 4.6±21.2) 7/490 (1.1 70.5) 8/269 (3.3±0. 5.9) 13/63 (20. The rates of clinical pregnancy.3±9. both terminated in the second trimester.6) 7/307 (2. ectopic pregnancy.05) (Table 3).2) 4/234 (1.3 Clinical outcomes of ICSI A total of 1.831).6) 5/305 (1. 5. one boy with cryptorchidism and another with epilepsy.

b Values in parentheses are percentages.7) 87/183 (0.6) (20. 2008. more detailed sperm selection may help to improve further the ICSI outcomes.. 1994. and chromosomal aneuploidy are significantly higher in the spermatozoa of OAT males (Liu et al.49) 1 Group 7 38 9 0 29/71 (40. High rates of DNA fragmentation (Tavalaee et al.8 2757.) 4 Discussion 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. sperm morphology is a crucial factor for sperm quality. un ed it ed . More importantly.53) 0 Group 2 99 21 0 78/305 (25..9) 52/107 (0. premature birth rate. Wilding et al.1 9/58 (15.6) 20/78 (25. Yanagimachi. c Values in parentheses are ratios.. 1992. No statistical differences were observed for any group (P > 0.6 Ren et al. the frequency of chromosomal disomy and diploidy was reduced 4. to guarantee that the sperm selected had a relatively normal form and good motility. However. 2011).1 7/38 (18...1±1.0±2.4) 20/38 (0. 2011). 2004.1 32/107 (29. 1997. ICSI outcomes were significantly improved (Antinori et al. 2004. 2009).8±1. ICSI can be successfully used for even the most severe cases of OATs or azoospermia.0) 37/140 29/98 10/48 (26..9± 702.8) 33. 1994).1± 671. Rates of DNA fragmentation.8 2799. (2007) have reported that in spermatozoa selected with hyaluronic acid. acrosomal defects (Cummins et al.45) 1 Group 6 107 30 0 77/234 (32.5) 26/58 (0.1 2965.53) 0 34.36) 69/130 (0. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press No. at the periphery of the PVP micro-droplet.. The wider application of ICSI and improvements in our understanding of sperm biology have encouraged more detailed investigations into sperm-derived effects on ICSI outcomes and children’s health.. (Live-birth rate..3 21/104 (21. weight of newborns was compared by one-way ANOVA.5±1. 1991). of triplets Live-birth rate/ET cycleb (%) Premature birth rateb (%) Weeks of premature birtha (w) Birth weighta (g) Low birth weight rateb (%) Sex ratioc (M/M+F) Malformations (n) a Group 1 104 23 1 79/269 (29.. There are published reports that when intracytoplasmic injection of morphologically selected spermatozoa (IMSI) was performed with sperm selected under a magnification level above 6000× (Antinori et al.4) (29. 2010). 1998). 1999). Parmegiani et al. Oliveira et al. Therefore.45) 3 Table 4 Results of ICSI children Group 3 Group 4 Group 5 183 130 58 43 30 10 0 1 0 140/490 98/307 48/155 (28.4±1.48) 4 34.7± 639.8) 6/29 (20. 1988.6) (31. Dubey et al.2 2891. 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.2) 55/104 (0..2± 623.5± 596.5) 34.4) 35..0± 715.1 18/99 (18.9±2. Currently. low birth weight rate and sex ratio were compared by the Chi-square test... of newborns (n) No. 2008. A tendency towards reduced fertilization rates was observed in Groups 1 to 5 as the sperm quality decreased.9) 25/77 (32. In our view. a normal-looking sperm could still contain serious nuclear malformations and chromosomal aneuploidies (Huszar et al.6) 33.. and epigenetic factors such as sperm-specific phospholipase C zeta (PLCζ) (Swann et al.7±3.05). 2010. weeks of premature birth were compared by the K-W test.8 42/130 (32.0 47/183 ( 6-fold.9) (31. indicating that sperm quality can affect the fertilization process. 2007.53) 0 Values are means ± SD. 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. We selected sperm under 400× magnification. Heytens et al. the rate of sperm nucleus normality was significantly higher.7± 652. Dubey et al.0 2898. Huszar et al. with these two methods.4) 24/79 (30.. Schatten. In addition.2 2981. We compared the outcomes of ICSI based on the sperm source and the quality of the ejaculates. mitochondrial dysfunction. Fenichel et al..6 3042. 2007).2) 45/99 (0. of twins No. 1995. Wilding et al.7) 35. 1991.. 2009) in poor quality sperm are all considered to be possible reasons for fertilization failure.. Perreault. Derijck et al.

