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Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles: a prospective, randomized study
RBMOnline - Vol 16 No 4. 2008 589-596 Reproductive BioMedicine Online; www.rbmonline.com/Article/3104 on web 4 February 2008
Article Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles: a prospective, randomized study Hong-shan Ge received his Bachelor and Master degrees in biology from Yangzhou University, PR China. From 2002 to 2006, he worked as a clinical embryologist in the Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical College. He is currently working in the Reproductive Health Center, The Second Affiliated Hospital of Wenzhou Medical College. He has published eight journal papers. His current research interests are focused on gamete and embryo morphology, implantation and cryobiology.
Dr Hong-shan Ge Hong-Shan Ge1,3, Wei Zhou2, Wei Zhang2, Jin-Jun Lin2 Reproductive Health Center, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, Wenzhou, Zhejiang Province, 325009 PR China; 2Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical College. Wenzhou, Zhejiang province, PR China 3 Correspondence: e-mail: [email protected]
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Abstract The aim of this study was to determine if assisted hatching (AH) could improve the rates of pregnancy and implantation for both fresh and frozen–thawed embryo transfer cycles. A total of 760 fresh embryo transfer cycles and 200 frozen–thawed embryo transfer cycles were randomly assigned to either the treatment group (AH) or the control group (no AH). Zona thinning by laser was performed just before embryo transfer. In fresh embryo transfer cycles, the AH group and control group results were comparable. There were no significant differences in the rates of positive human chorionic gonadotrophin (HCG; 47.5 versus 48.8%), clinical pregnancy (42.4 versus 42.6%), or implantation (26.3 versus 25.2%) between the two groups. However, in frozen–thawed embryo transfer cycles, the rates of positive HCG (32.0 versus 17.0%), clinical pregnancy (25.0 versus 14.0%) and implantation (16.7 versus 7.3%) were significantly greater in the AH group than in the control group (P < 0.05). The results of this investigation show that in the fresh embryo transfer cycles, laser-assisted hatching by zona thinning has no impact on the rates of positive HCG, clinical pregnancy and implantation, whereas in frozen–thawed cycles, assisted hatching by zona thinning significantly increases all three of these rates. Keywords: assisted hatching, fresh cycle, frozen–thawed cycle, implantation, pregnancy rates
Introduction What leads to the poor implantation rate of embryos in IVF/ intracytoplasmic sperm injection (ICSI) is now a hot issue in current studies of human assisted reproduction techniques. Besides intrinsic embryo abnormalities or defective uterine receptivity, hatching failure could also partly explain the low implantation rate in IVF/ICSI (Mantoudis et al., 2001). The successful hatching of embryos is thought to be a key event in the implantation process. If this hatching does not take place, further embryo development will not occur.
Embryo hatching and implantation may be impaired in some patients when the zona pellucida (ZP) is considered too thick or hard (Cohen et al., 1992a). Such thickening of the zona was thought to be correlated with basal FSH concentration and preovulatory oestradiol (Loret De Mola et al., 1997). Secondly, zona hardening is also thought to occur during in-vitro culture (De Felici and Siracusa, 1982) or after cryopreservation (Carroll et al., 1990). In addition, suboptimal culture conditions or cryopreservation procedures may also adversely affect
589 © 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
quantitative or qualitative trophectoderm-produced zona lysin secretion, which has been proposed as the primary mechanism of embryo hatching (Schiewe et al., 1995). Assisted hatching is defined as an artificial breaching of the ZP and was first introduced in clinical IVF by Malter and Cohen (1989) and Cohen et al. (1990) as an artificial means for the embryos to hatch to improve clinical pregnancy rates. Since then, several techniques have been developed. Zonal dissection, drilling and thinning, with the use of acidic solutions, proteinases, peizon vibrators and lasers, can all perform assisted hatching. However, the clinical relevance of assisted hatching remains controversial and elusive. Some research demonstrated that assisted hatching could increase implantation and pregnancy rates, especially in women with a poor prognosis, such as advanced age (Magli et al., 1998), repeated failures (Petersen et al., 2005), poor quality embryos, or frozen– thawed embryos (Gabrielsen et al., 2004). In contrast, other studies found that assisted hatching did not increase rates of implantation and clinical pregnancy, not only in nonselectively infertile couples (Hellebaut et al., 1996), but also in those patients with poor prognosis (Rufas-Sapir et al., 2004) and in frozen embryo transfer cycles (Ng et al., 2005). These conflicting results might arise from different experimental designs, patient characteristics, selection criteria and sample numbers, or from different assisted hatching techniques. Therefore, ascertaining the true effects of assisted hatching on patients with different characteristic, and how assisted hatching affects their clinical results, would surely help to improve the clinical results of those patients with hatching difficulties. In addition, ascertaining these effects provides us with another way of finding out the causes of embryo implantation failure. Based on the above evidence, a prospective, randomized clinical trial was conducted to test the efficacy of assisted hatching on both fresh and frozen– thawed embryo transfer cycles during the same period.
