Selective Short-term Fertilization Combined with Early Rescue ICSI: An Optimal Strategy for Patients at High Risk for Fertilization Failure

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1 Journal of Reproduction & Contraception doi: /j.issn Dec.; 25(4): Selective Short-term Fertilization Combined with Early Rescue ICSI: An Optimal Strategy for Patients at High Risk for Fertilization Failure Yu-ling HUANG 1,2*, Ai-hua WU 2*, Jian-qiao LIU 2 1. Center for Reproductive Medicine, the Third Affiliated Hospital of Guangzhou Medical University; Key Laboratory for Reproductive Medicine of Guangdong Province; Key Laboratory for Major Obstetric Diseases of Guangdong Province, Guangzhou , China 2. Obstetrics and Gynecology Department, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou , China Objective To investigate clinical outcomes in patients who were at more precise criteria risks for fertilization failure and were treated with selective, short-term fertilization (oocytes and sperm co-incubated for 4 h) and early rescue intracytoplasmic sperm injection (ICSI). Methods A retrospective analysis was performed on women undergoing assisted reproductive technology (ART). They were assigned to 4 groups: short-term in vitro fertilization (short-term IVF, group A, n=217), regular IVF (oocytes and sperm coincubated overnight, group B, n=1 475), short-term IVF and early rescue ICSI (shortterm ICSI, group C, n=94), and regular ICSI (group D, n=237). Results In group A, 69.8% (217/311) achieved normal fertilization rates, and the complete fertilization failure rate (fertilization rate was 0%) was 12.9% (40/311). But all of the fertilization failure oocytes got rescue ICSI. In group B, the complete fertilization failure rate was 1.1% (19/1 692). The fertilization rate, 2 PN (pronucleus) rate, and 1 PN rate were significantly lower in group A than those in group B (70.9% vs 80.8%, 57.8% vs 66.3%, and 3.5% vs 6.2%, respectively). No significant differences were observed in clinical pregnancy rates and birth defect rates between groups A and B. The fertilization rates in groups C and D did not significantly differ (77.9% vs 76.2%), which was also true for birth defect rates. The clinical pregnancy rate of group C was higher than that of group D (51.2% vs 42.3%), but this difference was not significant (P). This work was supported by Science and Information Technology of Guangzhou (2012Y ) Corresponding author: Jian-qiao LIU; ljq88gz@qq.com *: These authors contributed equally to this work 207

2 Conclusion These results suggested that selective, short-term fertilization can result in effective outcomes for patients who were at high risk for fertilization failure. Key words: in vitro fertilization and embryo transfer (IVF-ET); short-term fertilization; early rescue intracytoplasmic sperm injections (ICSI); pregnancy Fertilization failure can occur in 3.52% 20% of patients who have been treated with in vitro fertilization and embryo transfer (IVF-ET) [1-4]. However, there is no effective indicator to precisely predict the chance of fertilization failure. In the event of failure, patients may have to surrender the cycle because there are no more embryos to transfer, which causes financial loss and psychological trauma. Intracytoplasmic sperm injections (ICSI) can be performed within h to rescue the fertilization failure, thereby potentially reducing the financial loss and providing the patients with reassurance. This remedial measure can increase fertilization rates by 30% 76% [5]. However, the pregnancy rates remain low because 1-day-old oocytes age rapidly and lose their embryo development potentials [6-8]. ICSI is not currently widely used because of the low associated pregnancy rates of the 24 h eggs. Some authors have used a half-icsi method (half-ivf and half-icsi) to avoid the complete failure of fertilization and achieved normal fertilization in a substantial number of patients. In fact, Plachot et al. and Wang et al. reported [9,10] that 67.2% and 63.6% of patients, respectively, achieved normal fertilization. However, the half-icsi method does not appear to be advantageous in patients who achieve fertilization