Ensieh Shahrokh Tehraninejad 1,2, Elham Azimi Nekoo 2, Firouzeh Ghaffari 1, Maryam Hafezi 1, Leila Karimian 3 and Arezoo Arabipoor 1.

Transcription

1 doi: /jog J. Obstet. Gynaecol. Res. Vol. 41, No. 11: , November 2015 Zygote intrafallopian tube transfer versus intrauterine cleavage or blastocyst stage transfer after intracytoplasmic sperm injection cycles in patients with repeated implantation failure: A prospective follow-up study Ensieh Shahrokh Tehraninejad 1,2, Elham Azimi Nekoo 2, Firouzeh Ghaffari 1, Maryam Hafezi 1, Leila Karimian 3 and Arezoo Arabipoor 1 1 Departments of Endocrinology and Female Infertility and 3 Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR and 2 Obstetrics and Gynecology Department, Faculty of Medicine, Tehran University of Medical Science, Tehran, Iran Abstract Aim: This study aimed to compare the outcomes between zygote intrafallopian transfer (ZIFT) with intrauterine day-3 (cleavage stage) embryo transfer and intrauterine day-5 (blastocyst stage) embryo transfer in patients undergoing intracytoplasmic sperm injection. Material and Methods: This prospective study was performed at Royan Institute, Tehran, Iran, between January 2012 and January Two hundred fifty women with more than three unexplained implantation failures were divided non-randomly into three groups according to embryonic age and methods used as follows: (i) intrauterine cleavage-stage embryo transfer (n = 100); (ii) intrauterine blastocyst-stage embryo transfer (n = 50); and(iii) ZIFT (n = 100). Implantation, clinical pregnancy, miscarriage and live birth rates were our main outcomes. Results: Patients characteristics and ovarian response were comparable among the three groups. Implantation rate (56.1% vs 27.9%) was significantly higher in the blastocyst group as compared to the ZIFT group; however, clinical pregnancy rate (38% vs 23%) was not statistically significantly different between the two groups, but due to the significantly higher miscarriage rate (34.7% vs 5.3%) in the ZIFT group, the live birth rate was significantly higher in the blastocyst group (P = 0.04).Nosignificant differences were found between the cleavage-stage and blastocyst-stage groups in terms of implantation, clinical pregnancy, miscarriage and live birth rates. Conclusion: We do not recommend the use of the ZIFT procedure for patients with repeated implantation failures. It seems that replication of cleavage- or blastocyst-stage embryo transfer is more efficient and affordable. Key words: blastocyst-stage embryo, cleavage-stage embryo, intracytoplasmic sperm injection outcome, zygote intrafallopian transfer. Introduction Repeated implantation failure (RIF) following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is still a continued challenge in assisted reproductive technologies (ART) centers. 1 Different approaches have been reported in these patients to improve the success rates. 1 Assisted hatching, blastocyst transfer, zygote intra-fallopian transfer (ZIFT) and co-culture system are some of the treatment options used. 1 Zygote transfer has Received: December Accepted: May Reprint request to: Mrs Arezoo Arabipoor, Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Number 12, East Hafez Avenue, BaniHashem Street, Resalat Highway, PO Box , Tehran , Iran. arezoo.arabipoor@gmail.com 2015 Japan Society of Obstetrics and Gynecology 1779

2 E. Shahrokh Tehraninejad et al. some advantages: (i) an ideal and natural intrafallopian milieu that leads to a better synchronization between embryonic and endometrial development; 2 (ii) a rapid scheduling that causes alleviation of the patient s stress; 3 and (iii) a simple culture of human zygotes that avoids the extended culture s disadvantages, including fragmentation and complete block of development. 3 However, the ZIFT method also has some disadvantages, such as the need for general anesthesia, laparoscopy and a potential greater risk of ectopic pregnancy, 2 so it may be a costly procedure. 