two cases of stillbirth. Tepla et al.. and the initiation of mitotic division. the same was true for the perinatal parameters in all seven groups. 1998. chemical pregnancy. and sources of sperm have no obvious effects on pronuclei syngamy. 2002)..9.. epididymal obstruction also can cause sperm damage (Shapiro et al. 1999). and nine cases of malformation occurred. multiple pregnancies. or miscarriage. was significantly higher than in other groups. while others do not (Ludwig et al. in accordance with China’s ART Regulations. Hence. 2004. Although we did not observe statistically significant differences in cleavage rates among the different groups. Our data seem to support the notion that sperm from the testis or epididymis have decreased fertility potential after ICSI (Loutradi et al.. Some investigators believe there is some effect (Bonduelle et al. our results demonstrate that sperm quality and source affect the fertilization process and the formation of high-quality embryos un ed it ed . Aside from this factor. The malformation rate was 1. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press 7 Significantly lower fertilization rates were observed when testicular or epididymal sperm from men with OA or ejaculation failure were used for ICSI.. This result indicates that although different sources and different qualities of sperm could influence the fertilization process and the formation of high-quality embryos. Aside from the perinatal parameters. 1988). and a high percentage of good-quality sperm may be advantageous for generating a high percentage of high-quality embryos. 5. 2004). the data on ICSI clinical outcomes did not show statistically significant differences in the rates of clinical pregnancy. This result suggests that after completing fertilization.. ectopic pregnancy. Definite male-factor infertility in Groups 4. FIVNAT et al. Therefore. 2007. Kolettis et al. Possible explanations for this result may be that testicular and epididymal spermatozoa are less mature and subsequently less competent at fertilization than ejaculated sperm. which is consistent with previously reported malformation rates ranging from 1. those factors seem able to affect the formation of high-quality embryos. 2003. Sperm DNA fragmentation is a specific paternal factor that can influence embryo development when it increases above a certain level (Sakkas et al. These factors might explain the discrepancy between the effects of ICSI on cleavage and the formation of high-quality embryos.. and then the paternal influences on embryo development begin to be apparent (Braude et al. Fedder et al. However. Seli et al. In the present study. two cases of perinatal death. The development of a human embryo in the early stages is controlled by maternally inherited mRNA.. 2004). the percentages of female patients younger than 35 years old were much higher in these groups.25% (9/718). different quantities.. The embryonic genome is activated at approximately the 8-cell stage.. qualities. 2008). Verza et al. 2006). embryonic genome formation. 2006).. the potential viability of ICSI embryos after transfer is nearly equal no matter what type of sperm is used. 2002. the malformation rate was another important indicator to evaluate the safety of ICSI. 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... 2007). Further multi-center studies or meta-analyses will be required to obtain a definitive answer. In conclusion.Lu et al. 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. our cases were too few for a precise statistical analysis.. and 6. and 6 led to the couples visiting a fertility clinic at younger ages. The numbers of transferred embryos were significantly lower in Groups 4.. 5. There is an ongoing debate about whether sperm quality and source affects the malformation rate and genetic materials of ICSI children. with normal semen parameters. resulting in lower numbers of transferred embryos.7% for ICSI (Rimm et al. 2003). Furthermore. Decreased fertilization rates and lower percentages of high-quality embryos could also contribute to the reduced numbers of transferable embryos. 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 et al. when compared with all of the groups that used ejaculated sperm. 1999. Virro et al. Furthermore. morphologies. the high-quality embryo rate in Group 1...1% . Unfortunately. 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.