via both IVF and ICSI because it increases the financial burden and the proportion of ICSI cases in assisted reproductive technology (ART), which is not in accordance with relevant Chinese policies. Currently, the success rates of ICSI and conventional IVF are similar. However, the long-term risks that are associated with ICSI [11] raise doubts regarding its ability to replace conventional IVF treatments, and more evidence is needed. A previous study assessing ICSI reported the presence of a second polar body (Pb2) in 78.3% of the eggs at 3 h after injection, and assessment diodes successfully predicted egg fertilization using specificity and sensitivity analyses during the first 3 h after injection [12]. A longer duration of fertilization has been associated with higher proportions of Pb2, and in fact, Suppinyopong et al. [13] observed that 84.4% of fertilized oocytes formed Pb2 at 6 h after ICSI. Additionally, Jin et al. [14] observed that 84.5% of M II oocytes formed Pb2 at 4 h after IVF, 93.3% of which were fertilized. The short co-incubation of the gametes has been demonstrated to reduce this time to 1 6 h, and even as short as 30 s, to achieve fertilization [15]. Preliminary studies in patients that have been treated with conventional IVF revealed that no oocytes contained the second polar bodies after the granulosa cells were stripped for 6 h, 208

3 but a timely rescue by ICSI was achieved [16]. This injection improved the fertilization, pregnancy, and implantation rates of early rescue ICSI at 4 h or 6 h after insemination compared with rescue ICSI at 22 h after insemination, which is after the oocytes have aged [14,16]. Previous studies have demonstrated [14,16] that removing the cumulus cells at 4 h or 6 h after insemination and combining early rescue ICSI is an efficient back-up option for unfertilized oocytes, and it may prevent the complete fertilization failure that is often observed with conventional IVF. In fact, some centers have adopted short-term fertilization for all patients (except ICSI) to prevent complete fertilization failure. The window of opportunity for polar body detection and cumulus cell removal in short-term fertilization is crucial for pronucleus formation, and therefore, its long-term safety has been questioned. Too much external stimulation may increase the risks of detrimental epigenetic events in the offspring. Moreover, conflicts with routine work schedules increase the workloads of the embryologists and the operating costs of the centers. Adopting short-term fertilization combined with rescue ICSI for patients who are at high risk for fertilization failure can circumvent the unnecessary use of ICSI and enable fertilization before the oocytes lose their developmental potential [14,17]. However, controversies over the standards that must be set for the prevention of these failures remain. Primary infertility, males with seminal thresholds, and unexplained infertility are all generally associated with high risks for fertilization failure [14,17], but primary infertility in women is controversial. To date (unpublished data), quite a few male patients have been observed to exhibit indicators for ICSI as reported by the World Health Organization (WHO) Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction (5th edition), including teratospermia rates 99%, sperm deformity index (SDI) 1.6, and acrosin<52 U/L. However, positive anti-sperm antibodies in seminal plasma (as detected using an immune bead assay) may allow for normal fertilization if the sperm motilities and densities are normal. Thus, these patients were selected as subjects in this study together with the other 2 aforementioned high-risk groups, and the resulting clinical effects, IVF failure rates, and ICSI proportions were observed at our reproductive center. Materials & Methods Patient selection and characteristics A total of patients were studied from July 2012 to April 2013 at the Assisted Reproductive Center of the Third Affiliated Hospital of Guangzhou Medical University. The female patients were years old, and >5 oocytes were obtained from each patient. Among them, 311 patients experienced the following characteristics: 1) primary infertility and male sperm densities ranging (5 15) 10 6 /ml with sperm motilities of (a+b) 10% 35%; 209