Materials and methods Patient selection
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A total of 831 freshly retrieved oocyte cycles and 245 frozen–thawed cycles were performed at the Reproductive Medicine Centre, the First Affiliated Hospital of Wenzhou Medical College during this study. Patients for both fresh and frozen–thawed embryo transfer cycles were included if they met the following criteria: (i) normal baseline FSH concentration (3–12 IU/l); (ii) fewer than five failed cycles of assisted reproduction treatment, including fresh IVF/ ICSI embryo transfer cycles and/or frozen–thawed embryo transfer cycles. Those patients with uterine abnormality or low fertilization capacity (rate of fertilization <20% and late ICSI following fertilization failure of IVF) were excluded. In addition to above criteria, frozen–thawed cycle patients also had to meet following two requirements: (i) survival embryo number ≥1; (ii) total number of living blastomeres for embryo transfer ≥3 on day 2 or ≥5 on day 3.
Finally, a total of 760 fresh IVF/ICSI-embryo transfer cycles and 200 frozen–thawed embryo transfer cycles that met the above criteria were recruited in the study. A written informed consent to participate was obtained before starting the treatment. The study was approved by the Research Ethics Board of the hospital.
Ovarian stimulation, oocyte retrieval and embryo transfer procedures Ovarian stimulation was performed with the use of recombinant FSH (Gonal-F; Serono) and human chorionic gonadotrophin (HCG; Profasi; Serono) after pituitary suppression with gonadotrophin-releasing hormone (GnRH) agonist that was started in the late luteal phase of the previous cycle. Monitoring was performed by means of ovarian ultrasonography and serum oestradiol measurement. The dose of FSH was adjusted according to individual response. Ovulation was triggered with HCG (10,000 IU) and oocyte retrieval was carried out 36 h later by transvaginal ultrasoundguided follicular aspiration. ICSI or IVF was performed 4–6 h after oocyte retrieval. Fertilization was assessed 16–18 h after IVF or ICSI for the appearance of two pronuclei and two polar bodies. Embryo morphology was evaluated according to the number and shape of blastomeres, and the degree of fragmentation and multinucleation, as described previously (Ge et al., 2008). Good embryos were defined as those having regular blastomeres, ≤20% fragments and no multinucleated blastomeres and those containing at least three cells on day 2 or six cells on day 3. After embryo transfer, surplus good embryos were cryopreserved.
Embryo freezing and thawing protocols Embryos were cryopreserved using a programmable freezer (Planer Products Ltd, Sunbury-On-Thames, UK) with a freezing media kit (Quinn’s; SAGE, USA), which contained the following solutions: phosphate buffered saline (PBS), 1.5 mol/l propanediol (PROH) and 1.5 mol/l PROH plus 0.1 mol/l sucrose. The freezing programme for embryos in the study unit was as follows: starting temperature 20°C; rate of cooling 2°C/min from 20°C to –7°C; soak at –7°C for 5 min; manual seeding; hold the temperature at –7°C for 10 min; rate of cooling 0.3°C/min from –7°C to –30°C; rate of cooling 3°C/min from –30°C to –120°C. The frozen straw was quickly transferred from the freezing chamber to a reservoir of liquid nitrogen. Frozen embryos were thawed on the day of embryo transfer at room temperature for 30 s and then at 30°C in a water bath for 30 s. The retrieved embryos were then transferred to a plastic five-well dish where they were washed from the cryoprotectant at room temperature by stepwise dilutions (Quinn’s). Embryos were classified as fully intact (100% cells survived), partially damaged (≥50% cells survived) or degenerated (<50% cells survived). Frozen–thawed embryos were considered to have survived if ≥50% of the blastomeres were intact. Embryos were cultured in the CO2 incubator for a short period before transfer (about 30–60 min).