De Vos.. Derijck.. Van Steirteghem. Gurgan. Technical and physiological aspects associated with ed 5 Acknowledgements This work was supported by National Natural Science Foundation of China (No. C. 36(5):321-326..L. O.1038/332459a0] . 86(3):606-611. Licata. [doi:10. Braude. Van Assche... frozen biopsy samples. [doi:10. Yarali... Pregnancy achieved by intracytoplasmic sperm injection using cryopreserved semen from a man with testicular cancer. Ho.. P. A. Verheyen. and/or other relationship with manufacturers of pharmaceuticals. 10. Joris. 2001. G. Eur J Obstet Gynecol Reprod Biol.. 16(6):835-841. [doi:10.. D. A..10.S.. Fertil Steril. Ying-Hui Ye. T. H. A. Antinori. L. Devroey.V. 1988. He-Feng Huang participated in the design of the study and revised the manuscript. de Boer.00626. E. and the Zhejiang Provincial Natural Science Foundation of China (No..1016/j.F. All authors read and approved the final manuscript. 68(4):714-717..S. The origin of spermatozoa does not affect intracytoplasmic sperm injection outcome.. I. H.. I. Reindollar.M. R.. T.T. A. Jequier. Bildirici. V. statistical analysis. Diamond. 2004. Chatterjee. J. and drafted the manuscript.8 Ren et al. [doi:10.D. A. A. 94(2):250-255.. S. A.05. 1997. E. 59(4):451-458.. Bonduelle.... Hum Reprod.A.. 2007CB948104). Nature. laboratory supplies. 1991. R.. Tournaye. 2008.R. C. 2007.fertnstert.1052] Chen. Emmi.. Fertil Steril. S.1002/mrd. G..and eight-cell stage of preimplantation development. F. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Yucel.... Hartmann.. H. A test of the human sperm acrosome reaction following ionophore challenge: relationship to fertility and other seminal parameters.E. M. Abdelmassih. 1996. 2004. financial interest. Aoki. Andrologia. Carrell. Neme...Y. Staessen. M. Production of reactive oxygen species by spermatozoa undergoing cooling. [doi:10. Chen.. Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation.2004. 1998. A. 62(3):639-641. V... and thawing. Tournaye.2600] Bukulmez. J..C. D. S. V. H.H. Abdelmassih. Abdelmassih. the potential viability of ICSI embryos is nearly equal regardless of the type of sperm used.036] Dubey. and/or medical devices or with commercial providers of medically related services. S..P. A. Nagy. M. Moore. van der Heijden. A.1111/j.fertnstert. After embryo transfer. 1999. Hum Reprod....L. Oliveira. Dozortsev.U. Yu-Li Qian and Chen-Ming Xu participated in the data collection. Fertil Steril. Hum Reprod.. S. Layman.1016/j. Liebaers.. 2002. P. A. Versaci. F. Ducibella. freezing.. Yovich. Fertil Steril. A. Bolton.. Y.. participated in the data collection. Lee. M. P. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Larger databases or a meta-analysis would be necessary to find a definitive answer as to whether the quality and source of sperm affects the malformation rate and genetic material of the offspring...C...1093/humrep/17. A.. T. Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence. Steirteghem. 332:459-461. Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia.01. C.K..M. Bonduelle.S. Kremer. S. 82(6):1532-1535. S. J. Giele. Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreserva[doi: tion.J. Pember. R. 12(2):98-103. the National Basic Research Program of China (No. Ducibella. ed it Author contributions: Yue-Hong Lu and Hui-Juan Gao designed the study.1016/S0301-2115(00)00347-X] un References [doi:10. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. A.M. Reindollar.. Cerusico. and revised the manuscript.. 81070532.T. 11(12):2645-2647. T. Griffin. M. B. E.. Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial. M. L. Hum Reprod. 2006. J Androl. [doi:10. Bai-Jia Li and Ying-Ming Zheng participated in the data collection and statistical analysis.. 14 Suppl 1:243-264..2006..W. I. Human gene expression first occurs between the four. P.. Hamilton. Gagnon. D.. H... K. P.. Liu.E.. Huang..1016/S0015-0282(97)00313-0] Dubey...C. C.1093/humrep/dem166] Devroey..... Thorp. d'Angelo..N. J. Keymolen. K.T. Mol Reprod. 17(10):2600-2614...... Penzias. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press after ICSI.K..x] Cummins. [doi:10. Yang. R.2004. Antinori. Wilcox.1439-0272. S.. J.. Carrell... Dani... Reprod Biomed Online. P. Penzias.H. Chohan.092] Competing financial interests statement: Neither I nor my spouse/partner has a commercial interest.. 2001. 1994. Van Assche.1016/S1472-6483(10)60150-2] [doi:10. participated in its design and coordination. Van Steirteghem.. Liebaers. 22(9):2368-2376. Z207021).... H.W. Silber...A.. Fan Jin conceived of the study... Camus. D.. Ramos. Improved in vitro fertilization embryo quality and pregnancy rates with intracytoplasmic sperm injection of sperm from fresh testicular biopsy samples vs. Devroey. G..