4 Some previous studies reported acceptable pregnancy and implantation rates by the ZIFT procedure in patients with RIF 2,3,5 7 andarecentstudybyweissman et al. stated that ZIFT is a powerful method in the clinical management of selected patients with high-order RIF. 8 On the other hand, in studies by Ozkaya et al. andaslan et al., the authors could not find any superiority of the ZIFT procedure compared to transcervical uterine embryo transfer (ET). 9,10 Recent advances in embryo culture systems and laboratory conditions have provided a better environment for embryo development. 11 Potential advantages of blastocyst transfer are a better selection of viable embryos and synchronization of embryonic stage with theendometriuminthepresenceofreduceduterinecontractions. 11 However, blastocyst transfer may have some disadvantages, such as cancellation of ET due to block of development after 5 days in extended culture, a lower embryo freezing rate and a higher laboratory workload. 11 Although few studies have been done to compare these two methods in this field, 6,12,13 it seems that for making a decision in this regards, further studies are needed. Therefore, this study was designed to evaluate and to compare the implantation, clinical pregnancy, miscarriage andlivebirthratesamonganicsi+ziftgroup,an ICSI + cleavage-stage group and an ICSI + blastocyststage group. Methods Patient selection This prospective study was performed at Royan Institute, Tehran, Iran, between January 2012 and January This study was approved by the Institutional Review Board and Ethics Committee of Royan Institute. The inclusion criteria were as follows: (i) a minimum of three previous failed IVF/ICSI-ET cycles, excluding frozen-thawed ET; (ii) women aged 43 years; (iii) normal response to controlled ovarian stimulation in previous and present cycles, at least five eggs retrieved or at least three zygotes obtained; (iv) the presence of at least one patent, documented by hysterosalpingography (HSG); and (v) a normal uterine cavity, as evidenced by hysteroscopy. If recent (<1 year) document on tubal status was not available, tubal patency were reevaluated by HSG. Hydrosalpinges, bilateral tubal obstruction and severe male factor infertility cases were excluded. Eligible patients and their husbands gave a signed informed consent before enrolling in the study. The controlled ovarian stimulation was performed according to standard long-agonist protocol as described in detail elsewhere. 14 The starting dose of gonadotrophins was based on the patients previous treatment cycles. Oocyte retrieval was carried out h after injection of human chorionic gonadotrophin (hcg), using transvaginal-ultrasoundguided needle aspiration under general anesthesia. The ZIFT procedure was performed by a physician on even days; therefore, patients were also divided into two sub-groups based on the physician s decision and day of ovum pick-up. Laboratory procedures All semen samples were prepared by the density gradient method. Each of the MII oocytes was placed in a Petri dish containing 10 drops of G1 V5 (Vitrolife) for injection. Sperms were immobilized in 10% polyvinylpyrrolidone (Global) droplets followed by breaking the tail by an injection pipette. Then, a single sperm was aspirated tail-first into the pipette and injected into the target oocyte that had a visible first polar body (MII). Normal fertilization was recognized by the appearance of two pronuclei and of the second polar body, h after microinjection. Fertilized oocytes were cultured in fresh 50 μl of G1 V5 supplemented with 10% recombinant human serum albumin covered with mineral oil at 37 C in an atmosphere of 5% CO2 for up to the stage of ZIFT or up to day 3 in order to transfer the embryos designated for the extended culture to G2 V5 (Vitrolife) for another 48 h (day 5). Embryo quality was assessed daily until the moment of transfer. Transfer procedures and luteal support The ZIFT procedure was performed 24 h after oocyte retrieval with the use of a three- puncture laparoscopy method. In patients without tubal factor, the transfers were performed in both fallopian tubes, while in patients with tubal factor; transfer was performed to the tube with proved patency according to laparoscopy or HSG, done during infertility evaluation. Three to six zygotes were loaded into the delivery catheter (Laboratoire C. C.D) and transferred through a third paraumbilical puncture deep into the fallopian tube to the ampulla region Japan Society of Obstetrics and Gynecology