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Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
Assignment On the day of embryo transfer, just before transfer, eligible patients were randomized to either the treatment group (assisted hatching) or the control group (no assisted hatching) according to a randomization list based on sequential numbers in sealed envelopes. To eliminate potential bias, group allocation was performed just before embryo transfer. By that time, the work of embryo selection had been done and it had already been decided which embryos would be transferred, cryopreserved or discarded. For frozen–thawed cycles, group allocation was randomized after embryos were thawed among those patients who met the study’s criterion. To avoid bias from different skills among the operators, the procedures of zona thinning by laser, embryo thawing and embryo transfer, were performed by specific individuals. Both patients and the clinician were blinded to the group allocation.
Assisted hatching by laser thinning of the zona pellucida On the day of embryo transfer, immediately prior to transfer, embryos undergoing laser-assisted hatching received the following treatment. The ZP was thinned using a 1.48 μm wavelength diode laser (OCTAX EyeWareTM, Germany). A few milliseconds of laser irradiation achieved a ZP thinning to about 50% of the initial ZP thickness. This irradiation was initiated at one point and continued until 25% of the ZP was thinned. That is, laser drilling was initiated at the 9 o’clock position and consecutive irradiations were generated until the 12 o’clock position was reached.
Statistical analysis Clinical pregnancies were defined by the presence of one or more gestational sacs. The implantation rate was the proportion of embryos transferred, which resulted in an intrauterine gestational sac. The primary outcome measure was implantation rate. Secondary outcome measures included pregnancy rates (rates of positive HCG, clinical pregnancy and multiple pregnancies) and live birth rate. Results were analysed using Student’s t-test for numerical variables and chi-squared test for categorical variables. A difference of P < 0.05 was considered as significant.
Results Fresh embryo transfer cycles A total of 831 IVF/ICSI cycles were performed during the study period. Of these, 772 met the inclusion criteria, but 12 patients abandoned embryo transfer for various reasons,
such as avoiding potential risks of ovarian hyperstimulation syndrome. The remaining 760 fresh cycles were finally randomly allocated into either the assisted hatching group or the control group (no assisted hatching). Patient characteristics in the assisted hatching and control groups are summarized in Table 1. The mean age, duration of infertility, causes of infertility, number of pervious IVF/ ICSI cycles, rate of ICSI performed and number of previous failed cycles were similar in both the groups. Embryology data and outcomes are summarized in Table 2. The number of oocytes retrieved, number of oocytes fertilized, number of good quality embryos and number of embryos transferred were all comparable between the two groups. No significant difference in the rates of positive HCG tests (47.5 versus 48.8%), clinical pregnancy (42.4 versus 42.6%), multiple pregnancies (40.2 versus 35.8%), implantation (26.3 versus 25.2%) and live birth (34.9 versus 35.4%) were found between the assisted hatching and control groups. Patients in each group were further subdivided into two groups according to the women’s ages (<35 years versus ≥35 years). No significant differences in pregnancy rates (rates of positive HCG tests, clinical pregnancy and multiple pregnancies), implantation or live birth rates were detected between these two groups of women (Table 3).
Frozen–thawed embryo transfer cycles A total of 245 frozen–thawed cycles were also performed, of which 45 were excluded either because they didn’t meet the criteria of the study or embryo transfer was abandoned. Therefore, a total of 200 patients were recruited and were also randomly allocated into either the assisted hatching group or the control group (no assisted hatching). No significant differences were detected in the mean patient age at embryo thawing, duration of infertility, causes of infertility, scheme of transfer and number of previous fresh IVF/ICSI cycles between the assisted hatching and control groups (Table 4). There were also no significant differences in the number of embryos thawed, embryo survival rate, number of embryos fully intact and number of embryos transferred between two groups (Table 5). The rates of positive HCG tests (32.0 versus 17.0%), clinical pregnancy (27.0 versus 15.0%) and implantation (16.7 versus 7.3%) were significantly greater in the assisted hatching group than in the control group (P < 0.004; P < 0.04; P < 0.001, respectively) (Table 5). Although the multiple pregnancy rate (44.0 versus 28.6%) and the live birth rate (21.0 versus 12.0%) of the assisted hatching group appeared to be higher than those of the control group, the difference was not statistically significant.