4 2) unexplained infertility; 3) normal sperm motilities and densities but one of the following: teratospermia rates 99%, SDI 1.6, acrosin<52 U/L, and positive anti-sperm antibodies in the seminal plasma (coated sperm>10% as shown by immune bead assay). A total of 217 patients (short-term IVF, group A) underwent normal fertilization, which was defined as 30% of mature oocytes showing 2 polar bodies (Pb), and 94 underwent early rescue ICSI (shortterm ICSI, group C). A total of patients who possessed IVF indicators without the aforementioned conditions were treated with regular overnight fertilization (regular IVF, group B). A total of 237 patients for the male factor infertility factors had regular ICSI (regular ICSI, group D), excluding the surgical sperm aspiration patients. All of these procedures were approved by the ethics committee of our hospital. All participants provided their written consent to participate in this study. Superovulation scheme A routine procedure of long-programmed ovarian stimulation was performed. On the 19th to 21st day of the last menstrual period, mg of Diphereline (triptorelin acetate for injection, IPSEN Co., France) was injected to down-regulate the hormones. On the 2nd 3rd day of menstruation, when the serum LH and E2 reached standard levels, Gonal-F (Serono Co., Switzerland) was injected to stimulate ovulation. When over 3 follicles were present with the diameter 18 mm, which could be observed by ultrasound inspection, U hcg (Livzon Co., China) was injected to promote follicular maturation. The oocytes were harvested and guided by transvaginal ultrasound h later. Sperm mixed agglutination surface antibody The sperm membrane surface antibody IgG was detected using a sperm membrane surface antibody IgG detection kit (Kang Biomedical Engineering Co., Shenzhen, China). When more than 10% of the sperm were coated, the mixed agglutination reaction (MRA) test was considered to be positive. Test for teratospermia rate The teratospermia rate was tested according to the WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction (5th edition). Modified Papanicolaou staining was used for the spermatozoa, which was suggested for sperm morphology analyses detection. IVF and Pb observation The oocytes were harvested and fertilized according to previously described methods [18]. Oocytes in group A were fertilized at h after hcg injection and co-incubated for 4 h. In group B, the fertilization time was h after hcg injection, and co-incubation was performed for h. Four hours after fertilization, the granulosa cells could be stripped to observe Pb2 in group A. Fertilization was identified as the appearance of two separate Pb or Pb2 in the dynamic observation. Normally, the 2 separate Pb were present in over 30% of 210

5 the mature oocytes within 6 h of fertilization. Otherwise, rescue ICSI was applied to the oocytes that lacked a Pb2. Available embryo evaluation We performed the standard evaluations at our center. The cleavage available embryos were transferred and frozen if cell counts of >5 could be obtained at h after oocyte harvest with cell fragments of 20%, and a ratio of maximal blastomeres to minimum blastomeres of <2. Embryo transfer and patient preparation The embryos were transferred at 72 h after oocyte harvest. The number of transferred embryos was decided according to the standards of the Ministry of Health and our center to lower the multiple pregnancy rate. For patients who were <38 years old, 2 embryos were transferred, and for those who were 38 years old or with 3 treatment cycles, 3 embryos were transferred. Progesterone or hcg was routinely used to support the corpus luteum. Serum hcg was tested to identify pregnancy at 14 d after transplantation. When the gestational sac and embryo bud were detected by ultrasound at 4 weeks after transplantation, the patient was diagnosed as pregnant. Statistical analysis The data are shown as mean standard deviation (x s). Student s t test and χ 2 test were used for the statistical analyses using the