3 ZIFT vs. uterine ET in patients with RIF Transfers at the cleavage- and blastocyst-stage were performed on day 3 or day 5, respectively, after oocyte retrieval, using a Labotect catheter (Labotect) in a standard technique by an expert gynecologist and embryologist. Excess good-quality embryos were cryopreserved using vitrification technique in two groups. In two groups, the luteal phase was supported by 400 mg vaginal progesterone twice a day until the day of β-hcg assay (16 days after ET). Primary outcome measures included implantation, clinical pregnancy, and live birth rates. Implantation rate is defined as the number of visualized intrauterine gestational sacs divided by the number of transferred embryos. A clinical pregnancy is defined as ultrasonographic observation of an intrauterine gestational sac with fetal heart beat. Spontaneous abortion is considered as a clinical pregnancy lost before 20 weeks gestation. Live birth is defined as delivery of a fetus with signs of life after 20 completed weeks of gestational age. Miscarriage and live birth rates are reported, respectively, as total spontaneous abortion and total live birth per total number of patients with ET in each group. Statistical analysis Obtained result was expressed as mean ± standard error. Statistical analysis was accomplished using SPSS 20. Normality of data was checked by Kolmogorov Smirnov test. Inter-group differences were assessed through one-way ANOVA and differences between the two groups were evaluated using Tukey s test in quantitative normal variables. Inter-group differences were evaluated by χ 2 -test in qualitative variables. Statistical significance was defined at P < Results During the study period, 100 ICSI + ZIFTcycles and 150 ICSI + trans-cervical embryo-transfer cycles were conducted for 100 and 150 couples, with RIF, respectively. The ICSI + trans-cervical embryos transfer group was divided into two other main groups based on day of transferred embryos as follows: day-3 cleavage-stage embryo transfer (n = 100 cycles) and day-5 blastocyststage embryo transfers (n = 50 cycles). Patients characteristics were similar among the three groups, as shown in Table 1. There were no significant differences in terms of age, body mass index, duration of infertility, number of previous failed cycles, proportion of patients with primary infertility, cause of infertility, number of gonadotrophin ampoules, duration of stimulation, number of retrieved oocytes and endometrial thickness (P < 0.05). As expected, there were significant differences in number of embryos transferred between the ZIFT group and the other two groups (P < 0.001) (Table 1). Table 1 Comparison of study population characteristics among three groups Variables ICSI + ZIFT (n = 100) ICSI + cleavage-stage transfer (n =100) ICSI + blastocyst-stage transfer (n =50) P-value Age (years) 33.1 ± ± ± BMI ( kg/m 2 ) 25.8 ± ± ± Type of infertility n (%) 0.2 Primary 73(73%) 79 (79%) 42 (84%) Secondary 27 (27%) 21 (21%) 8 (16%) Cause of infertility n (%) 0.1 Ovulatory 12 (12%) 18 (18%) 12 (24%) Male factor 57 (57%) 57 (57%) 31 (62%) Unexplained 31 (31) 25 (25) 7 (14%) Infertility duration (years) 8.6 ± ± ± No. previous ART cycles 3.3 ± ± ± Stimulation duration 10.2 ± ± ± (days) No. of gonadotrophin 31.9 ± ± ± ampoules No. of retrieved oocytes 11.1 ± ± ± Total no. of embryos 5.6 ± ± ± No. of transferred zygotes 4.1 ± ± ± 0.6 <0.001* or embryos Endometrial thickness on transfer day 9.5 ± ± ± *ZIFT vs blastocyst-stage (P < 0.001), ZIFT vs cleavage-stage (P < 0.001), and blastocyst-stage vs cleavage-stage (P = 0.08) Japan Society of Obstetrics and Gynecology 1781