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Table 1. Comparison of the characteristics of patients undergoing fresh embryo transfer cycles, with either assisted hatching or no assisted hatching. Characteristic
Assisted hatching
No assisted hatching
No. of cycles Age (years) Duration of infertility (years) Cause of infertility Male factor Tubal Mixed Unexplained ICSI performed Previous failed IVF/ICSI cycles
387 31.08 ± 4.68 4.6 ± 2.5
373 30.44 ± 4.15 4.2 ± 2.9
92 (23.8) 240 (62.0) 42 (10.9) 13 (3.4) 134 (34.6) 1.39 ± 0.82
79 (21.2) 234 (62.7) 44 (11.8) 16 (4.3) 145 (38.9) 1.34 ± 0.75
Values are either mean ± SD, or number (%). ICSI = intracytoplasmic sperm injection. There were no statistically significant differences between the two groups.
Table 2. Comparison of the outcomes of fresh embryo transfer cycles between the assisted hatching and no assisted hatching groups. Outcome
Assisted hatching
No assisted hatching
No. of cycles Oocytes retrieved Fertilized oocytes Good quality embryos Embryos transferred Positive HCG tests Clinical pregnancies Multiple pregnancies Implantations Live births
387 5194 (13.42 ± 7.39) 3399 (8.78 ± 5.71) 1685 (4.35 ± 3.72) 870 (2.25 ± 0.57) 184 (47.5) 164 (42.4) 66 (40.2) 229 (26.3) 135 (34.9)
373 5398 (14.47 ± 7.92) 3521 (9.44 ± 5.37) 1791 (4.80 ± 3.77) 846 (2.26 ± 0.53) 182 (48.8) 159 (42.6) 57 (35.8) 213 (25.2) 132 (35.4)
Values are either number (mean ± SD) or number (%). HCG = human chorionic gonadotrophin. There were no statistically significant differences between the two groups.
Table 3. Comparison of outcomes in fresh embryo transfer cycles by age between the assisted hatching and no assisted hatching groups. Outcome
Embryos transferred Positive HCG test Clinical pregnancies Implantations Live births
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<35 years of age Assisted No assisted hatching hatching (n = 297) (n = 317)
Assisted hatching (n = 90)
≥35 years of age No assisted hatching (n = 56)
641 153 (51.5) 138 (46.5) 192 (30.0) 119 (40.1)
226 31 (34.4) 26 (28.9) 37 (16.4) 20 (22.2)
140 17 (30.4) 13 (23.2) 19 (13.6) 10 (17.8)
703 165 (52.0) 148 (46.7) 194 (27.6) 122 (38.5)
Values are number, or number (%). HCG = human chorionic gonadotrophin. There were no statistically significant differences between outcome measures with or without assisted hatching for either age group.
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Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
Table 4. Comparison of the characteristics of patients undergoing frozen–thawed embryo transfer cycles, with either assisted hatching or no assisted hatching. Characteristic
Assisted hatching
No assisted hatching
No. of cycles Age at embryo thawing (years) Duration of infertility (years) Cause of infertility Male factor Tubal Mixed Unexplained Scheme of transfer Natural HRT Previous failed IVF/ICSI cycles
100 31.84 ± 3.85 4.7 ± 3.4
100 30.66 ± 4.42 4.3 ± 2.7
32 (32.0) 48 (48.0) 11 (11.0) 9 (9.0)
25 (25.0) 52 (52.0) 16 (16.0) 7 (7.0)
60 (60.0) 40 (40.0) 1.28 ± 0.82
59 (59.0) 41 (41.0) 1.33 ± 0.82
Values are either mean ± SD or number (%). HRT = hormone replacement therapy; ICSI = intracytoplasmic sperm injection. There were no statistically significant differences between the two groups.
Table 5. Comparison of outcomes in frozen–thawed embryo transfer cycles between the assisted hatching and no assisted hatching groups. Outcomes
Assisted hatching
No assisted hatching
P-value
No. of cycles Thawed embryos Embryo survival rate No. of fully intact embryos Embryos transferred Positive HCG tests Clinical pregnancies Multiple pregnancies Implantation Live births
100 344 (3.44 ± 1.61) 249 (72.4) 83 233 (2.33 ± 0.67) 32 (32.0) 25 (25.0) 11 (44.0) 39 (16.7) 21 (21.0)a
100 353 (3.53 ± 1.68) 265 (75.1) 75 246 (2.46 ± 0.76) 17 (17.0) 14 (14.0) 4 (28.6) 18 (7.3) 12 (12.0)
– NS NS NS NS 0.004 0.04 NS 0.001 NS
Values are either number (mean ± SD) or number (%), unless otherwise stated. HCG = human chorionic gonadotrophin; NS = not statistically significant. a One case of monozygotic twinning.