SPSS 17.0 software. P<0.05 was considered to be statistically significant. Results The basic information describing the patients in groups A and B was compared (Table 1). As shown in Table 1, the patients ages, BMI, mean duration of infertility, and basic E 2 and FSH levels did not significantly differ between the two groups. Primary infertility in group A was significantly higher than that in group B (64.1% vs 44.0%). These two groups also had significantly different unexplained infertility rates, male factor rates, and complex factor rates (P<0.05). The fertilization rates, the 2 PN rates, and the 1 PN rates of group A were significantly lower than those of group B (P<0.05). Notably, 3 PN rates were significantly higher than those of group B (P<0.05). Significant differences in the available embryo rates were also observed between the two groups (P<0.05) (Table 2). However, the rates of clinical pregnancies, miscarriages, ectopic pregnancies, live births, and birth defects were not significantly different (P). A total of 94 patients underwent rescue ICSI (group C) (Table 3), and all of them transfered their embryos from ICSI. Compared with the 237 regular ICSI patients (group D), 211

6 Table 1 Clinical data for the short-term IVF group (group A) and regular IVF group (group B) (x s, %) Indicator Short-term IVF Regular IVF P value No. of cycles (n) Age (year) BMI (kg/m 2 ) Mean duration of infertility (a) Primary infertility (%) Main cause of infertility Tubal disease (%) Endometriosis (%) Anovulation (%) Unexplained (%) Male factor (%) Complex (%) Basic hormone 64.1 (139/217) 24.9 (54/217) 5.5 (12/217) 2.3 (5/217) 11.5 (25/217) 22.1 (48/217) 33.7 (73/217) 44.0 (649/1 475) 65.5 (966/1 475) 3.6 (53/1 475) 5.6 (82/1 475) 2.7 (40/1 475) 2.3 (34/1 475) 20.3 (300/1 475) <0.05 E2 (pmol/l) FSH (U/L) Table 2 Clinical outcomes of the short-term IVF group (group A) and regular IVF group (group B) (x s, %) Indicator Short-term IVF Regular IVF P value n ET cycles (n) No. of oocytes retrieved (n) Fertilization (%) 1 PN rate (%) 2 PN rate (%) Polyspermy rate (%) Complete fertilization failure (%) Cleavage rate (%) Useful embryo rate (%) Useful oocyte rate (%) 70.9 (1 984 /2 797) 3.5 (99/2 797) 57.8 (1 618/2 797) 9.6 (267/2 797) 0.0 (0/217) 97.8 (1 942/1 984) 55.3 (1 097/1 984) 39.2 (1 097/2 797) 80.8 (15 280/18 909) 6.2 (1 173/18 909) 66.3 (12 547/18 909) 8.3 (1 560/18 909) 1.3 (19/1 475) 97.6 (14 916/15 280) 52.3 (7 994/15 280) 42.3 (7 994/18 909) <0.05 <0.05 Mean No. of embryos transferred (n) <0.05 Implantation rate (%) Clinical pregnancy rate (%) Multiple pregnancy rate (%) Miscarriage rate (%) Ectopic pregnancy rate (%) Live birth rate (%) 35.0 (143/408) 52.3 (103/197) 36.9 (38/103) 13.6 (14/103) 2.9 (3/103) 44.2 (87/197) 35.0 (918/2 618) 51.1 (667/1 306) 37.8 (252/667) 15.3 (102/667) 2.7 (18/667) 41.9 (547/1 306) Rate of birth defects ( ) 0.0 (0/126) 11.6 (9/775) no significant differences were observed in their ages, BMI, infertility duration and basic hormone levels. Notably, infertility as caused by the male factor in group D was higher than that in group C (P). The fertilization rates, the cleavage rates, the available embryo 212

7 Table 3 Clinical data for the rescue ICSI group (group C) and regular ICSI group (group D) (x s, %) Indicator Rescue ICSI Regular ICSI P value No. of cycles (n) Age (year) BMI (kg/m 2 ) Mean duration of infertility (a) Primary infertility (%) Main cause of infertility Tubal disease (%) Endometriosis (%) Anovulation (%) Unexplained (% ) Male factor (%) Complex (%) Basic hormone 78.7 (74/94) 12.8 (12/94) 1.1 (1/94) 3.2 (3/94) 5.3 (5/94) 42.6 (40/94) 35.0 (33/94) 69.6 (165/237) 6.3 (15/237) 2.1 (5/237) 1.3 (3/237) 1.3 (3/237) 59.5 (141/237) 29.5 (70/237) E2 (pmol/l) FSH (U/L) rates, the miscarriage rates, the ectopic pregnancy rates, the live birth rates, and the birth defect rates of groups C and D did not significantly differ (P) (Table 4). However, the Table 4 Clinical outcomes of the rescue ICSI group (group C) and regular ICSI group (group D) (x s, %) Indicator Rescue ICSI Regular ICSI P value n ET cycles (n) No. of oocytes retrieved (n) Comepletely rescue ICSI (%) No. of oocytes