4 E. Shahrokh Tehraninejad et al. As shown in Table 2, fertilization, chemical and clinical pregnancy rates were similar among the three groups with P =0.1,P =0.08andP = 0.2, respectively. Implantation rate in the blastocyst-stage group was higher (56.1%) than that in the ZIFT group (27.9%) (P < 0.001), but it was similar to that of the cleavage-stage group (57.7%) (P = 0.9). Significant differences were found in terms of miscarriage and live birth rates between the ZIFT and blastocyst-stage groups (8% vs 2% and 14% vs 36%, respectively; P =0.02,P = 0.003). Multiple pregnancy rates in the blastocyst- and cleavage-stage groups were higher (34.4% and 31.5%) than that in ZIFT group (13%), but these did not reach to statistically significant differences (P =0.1). Discussion The results of our study suggest that the potential benefits of blastocyst-stage transfer are higher than those for the ZIFT procedure in couples with previous implantation failures. The effect of the ZIFT procedure in the management of patients with RIF was evaluated in several studies with compatible results. Although a better outcome was reported in some studies, 5 7 others did not demonstrate the beneficial effect of ZIFT in these patients. 2,3,9,10 Most of the previous studies in these regards were performed at infertility centers in Israel because ART costs in that country are covered by government health insurance, so seven or even more attempts of ART cycles are common among infertile couples living in that country. In a recent study by Weissman et al., the authors suggested that the ZIFT procedure is a powerful tool in the management of patients with high-order RIF, but the results cannot be extrapolated as the benefit of ZIFT, due to the lack of a control group. Elsewhere, Ozkaya et al. reported that rapid ICSI + ZIFT is associated with similar implantation and take-home baby rates compared to intrauterine ET after ICSI in unexplained infertile couples, so according to the risk of laparoscopy and the unavoidable higher risk of multiple pregnancies, rapid ICSI + ZIFT seems unsuitable for clinical use. 9 In contrast to the study by Levran et al., we found that the ZIFT procedure in RIF patients was not superior to trans-cervical uterine ET, and even the outcome of blastocyst-stage transfer was better than that of ZIFT. The differences between studies could be due to the following reasons: (i) differences in means of previous failed IVF or ICSI cycles; (ii) differences in race and studied population; (iii) differences in sample size; and (iv) differences in study design (retrospective, prospective or randomized clinical trial). According to the study by Gat et al., it appears that selected patients with specific characteristics can benefit from ZIFT methods, so the researchers concluded that young patients ( 31 years), who underwent 6 previous IVF cycles, yielding over eight 2-pronuclear (2-PN) embryos with a low ( 0.4) ratio of top-quality embryos to total 2-PN embryos are suitable for ZIFT procedure, 15 but it seems that further large prospective studies or clinical trials are needed to identify the specific characteristics of RIF women who may benefit from the ZIFT procedure. 15 Based on our results, we do not offer the ZIFT procedure for patients with history of less than six times failed IVF or ICSI cycles because it is more expensive and needs general anesthesia and laparoscopy for transfer, while its performance is not superior to cleavage- or blastocyst-stage transfers. The extended embryo culture has been used since 1994, and several studies have evaluated the effect of day-5 ET in different groups of patients. 13,16 22 Hypothetically, the blastocyst culture provides an opportunity to select viable embryos and improves Table 2 Comparison of cycle outcomes among three groups Outcomes ICSI + ZIFT (n = 100) ICSI + cleavage stage transfer (n =100) ICSI + blastocyst stage transfer (n =50) P-value Fertilization rate (mean ± SD) 69.7 ± ± ± Implantation rate (mean ± SD) 27.9 ± ± ± 25.5 <0.001* Chemical pregnancy rate n (%) 24 (24) 35 (35) 20 (40%) 0.08 Clinical pregnancy rate n (%) 23 (23) 29 (29) 19 (38%) 0.1 Ectopic pregnancy rate n (%) 1 (4.3) 1 (3.4) 0 (0) NS Multiple pregnancy rate n (%) 3 (13) 10 (34.4) 6 (31.5) 0.1 Miscarriage rate n (%) 8 (8) 5 (5) 1 (2) 0.04** Live birth rate n (%) 14 (14) 23 (23) 18 (36) 0.009*** *ZIFT vs blastocyst-stage (P < 0.001), ZIFT vs cleavage-stage (P < 0.001), and blastocyst-stage vs cleavage-stage (P =0.9). **ZIFT vs blastocyst-stage (P =0.02), ZIFT vs cleavage-stage (P =0.1), and blastocyst-stage vs cleavage-stage (P = 0.3). ***ZIFT vs blastocyst-stage (P =0.003), ZIFT vs cleavagestage (P = 0.1), and blastocyst-stage vs cleavage-stage (P = 0.1). NS, not significant; SD, standard deviation Japan Society of Obstetrics and Gynecology