. Tarlatzis.. Ayraud.. H. J..F.C. D.P.. [doi:10. B. 125(1):181-186.. Sasaki. C.C. 24(10):2417-2428..L.F.T. Hum Reprod Vol. Pagou. H. Barbee. Cayli.. Effect of seminal oxidative stress on fertility after vasectomy reversal.22787. N. Thomas.. G. D.. P. 20(1):5-13...G.. A. Sharma.03.H. C.. Wang. Waldenström...D. C. Bernardi.N. Hellberg. A. Zepiridis.. 8:56.83] Loutradi.. Rizvi.. [doi:10.. DNA Fragmentation. Van Steirteghem. Meuleman. C. Malformation rate and sex ratio in 412 children conceived with epididymal or testicular sperm. R..D. D.. [doi:10. Coward. Cognigni. C. T. Sperm preparation methods. Comparison of the ICSI outcome of ejaculated sperm with normal. Fertil Steril...-Y. J Assist Reprod Genet. Reprod Biol Endocrinol.. percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE).. 340(8810):17-18. Ather..033] Pegg. Sato. 2000.Lu et al.. W. M.. J.. 2001.fertnstert.. J Reprod Fertil.. 15(10):2160-2164. resulting in improvement of embryo quality..C....D. Nilsson.. Huszar. A.. E.. Agarwal A.. Liu. G. His. [doi:10. D. Delpiano.. S.... 16(5):918-924. S.. H. Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy. Bachelot.. 104(2):129-136.... A.F.1007/s10815-006-9091-8] Oliveira..1016/j. Sendag.. Huang. North. Fertil Steril. Papadimas.. S.. Cryopreservation.G. 2007. P.A.C. 296(1-2):43-55.. Semin Reprod Med.. [doi:10. R.1093/hmg/ddm187] Kolettis. M.J. Jakab. Lee. Huang. Hamer. [doi:10. H. et al. G.S. 71(2):249-255. Lancet.C.2009. M. Virk.N.. 2000. Ozenci. Gabrielsen. A..1007/s10815-006-9022-8] . [doi:10. Reduced amounts and abnormal forms of phospholipase C zeta (PLCζ) in spermatozoa from infertile men... J Assist Reprod Genet. M.. A. R. Parrington.. actin localization and thermotropic phase transitions in ram spermatozoa. R. Semen characteristics and quality of the conceptus in fertilization in vitro.. E. [doi:10. 18(2):351-357. C.E. Kuo. L. Levi.... [doi:10. Franco.10. Engström.E.. Dynamics of human sperm acrosome reaction: relation with in vitro fertilization.1093/humrep/16.0910451] un [doi:10.2160] Göker. Chen. Filicori. S.1086] [doi:10.. Fertil Steril. R. Biol Reprod. J.. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Giraud. Ruas.1055/s-2002-23515] Perreault. 35(3):216-223. Ciampaglia. Percutaneous epididymal sperm aspiration ed it Flaherty..P. 1991. Bontis.C. Humaidan.. 2007.V.B.S. E.. S. Ludwig.. de Mouzon.. Naru.01. Boucher.. 10(10):2623-2629. D.2007.. D. T. ed Fenichel. [doi:10.. Waqar. M... J. M. G. Utsunomiya. Baruffi..K.1016/S0093-691X(97)00400-7] Fedder.. Z. 2007.abnormal parameters and testicular sperm. Chromatin remodeling in mammalian zygotes. Katalinic. S. Young.1023/B:JARG. E. Sendag. Basteris. Single blastocyst transfer after ICSI from ejaculate spermatozoa. E.. L. Hsu.. Deane. Yaegashi. D. G. P. 2010. M. Kremer. [doi:10. versus microsurgical epididymal sperm aspiration for irreparable obstructive azoospermia--experience with 100 cases.0000029495.. Mutat Res. 21(4):119-126.1016/0140-6736(92)92425-F] Parmegiani.C..1016/0165-1110(92)90031-4] Ramos. [doi:10. Lambrecht.. The history and principles of cryopreservation... N.. 1992. A. 93(2):598-604.. Mauri. FIVNAT. Reprod Biomed Online.N.. E. Farahifar. S..1016/0012-1606(88)90070-X] Perreault. van Kooy. J. W.I. 49(1):33-41. Lin. Donzeau. 