for rescue ICSI (n) Fertilization (%) 1 PN rate (%) 2 PN rate (%) Polyspermy rate (%) Cleavage rate (%) Useful embryo rate (%) Useful oocyte rate (%) 43.5 (40/92) (640/822) 4.5 (37/822) 66.4 (546/822) 7.0 (57/822) 96.9 (620/640) 53.9 (415/770) 50.5 (415/822) / / 76.2 (1 727/2 267) 3.0 (69/2 267) 71.3 (1 616/2 267) 1.9 (42/2 267) 97.6 (1 686/1 727) 55.0 (949/1 727) 41.9 (949/2 267) <0.05 Mean No. of embryos transferred (n) Clinical pregnancy rate (%) Implantation rate (%) Multiple pregnancy rate (%) Miscarriage rate (%) Ectopic pregnancy rate (%) Live birth rate (%) 51.2 (42/82) 35.1 (59/168) 40.5 (17/42) 11.9 (5/42) 0.0 (0/42) 45.1 (37/82) 42.3 (88/208) 27.0 (112/415) 27.3 (24/88) 11.4 (10/88) 2.3 (2/88) 36.5 (76/208) Rate of birth defects ( ) 19.2 (1/52) 10.0 (1/99) 213

8 multiple PN rate of group C was significantly higher than that of group D (P). The clinical pregnancy rate of group C was higher than that of group D, but this difference was not significant (P). In this study of short-term fertilization patients, 69.8% (217/311) achieved normal fertilization rates. To them the complete fertilization farlure rate (fertilization rate was 0%) was 12.9% (40/311) and part fertilization farlure rate (fertilization rate <30%) was 17.4 % (54/311). But all of the fertilization failure oocytes got rescue ICSI. To the regular IVF patients the complete fertilization failure rate was 1.1% (19/1 692). Discussion For short-term fertilization, the mechanical stimulation that is associated with the stripping of granulose cells may pose epigenetic risks to embryo development. Therefore, it is offered as a complementary option to ART patients. In this study, patients who were at high risk for fertilization failure were treated with short-term fertilization, and 69.8% (217/311) achieved normal fertilization rates. The complete fertilization failure rate in the IVF patients was 1.1% (19/1 692), which was much lower than the lowest rate of 3.52% that was previously reported [4]. The proportion of ICSI was 16.36% (331/2 023), which was also lower than the rate that was reported in a previous study of 19.97% [17]. The former criteria for short-term fertilization were primary infertility, unexplained infertility, and seminal threshold. We adopted more precise inclusion criteria as follows: 1) primary infertility, including males with a threshold of sperm motility and density; 2) unexplained infertility; and 3) normal sperm motility and density with only one of the following: a teratospermia rate of 99%, SDI 1.6, acrosin <52 U/L, and positive anti-sperm antibodies in the seminal plasma as detected by an immune bead assay. These new criteria did not expand the scope of short-term fertilization, but did help to avoid unnecessary short-term fertilization and decreased the workload at the center. Moreover, these new criteria decreased the complete fertilization failure rate and lowered the proportion of ICSI, which could aid in the optimization of IVF strategies. The fertilization and 2 PN rates of the short-term IVF group were significantly lower than those of the regular IVF group, which was not consistent with the lack of difference that was previously reported [14,17]. However, we selected high-risk patients for fertilization failure using more precise inclusion criteria, while previous studies did not use these criteria. However, the 3 PN rate was significantly higher in the short-term IVF group than in the regular group. This may have been due to the removal of the granulosa cells within 4 h after fertilization, which could have damaged the zona pellucida, which is a natural barrier that prevents polyspermy. Additionally, mechanical stripping could damage the microstructures of the oocytes and affect spindle bodies, for example, resulting in the abnormal formations of polar bodies [14]. Nevertheless, if granulosa cells are removed within 6 h, the polyspermy 214