5 ZIFT vs. uterine ET in patients with RIF both uterine and embryonic synchronicity, resulting in higher implantation rate and better pregnancy outcomes. 23 The efficacy of blastocyst transfer on patients with repeated unexplained IVF failure was assessed in many studies. Although some reported a better outcome, 12,24,25 others found unsatisfactory results in RIF patients, 6,11,26,27 so there are still conflicting results in this regard. In agreement with a recent study by Karacan et al., we found no difference between blastocyst- and cleavagestage transfer groups; however, in our study, the pregnancy and live birth rates in the blastocyst-stage group were higher than in the cleavage-stage group, but the differences did not reach statistical significance. Also in similar ways in agreement with the study by Papanikolaou et al., 28 the rate of miscarriage in the blastocyst-stage group was lower than in the cleavagestage group, but it did not reach significant levels. Karacan et al. believed that due to some possible intrinsic factors expressing after blastocyst-stage and/or due to some endometrial factors being responsible for consecutive implantation failure, all the blastocyst embryos could not result in healthy pregnancies. 11 A potential limitation of our study is the inherent bias of non-randomized design, especially regarding the physician s attitude in counseling for stage of ET; however, statistical analysis showed that the three groups were comparable in terms of demographic and basic characteristics. We believed that the smaller sample size of the blastocyst group could be the reason that we did not reach any statistically significant differences. In a recent Cochrane review, Glujovsky et al. evaluated 23 randomized clinical trials (RCT) in order to compare the efficacy of cleavage-stage with blastocyst-stage transfers in ART treatments for different groups of patients. 23 They concluded that there is a small significant difference in live birth rate in favor of blastocyst transfer efficacy (day-5 to day-6 embryo) as compared to cleavage-stage transfer (day-2 to day-3 embryo). In agreement to this recent meta-analysis, we recommend a future RCT on RIF patients to compare miscarriage, live birth and cumulative live births in order to enable ART centers and service providers to make wellinformed decisions on the best management option for these patients. 23 In conclusion, based on our results, we do not recommend the use of the ZIFT procedure for patients with RIF. It seems that replication of cleavage- or blastocyststage ET is more efficient and affordable. Acknowledgments The authors would like to thank their colleagues at the Royan Institute. The authors appreciate the Deputy of Research at the Royan Institute, who supported this paper financially. Disclosure There is no conflict of interest in this article. References 1. Simon A, Laufer N. Assessment and treatment of repeated implantation failure (RIF). J Assist Reprod Genet 2012; 29: Vorsselmans A, Platteau P, De Vos A, Albano C, Van Steirteghem A, Devroey P. Comparison of transfers to fallopian tubes or uterus after ICSI. Reprod Biomed Online 2003; 7: Dale B, Fiorentino A, de Simone ML et al. Zygote versus embryo transfer: A prospective randomized multicenter trial. JAssist Reprod Genet 2002; 19: Alleyassin A, Mahmoodan A, Aghahosseini M, Safdarian L, Saeidabadi HS. Comparison of immediate and delayed transfer of micro-injected oocytes into fallopian tubes: A prospective, randomized clinical trial. Int J Fertil Steril 2008; 2: Levran D, Mashiach S, Dor J, Levron J, Farhi J. Zygote intrafallopian transfer may improve pregnancy rate in patients with repeated failure of implantation. Fertil