46(6):1086-1094... Kleingeld.. Wu. S. Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. P.5... F.. [doi:10. J.1093/humrep/15.. H.L. S... Hum Reprod. Sakkas. 2002. E. Ernst. R.M.D. 2008.L.. Pasqualotto. H. Kidwai. T. Ozkavukcu.E. C... K. J Assist Reprod Genet.. Motta. T. V. Troilo. T. L. M..... 16(21):2542-2551. 14(5):650-663. N. D. Levy.. Pregnancy course and health of children born after ICSI depending on parameters of male factor infertility.H.gyobfe. J Formos Med Assoc. C. Belaisch-Allart. 2004. T.L.1016/S0301-2115(02)00067-2] Heytens. R. Payne.J. 22(4):1080-1085. C.. 2000.. Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles.G.... K. Royère. Hum Mol Genet. "Physiologic ICSI": hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus. Hum Reprod.. E. Mattews. W. The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro..1016/j. [doi:10.6.. Goulis.1093/humrep/dep207] Holt.. 1992.F. Ricci.M. K.T.017] [doi:10. 1991. 2003. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes..1186/1477-7827-8-56] Palermo.. Eur J Obstet Gynecol Reprod Biol. Mitochondrial Dysfunction and Chromosomal Aneuploidy in the Spermatozoa of Oligoasthenoteratozoospermic Males. D. 1988. 2010.. Arima.D.1016/S0015-0282(98)00467-1] Holt.1530/jrf.. R.E....C. E. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press 9 the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human. Gynecol Obstet Fertil. J. F. 2002. 2007. Erb. [doi:10... A. 1995.. C. The effects of sperm quality on embryo development after intracytoplasmic sperm injection. Mourouvin.. Hum Reprod.B. Yoon.. Grimbizis. 5(2):106-110. I.M. [doi:10. Tavmergen. North.. Hum Reprod.. Pregnancies after intracytoplasmic injection of a single permatozoon into an oocyte. C.. M. Hiura. I.V. Fissore. 23(2):69-74. Cheng.. Sulaiman.. Swann. T... J. Petersen.M. K. Slott. P.. Silva. R. Tsao. Devroey.N.. 91(2):451-61.R. Massaro. 55(5):994-999.. A. F. Lin... Theriogenology. [doi:10.... 21(3):357-366. Tomatsu.. S. A. 2009.. Smith. R.918] Mortimer.. J. D. B.1093/humrep/dei238] Miller. Dev Biol.. Nelson.1093/humrep/del488] [doi:10.. Grizard. H... Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects. B. Y. Joris. Head. S. 2007.. J.1095/ biolreprod46. D. Hum Reprod.. U. A. 1999. Otsu.. 1992. 24(5):167-171. J Androl. Lin.E. L.H. Urol J. Intracytoplasmic sperm injection outcome using ejaculated sperm and retrieved sperm in azoospermic men.1016/S1472-6483(10)61060-7] Kobayashi. Chatziioannou.. 99(6):459-465.... S. Loft. Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection.0. 2006. [doi:10....