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Discussion It is well known that hatching failure is one of the important reasons for low embryo implantation rates in IVF/ICSI. It has been postulated that assisted hatching could increase embryo implantation rates. There are three possible mechanisms by which assisted hatching could improve embryo implantation. First, suboptimal culture conditions (De Felici and Siracusa, 1982) or cryopreservation (Carroll et al., 1990) procedures might make the ZP thicken and/or harden, which might cause hatching difficulty. By thinning, creating a hole or other means, assisted hatching could mechanically facilitate the hatching process. Second, studies in human (Liu et al., 1993) and animal models (Schiewe et al., 1995) found that assisted hatching resulted in earlier hatching than in non-assisted hatching embryos. Facilitation of earlier embryonic hatching might be particularly important given that the short window of endometrial receptivity appeared to be shifted 1–2 days earlier in cycles with ovarian stimulation for assisted reproduction treatment compared with natural cycles (Develioglu et al., 1999; Nikas et al., 1999). Third, the artificial gap produced by hatching may also serve as a channel for the exchange of metabolites, growth factors and messages between embryos and the endometrium (Cohen et al., 1992a). However, so far, the clinical results of performing assisted hatching are still conflicting. Several authors have reported that assisted hatching of cleaved embryos increases the implantation and pregnancy rate (Cohen et al., 1992b; Magli et al., 1998; Gabrielsen et al., 2004; Petersen et al., 2005; Ghobara et al., 2006). Other studies did not find a significant benefit from assisted hatching of embryos (Hurst et al., 1998; Rufas-Sapir et al., 2004; Ng et al., 2005; Frydman et al., 2006). However, among these studies, very few randomized studies were available and most reports were of retrospective analyses. This prospective randomized study failed to show any beneficial effect of laser ZP thinning when compared with control group in terms of implantation rates (26.3 versus 25.2%), clinical pregnancy rate (42.4 versus 42.6%) and live birth rates (34.9 versus 35.4%) in fresh IVF/ICSI cycles. This is consistent with some other studies in which assisted hatching was performed before transfer of embryos to patients with non-selective or good prognosis (Hellebaut et al., 1996; Tucker et al., 1996; Hurst et al., 1998; Sagoskin et al., 2007) Cohen and co-workers failed to demonstrate a significant benefit from non-selective assisted hatching in a randomized study of patients with normal FSH concentration (Cohen et al., 1992a). Hellebaut et al. (1996) evaluated implantation rates after assisted hatching using partial zona dissection, which included 120 randomized non-selective patients and found no increase in rates of pregnancy or implantation when compared with the control group. Tucker et al. (1996) also failed to demonstrate a significant benefit from non-selective assisted hatching in ICSI cycles.
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Recently, one randomized study specifically addressed good prognosis patients (defined as patients <39 years of age with normal FSH and oestradiol concentrations, no more than one previous unsuccessful cycle of IVF-embryo transfer and good embryo quality on day 3) and they also found no benefit
from assisted hatching among this patient population. Rates of implantation, pregnancy, spontaneous pregnancy loss and live birth were all very similar regardless of whether or not embryos underwent assisted hatching before transfer (Sagoskin et al., 2007). Another retrospective study with good prognosis patients arrived at the same conclusion that assisted hatching did not improve clinical results (Hurst et al., 1998). In addition, the effect of assisted hatching on two age subgroups was studied to further clarify the relationship between assisted hatching and maternal age. No significant difference was detected in clinical results for either older or younger subgroups. These conclusions are in agreement with other studies that have also failed to find any beneficial effect of assisted hatching in older patients (Ali et al., 2003; Frydman et al., 2006; Makrakis et al., 2006). However, other studies showed assisted hatching could significantly improve the outcome of IVF cycles of older women (Magli et al., 1998; Meldrum et al., 1998). Two meta-analyses demonstrated that assisted hatching increased implantation and pregnancy rates in older women (Edi-Osagie et al., 2003; Sallam et al., 2003). One of the reasons that have been suggested to explain the generally lower implantation rate after transfer of frozen– thawed embryos compared with fresh embryos is the alteration of the architecture of the ZP during the freezing–thawing process. This alteration causes the embryo to have ‘exacerbated abnormal zona hardness’, which results in failure of embryonic ZP rupture (Tucker et al., 1991; Balaban et al., 2006). Six very recent, well-designed prospective studies reported controversial issues regarding frozen–thawed embryos and assisted hatching (Gabrielsen et al., 2004; Primi et al., 2004; Ng et al., 2005; Balaban et al., 2006; Petersen et al., 2006; Sifer et al., 2006). Ng et al. (2005) failed to show any improvement in implantation or pregnancy rates after the transfer of laserthinned