Steril 1998; 69: Levran D, Farhi J, Nahum H, Royburt M, Glezerman M, Weissman A. Prospective evaluation of blastocyst stage transfer vs. zygote intrafallopian tube transfer in patients with repeated implantation failure. Fertil Steril 2002; 77: Farhi J, Weissman A, Nahum H, Levran D. Zygote intrafallopian transfer in patients with tubal factor infertility after repeated failure of implantation with in vitro fertilizationembryo transfer. Fertil Steril 2000; 74: Weissman A, Horowitz E, Ravhon A, Nahum H, Golan A, Levran D. Zygote intrafallopian transfer among patients with repeated implantation failure. Int J Gynaecol Obstet 2013; 120: Ozkaya O, Sezik M, Kaya H et al. Comparison of tubal transfer and transcervical uterine embryo transfer of immediate injected oocytes and 2 3 days old cultured embryos. Geburtshilfe Frauenheilkd 2006; 66: Aslan D, Elizur SE, Levron J et al. Comparison of zygote intrafallopian tube transfer and transcervical uterine embryo transfer in patients with repeated implantation failure. Eur J Obstet Gynecol Reprod Biol 2005; 122: Karacan M, Ulug M, Arvas A et al. Comparison of the transfer of equal numbers of blastocysts versus cleavage-stage embryos after repeated failure of in vitro fertilization cycles. JAssist Reprod Genet 2014; 31: Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999; 72: Japan Society of Obstetrics and Gynecology 1783

6 E. Shahrokh Tehraninejad et al. 13. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998; 13: Tehraninejad ES, Hafezi M, Arabipoor A, Aziminekoo E, Chehrazi M, Bahmanabadi A. Comparison of cabergoline and intravenous albumin in the prevention of ovarian hyperstimulation syndrome: A randomized clinical trial. JAssistReprod Genet 2012; 29: Gat I, Levron J, Yerushalmi G, Dor J, Brengauz M, Orvieto R. Should zygote intrafallopian transfer be offered to all patients with unexplained repeated in-vitro fertilization cycle failures? J Ovarian Res 2014; 7: Olivennes F, Hazout A, Lelaidier C et al. Four indications for embryo transfer at the blastocyst stage. Hum Reprod 1994; 9: Jansen R, Mortimer D. In vitro culture of human blastocysts. In Gardner D, Schoolcraft WB (eds). Towards Reproductive Certainty: Fertility and Genetics. Carnforth: Parthenon Press, 1999; Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B. Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil Steril 2000; 73: Coskun S, Hollanders J, Al-Hassan S, Al-Sufyan H, Al-Mayman H, Jaroudi K. Day 5 versus day 3 embryo transfer: A controlled randomized trial. Hum Reprod 2000; 15: Bungum M, Bungum L, Humaidan P, Yding AC. Day 3 versus day 5 embryo transfer: A prospective randomized study. Reprod Biomed Online 2003; 7: Papanikolaou EG, D Haeseleer E, Verheyen G et al. Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study. Hum Reprod 2005; 20: Utsunomiya T, Ito H, Nagaki M, Sato J. A prospective, randomized study: Day 3 versus hatching blastocyst stage. Hum Reprod 2004; 19: Glujovsky D, Blake D, Farquhar C, Bardach A. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev 2012; 11: 7.CD doi: / Guerif F, Bidault R, Gasnier O et al. Efficacy of blastocyst transfer after implantation failure. Reprod Biomed Online 2004; 9: Barrenetxea G, Lopez de Larruzea A, Ganzabal T, Jimenez R, Carbonero K, Mandiola M. Blastocyst culture after repeated failure of cleavage-stage embryo transfers: A comparison of day 5 and day 6 transfers. Fertil Steril 2005; 83: Levitas E, Lunenfeld E, Har-Vardi I et al. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2 3 embryo transfer cycles: A prospective, randomized study. Fertil Steril 2004; 81: Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Dramatic declines in implantation and pregnancy rates in patients who undergo repeated cycles of in vitro fertilization with blastocyst transfer after one or more failed attempts. Fertil Steril 2001; 76: Papanikolaou EG, Camus M, Fatemi HM et al. Early pregnancy loss is significantly higher after day 3 single embryo transfer than after day 5 single blastocyst transfer in GnRH antagonist stimulated IVF cycles. Reprod Biomed Online 2006; 12: Japan Society of Obstetrics and Gynecology