9 problem is resolved [14,19]. However, for the rescue ICSI patients, the clinical pregnancy rate, the implantation rate, and the live birth rate were higher in those individuals who had their granulosa cells stripped within 6 h compared with those whose cells were stripped at 6 8 h or after over 8 h [20]. Considering the advantages and disadvantages, we considered 4 h after fertilization to be the best time for granulosa cell removal. The available embryo rate of the short-term IVF group was significantly higher than that of the regular IVF group, and the clinical pregnancy rate was slightly higher. Reducing the binding time of the egg and the sperm may improve the quality of the embryo [21] because fewer reactive oxygen species (ROS) would be generated from dead sperm, which would otherwise harden the zona pellucida and reduce embryo vitality. Additionally, this method may diminish the burden of the sperm for supplying nutrients to support embryo development. Stripping the granulosa cells of the oocyte-corona-cumulus complex could also eliminate the adverse effects of hormones on the embryo [22]. Increased estrogen has been suggested to directly influence the development and implantation of the embryo, and high levels of external estrogen may have direct toxic effects [23]. Short-term fertilization can avoid the aforementioned problems, increase the developmental potential of the embryo, and improve the pregnancy outcome. In this study, which is consistent with previous reports, the miscarriage rates, the ectopic pregnancy rates, the live birth rates, and the birth defect rates did not significantly differ, confirming the positive effects of short-term fertilization on pregnancy outcomes. Rescue ICSI is a complementary treatment for fertilization failure that can provide hope for patients who are at high risk. Late rescue ICSI, which is carried out within h after complete fertilization failure, has only been reported to achieve a pregnancy success rate of 9.7% [5] ; in fact, the aged egg may be experience increased aneuploidy rates [23,24]. Increased aneuploidy was a confounding factor in the aforementioned study because the failed eggs had high aneuploidy rates that were independent of the rescue timing. Furthermore, embryological diagnoses were used to determine that only 17% of the embryos were normal diploids before implantation [25], and the increased aneuploidy rates were not isolated to the embryos that were used for the late rescue ICSI. Nevertheless, the aging of the egg may impact the cellular biochemistry, molecules and microstructure, and the arrangement of the spindle body and chromosomes [5,26], leading to unpredictable abnormalities in imprinting genes [23]. Short-term fertilization combined with early rescue ICSI can remedy fertilization failure before the oocytes aging. This method can increase the pregnancy success rate by up to 44% [5], which is a satisfying outcome. Additionally, no significant difference was observed in the fertilization rate; however, the multiple PN rate of the rescue ICSI group was significantly higher than that of the regular ICSI group. Polyspermy may also occur when sperm enters the oocyte without achieving activation. Therefore, sperm could be 215

10 injected into an oocyte that was already penetrated by a sperm during IVF. The signs of fertilization (oocyte activation) are difficult to identify in some oocytes, particularly in those with fragmented polar bodies, which may be another reason for the elevated polyspermy rates. The avoidance of polyspermy may be difficult [20]. Nevertheless, the available embryo rates did not significantly differ between the two groups. Therefore, this study suggests that short-term fertilization in combination with early rescue ICSI is an optimal strategy for patients who are at high risk for fertilization failure. This strategy may maximize chances of pregnancy success and reduce treatment costs. Selective short-term fertilization can prevent unnecessary ICSI treatment for high-risk patients who undergo normal fertilization procedures. Early rescue ICSI before the oocytes aging can achieve a higher number of gametes with development potentials and result in more satisfactory pregnancy outcomes in individuals who are experiencing complete fertilization failure and low fertilization rates. This method may be translated into clinical practice. References 1. Staessen C, Camus M, Clasen K, et al. Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen. Hum Reprod, 1999, 14(10): Liu DY, Baker HW. Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization. Hum Reprod, 2000, 15(3): Combelles CM, Morozumi K, Yanagimachi R, et al. Diagnosing cellular defects in an unexplained case of total fertilization failure. Hum Reprod, 2010, 25(7): Ming L, Liu P, Qiao J, et al. Synchronization between embryo development and endometrium is a contributing factor for rescue ICSI outcome. Reprod Biomed Online, 2012, 24(5): Beck-Fruchter R, Lavee M, Weiss A, et al. Rescue intracytoplasmic sperm injection: a systematic review. Fertil Steril, 2014, 101(3): Morton PC,Yoder CS, Tucker MJ, et al. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure. Fertil Steril, 1997, 68(3): Yuzpe AA, Liu Z, Fluker MR. Rescue intracytoplasmic sperm injection (ICSI)-salvaging in vitro fertilization (IVF) cycles after total or near total fertilization failure. Fertil Steril, 2000, 73(6): Kuczyñski W, Dhont M, Grygoruk C, et al. Rescue ICSI of unfertilized oocytes after IVF. Hum Reprod, 2002, 17(9): Plachot M, Belaisch-Allart J, Mayenga JM, et al. Outcome of conventional IVF and ICSI on sibling oocytes in mild male factor infertility. Hum Reprod, 2002, 17(2): Wang YQ, Yang J, Xu WM. Indications and clinical outcomes of half-icsi in 99 cases. Zhonghua Nan Ke Xue (in Chinese), 2009, 15(9): Ludwig M, Katalinic A, Gross S, et al. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet, 2005, 42(4): Van den Bergh M, Bertrand E, Englert Y. Second polar body extrusion is highly predictive for oocyte 216

11 fertilization as soon as 3 hr after intracytoplasmic sperm injection (ICSI). J Assist Reprod Genet, 1995, 12 (4): Suppinyopong S, Choavaratana R, Karavakul C. Timing of second polar body extrusion and pronuclear formation after intracytoplasmic sperm injection (ICSI). J Med Assoc Thai, 2000, 83(5): Jin H, Shu Y, Dai S, et al. The value of second polar body detection 4 hours after insemination and early rescue ICSI in preventing complete fertilisation failure in patients with borderline semen. Reprod Fertil Dev, 2014, 26(2): Bungum M, Bungum L, Humaidan P. A prospective study, using sibling oocytes, examining the effect of 30 seconds versus 90 min gamete co-incubation in IVF. Hum Reprod, 2006, 21(2): Chen C, Kattera S. Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF. Hum Reprod, 2003, 18(10): Xiong S, Han W, Liu JX, et al. Effects of cumulus cells removal after 6 h co-incubation of gametes on the outcomes of human IVF. J Assist Reprod Genet, 2011, 28(12): Huang YL, Du HZ, Kang XJ, et al. Related factors of in vitro fertilization and embryo transfer patients with complete fertilization failure. J Reprod Contracep, 2013, 24(2): Lundqvist M, Johansson U, Lundkvist O, et al. Reducing the time of co-incubation of gametes in human invitro fertilization has no beneficial effects. Reprod Biomed Online, 2001, 3(1): Liu WW, Liu JX, Zhang XD, et al. Short co-incubation of gametes combined with early rescue ICSI: an optimal strategy for complete fertilization failure after IVF. Hum Fertil (Camb), 2014, 17(1): Dirnfeld M, Bider D, Koifman M, et al. Shortened exposure of oocytes to spermatozoa improves in-vitro fertilization outcome: a prospective, randomized, controlled study. Hum Reprod, 1999, 14(10): Kattera S, Chen C. Short coincubation of gametes in in vitro fertilization im proves implantation and pregnancy rates: a prospective, randomized, controlled study. Fertil Steril, 2003, 80(4): Valbuena D, Martin J, de Pablo JL, et al. Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo. Fertil Steril, 2001, 76(5): Miao YL, Kikuchi K, Sun QY, et al. Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Hum Reprod Update, 2009, 15(5): Fragouli E, Escalona A, Gutierrez-Mateo C, et al. Comparative genomic hybridization of oocytes and first polar bodies from young donors. Reprod Biomed Online, 2009, 19(2): Takahashi T, Igarashi H, Amita M, et al. Molecular mechanism of poor embryo development in postovulatory aged oocytes: mini review. J Obstet Gynaecol Res, 2013, 39(10): (Received on October 6, 2014) 217

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