thawed embryos (9.0% and 12.5%, respectively) compared with control embryos (6.8% and 15%, respectively). This result was very similar to the results obtained by three other randomized studies. In frozen–thawed embryo transfer cycles no benefits of ZP thinning by pronase (Sifer et al., 2006), by laser (Petersen et al., 2006) or drilling by laser (Primi et al., 2004) was demonstrated. In contrast, the present study indicated that ZP thinning using laser before frozen–thawed embryo transfer could significantly improve the rates of pregnancy and implantation. This result was consistent with two other prospective studies (Gabrielsen et al., 2004; Balaban et al., 2006). The study by Gabrielsen et al. (2004) showed that zona drilling using acid increased the implantation rate of frozen–thawed embryos. The implantation rate in the assisted hatching group was 11.4% while that in the control group was only 5.8%. Balaban et al. (2006) also showed that ZP thinning using laser significantly increased the implantation and pregnancy rates (20.1 and 40.9%, respectively) compared with control (9.9 and 27.3%, respectively). In this study, assisted hatching was performed about 20 h after thawing and only on embryos that showed evidence of cleavage. Several points may explain the difference between studies showing a positive effect of assisted hatching and those showing no effect of assisted hatching. Firstly, different methods of assisted hatching were used. Although many studies showed RBMOnline®
Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
that no significant difference was found between methods of assisted hatching (Balaban et al., 2002), different research groups might in fact have some discrepancies, since assisted hatching is technical work and needs operators that have some experience. Secondly, different patient characteristics and selection criteria were involved. Some centres use assisted hatching for either poor-prognosis patients, such as women with advanced age, poor-quality embryos, embryos with thick ZP or previous implantation failures, or for good prognosis patients. Others use assisted hatching non-selectively in all couples undergoing IVF. Even if patients were chosen with the same basic characteristics, diversity might still exist because centres may have different stimulation schemes and embryo culture systems, or may transfer different numbers of embryos, have different skill levels and even measure clinical outcomes differently, which all may affect the results. Thirdly, regarding freeze–thawing cycles, different embryo freezing criteria and stage are employed: some centres freeze all surplus embryos (Petersen et al., 2006) and others only freeze good quality embryos (Ng et al., 2005). Also, some freeze cleavage embryos and blastocysts (Hiraoka et al., 2007), and others freeze pronuclei stage embryos. Furthermore, whether embryos were cultured after thawing might also affect research results. Finally, the design methods were different. Thus, a prospective randomized study may result in a more credible conclusion than that of a retrospective study. In summary, two conclusions were reached in the present study. For fresh embryo transfer cycles, assisted hatching by zona thinning had no significant impact on the rates of implantation, clinical pregnancy or live birth. In contrast, in frozen–thawed embryo transfer cycles, ZP thinning could significantly increase the rates of positive HCG test (P = 0.004), clinical pregnancy (P = 0.04) and implantation (P = 0.001). ZP thinning should be performed routinely in frozen–thawed embryo transfer cycles.
Acknowledgements This study was supported by grants from Zhejiang provincial Natural Science Foundation of China (Y204272). The authors are very grateful to all personnel of the Reproductive Medical Centre, The First Affiliated Hospital of Wenzhou Medical College for their work in this programme, and to Professor Ji-Qiang Lu and Mrs Ya-Mei Xue for their review of the manuscript.
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transfer at the cleavage stage. Human Reproduction 20, 979–985. Nikas G, Develioglu OH, Toner JP, Jones HW, Jr. 1999 Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Human Reproduction 14, 787–792. Petersen CG, Mauri AL, Baruffi RL et al. 2006 Laser-assisted hatching of cryopreserved-thawed embryos by thinning one quarter of the zona. Reproductive BioMedicine Online 13, 668–675. Petersen CG, Mauri AL, Baruffi RLet al. 2005 Implantation failures: success of assisted hatching with quarter-laser zona thinning. Reproductive BioMedicine Online 10, 224–229. Primi MP, Senn A, Montag M et al. 2004 A European multicentre prospective randomized study to assess the use of assisted hatching with a diode laser and the benefit of an immunosuppressive/ antibiotic treatment in different patient populations. Human Reproduction 19, 2325–2333. Rufas-Sapir O, Stein A, Orvieto R et al. 2004 Is assisted hatching beneficial in patients with recurrent implantation failures? Clinical and Experimental Obstetrics and Gynecology 31, 110–112. Sagoskin AW, Levy MJ, Tucker MJ et al. 2007 Laser assisted hatching in good prognosis patients undergoing in-vitro fertilization-embryo transfer: a randomized controlled trial. Fertility and Sterility 87, 283–287. Sallam HN, Sadek SS, Agameya AF 2003 Assisted hatching – a meta-analysis of randomized controlled trials. Journal of Assisted Reproduction and Genetics 20, 332–342.