fertnstert. B...A. E. Voca Raton. Yanagimachi.. [doi:10. Lai.. Neil.1256] Seli.. Webster.. 8(6):807-812. 91(4):1119-1126. B. D. [doi:10. S. P.. blastocyst development. F.W. H.. 1999. p. 2006. Schoolcraft. D.. A Meta-Analysis of Controlled Studies Comparing Major Malformation Rates in IVF and ICSI Infants with Naturally Conceived Children. Katayama.. Joris.... 2004.1530/ror. Muller.A. 2008.. B. 2004. 2010..039] Shapiro. Mariethoz. R.. Katayama. Tarlatzis. R. T. D.. Hum Reprod. I.. R.019] Tournaye. Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new ed it Sarkar. H.. Mika. 86(1):113-120. di Matteo... Fertil Steril. M. A.... Speirs. H. 2009. Z.A. 21(12):437-443. L.1080/01443610701327339] Schatten. Influence of sperm chromatin anomalies on assisted reproductive technology outcome.M. [doi:10. C. J. M.. Yang.C. W. P.12.. E.. Pados. G. Nagy. [doi: ..353-369. WHO Laboratory Manual for the Examination and Processing of Human Semen. 1994. Bianchi. The morphological assessment of human oocytes and early conception. Cryopreservation of spermatozoa: a 1996 review. (eds). F. Chen. E.1590/S1677-55382008000200015] Redgment. M.1530/rep.. Koci. S. Evenson. 165(2):299-335. O. 5th ed. Bourne. Tsirigotis..N. N. G. F. J Assist Reprod Genet. L. Lissens. 1993.. A. K.. W. S. 28(3):253-262. Coppola. Wutthiphan. 1993. Van Steirteghem.. [doi:10. Mammalian Fertilization. C. Intracytoplasmic sperm injection: An assisted reproductive technique and its outcome to overcome infertility. C. Lansac...1016/j..C.fertnstert... (Eds). M.1007/s10815-010-9505-5] World Health Organization. Esteves.. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) in press J. Devroey. Hum Reprod.1016/j. J Obstet Gynaecol... Diaz.10 Ren et al. p. M. 2004. Nagy... Sperm chromatin structure assay (SCSA) parameters are related to fertilization. Physiology of Reproduction. P. Rimm. Nieto. p. M. ed 10. K... Palagiano.1. J..7-114. Smitz... Baker.H. 127(4):431-439. 4:31-37. 82(2):378-383. J Assist Re[doi: prod Genet.L.01.. D.B.1016/S0022-5347(01)62532-1] un [doi:10... 1994. J.0040031] [doi:10.12... 81(5):1289-1295.G. Berger.H. 1996. [doi:10. A. Fertil Steril. Manicardi.J.J. Veek. J Urol. D. In: Knobil. G.D... Hum Reprod... Larson-Cook... Fertil Steril. J....2008.. Dev Biol. Origin of DNA damage in ejaculated human spermatozoa. Van Steirteghem.. 1990.2003. 2011.... Experience with assisted fertilization in severe male factor infertility and unexplained failed fertilization in vitro. D.. Reproduction.. NewYork.. Hum Reprod Update. J. 1997.. Barthelemy. Hand-book of the Laboratory Diagnosis and Treatment of Infertility. D. G. 2002. Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia. Sakkas. 9(4):680-683. A.H. Ovarian stimulation with buserilin / HMG / HCG: prospective randomized study of short versus long protocol. Peknicova.. Bianchi. 2007. Bizzaro. W. Wisanto.P. J. [doi:10. In: Keel.R. Z. Fertil Steril. M. 2(6):553-559.063] Watkins.. S. 77(2):233-237. A. Gardner.1016/j. World Health Organisation Press.. U.2003. 10. Mantalenakis S... D.1994. Liu..W. The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLC . Sperm defect severity rather than sperm Source is associated with lower fertilization rates after intracytoplasmic sperm injection.... O. L.00169] Swann... Hamamah.. Devroey. CRC Press. Fertil Steril. Verza. Dale..L. H. Extend of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Razavi. High fertilization and implantation rates after intracytoplasmic sperm injection.fertnstert.. 27(4):347-353... Evaluation of reproductive potential after intracytoplasmic sperm injection of varied human semen tested by antiacrosomal antibodies. 1994. E. Vasectomy reversal associated with increased reactive oxygen species production by seminal fluid leukocytes and sperm. Gribizis..K.. A.P.. N. Larman. Elzeinova.G. 160(4):1341-1346. Wetzels.. B. Lagos. Wilding. G.... Testicular and epididymal sperm in a microinjection program;methods of retrieval and results.Fertil Steril. Raven Press.1006/dbio. 2004. Fusco. [doi:10.E.. M. A. Braat. S... [doi:10. Int Braz J Urol.C. Rev Reprod. Craft. 34(1):49-56..fertnstert.. Tavalaee..1016/j. Fertil Steril.1016/S0015-0282(01)02962-4] [doi:10... J. Moffatt. Saunders. A.063] Tepla. Nasr-Esfahani. K.C. 8(7):1061-1066. 61(6):1045-1051..L. C. G. 1998. Bontis. 67(3):527-535. The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa. Spanos.2005. Mrazek. C. Liu. Sakkas. B. Geneva. Staessen. Virro. and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. al Shawaf. Yazdani.09.. 1994..189-317.. effective approach to infertility as a result of congenital bilateral absence of the vas deferens. E.0. J.1007/s10815-004-8760-8] Royere.