Schiewe MC, Hazeleger NL, Sclimenti C, Balmaceda JP 1995 Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model. Fertility and Sterility 63, 288–294. Sifer C, Sellami A, Poncelet C et al. 2006 A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen–thawed embryo transfers. Human Reproduction 21, 2384–2389. Tucker MJ, Morton PC, Wright G et al. 1996 Enhancement of outcome from intracytoplasmic sperm injection: does coculture or assisted hatching improve implantation rates? Human Reproduction 11, 2434–2437. Tucker MJ, Cohen J, Massey JB et al. 1991 Partial dissection of the zona pellucida of frozen–thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. American Journal of Obstetrics Gynecology 165, 341–344; discussion 344–345.
Declaration: The authors report no financial or commercial conflicts of interest. Received 7 August 2007; refereed 3 September; accepted 14 November 2007.
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Article Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles: a prospective, randomized study Hong-shan Ge received his Bachelor and Master degrees in biology from Yangzhou University, PR China. From 2002 to 2006, he worked as a clinical embryologist in the Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical College. He is currently working in the Reproductive Health Center, The Second Affiliated Hospital of Wenzhou Medical College. He has published eight journal papers. His current research interests are focused on gamete and embryo morphology, implantation and cryobiology.
Dr Hong-shan Ge Hong-Shan Ge1,3, Wei Zhou2, Wei Zhang2, Jin-Jun Lin2 Reproductive Health Center, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, Wenzhou, Zhejiang Province, 325009 PR China; 2Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical College. Wenzhou, Zhejiang province, PR China 3 Correspondence: e-mail: [email protected]
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Abstract The aim of this study was to determine if assisted hatching (AH) could improve the rates of pregnancy and implantation for both fresh and frozen–thawed embryo transfer cycles. A total of 760 fresh embryo transfer cycles and 200 frozen–thawed embryo transfer cycles were randomly assigned to either the treatment group (AH) or the control group (no AH). Zona thinning by laser was performed just before embryo transfer. In fresh embryo transfer cycles, the AH group and control group results were comparable. There were no significant differences in the rates of positive human chorionic gonadotrophin (HCG; 47.5 versus 48.8%), clinical pregnancy (42.4 versus 42.6%), or implantation (26.3 versus 25.2%) between the two groups. However, in frozen–thawed embryo transfer cycles, the rates of positive HCG (32.0 versus 17.0%), clinical pregnancy (25.0 versus 14.0%) and implantation (16.7 versus 7.3%) were significantly greater in the AH group than in the control group (P < 0.05). The results of this investigation show that in the fresh embryo transfer cycles, laser-assisted hatching by zona thinning has no impact on the rates of positive HCG, clinical pregnancy and implantation, whereas in frozen–thawed cycles, assisted hatching by zona thinning significantly increases all three of these rates. Keywords: assisted hatching, fresh cycle, frozen–thawed cycle, implantation, pregnancy rates
Introduction What leads to the poor implantation rate of embryos in IVF/ intracytoplasmic sperm injection (ICSI) is now a hot issue in current studies of human assisted reproduction techniques. Besides intrinsic embryo abnormalities or defective uterine receptivity, hatching failure could also partly explain the low implantation rate in IVF/ICSI (Mantoudis et al., 2001). The successful hatching of embryos is thought to be a key event in the implantation process. If this hatching does not take place, further embryo development will not occur.
Embryo hatching and implantation may be impaired in some patients when the zona pellucida (ZP) is considered too thick or hard (Cohen et al., 1992a). Such thickening of the zona was thought to be correlated with basal FSH concentration and preovulatory oestradiol (Loret De Mola et al., 1997). Secondly, zona hardening is also thought to occur during in-vitro culture (De Felici and Siracusa, 1982) or after cryopreservation (Carroll et al., 1990). In addition, suboptimal culture conditions or cryopreservation procedures may also adversely affect
589 © 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
quantitative or qualitative trophectoderm-produced zona lysin secretion, which has been proposed as the primary mechanism of embryo hatching (Schiewe et al., 1995). Assisted hatching is defined as an artificial breaching of the ZP and was first introduced in clinical IVF by Malter and Cohen (1989) and Cohen et al. (1990) as an artificial means for the embryos to hatch to improve clinical pregnancy rates. Since then, several techniques have been developed. Zonal dissection, drilling and thinning, with the use of acidic solutions, proteinases, peizon vibrators and lasers, can all perform assisted hatching. However, the clinical relevance of assisted hatching remains controversial and elusive. Some research demonstrated that assisted hatching could increase implantation and pregnancy rates, especially in women with a poor prognosis, such as advanced age (Magli et al., 1998), repeated failures (Petersen et al., 2005), poor quality embryos, or frozen– thawed embryos (Gabrielsen et al., 2004). In contrast, other studies found that assisted hatching did not increase rates of implantation and clinical pregnancy, not only in nonselectively infertile couples (Hellebaut et al., 1996), but also in those patients with poor prognosis (Rufas-Sapir et al., 2004) and in frozen embryo transfer cycles (Ng et al., 2005). These conflicting results might arise from different experimental designs, patient characteristics, selection criteria and sample numbers, or from different assisted hatching techniques. Therefore, ascertaining the true effects of assisted hatching on patients with different characteristic, and how assisted hatching affects their clinical results, would surely help to improve the clinical results of those patients with hatching difficulties. In addition, ascertaining these effects provides us with another way of finding out the causes of embryo implantation failure. Based on the above evidence, a prospective, randomized clinical trial was conducted to test the efficacy of assisted hatching on both fresh and frozen– thawed embryo transfer cycles during the same period.
Materials and methods Patient selection
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A total of 831 freshly retrieved oocyte cycles and 245 frozen–thawed cycles were performed at the Reproductive Medicine Centre, the First Affiliated Hospital of Wenzhou Medical College during this study. Patients for both fresh and frozen–thawed embryo transfer cycles were included if they met the following criteria: (i) normal baseline FSH concentration (3–12 IU/l); (ii) fewer than five failed cycles of assisted reproduction treatment, including fresh IVF/ ICSI embryo transfer cycles and/or frozen–thawed embryo transfer cycles. Those patients with uterine abnormality or low fertilization capacity (rate of fertilization <20% and late ICSI following fertilization failure of IVF) were excluded. In addition to above criteria, frozen–thawed cycle patients also had to meet following two requirements: (i) survival embryo number ≥1; (ii) total number of living blastomeres for embryo transfer ≥3 on day 2 or ≥5 on day 3.
Finally, a total of 760 fresh IVF/ICSI-embryo transfer cycles and 200 frozen–thawed embryo transfer cycles that met the above criteria were recruited in the study. A written informed consent to participate was obtained before starting the treatment. The study was approved by the Research Ethics Board of the hospital.
Ovarian stimulation, oocyte retrieval and embryo transfer procedures Ovarian stimulation was performed with the use of recombinant FSH (Gonal-F; Serono) and human chorionic gonadotrophin (HCG; Profasi; Serono) after pituitary suppression with gonadotrophin-releasing hormone (GnRH) agonist that was started in the late luteal phase of the previous cycle. Monitoring was performed by means of ovarian ultrasonography and serum oestradiol measurement. The dose of FSH was adjusted according to individual response. Ovulation was triggered with HCG (10,000 IU) and oocyte retrieval was carried out 36 h later by transvaginal ultrasoundguided follicular aspiration. ICSI or IVF was performed 4–6 h after oocyte retrieval. Fertilization was assessed 16–18 h after IVF or ICSI for the appearance of two pronuclei and two polar bodies. Embryo morphology was evaluated according to the number and shape of blastomeres, and the degree of fragmentation and multinucleation, as described previously (Ge et al., 2008). Good embryos were defined as those having regular blastomeres, ≤20% fragments and no multinucleated blastomeres and those containing at least three cells on day 2 or six cells on day 3. After embryo transfer, surplus good embryos were cryopreserved.
Embryo freezing and thawing protocols Embryos were cryopreserved using a programmable freezer (Planer Products Ltd, Sunbury-On-Thames, UK) with a freezing media kit (Quinn’s; SAGE, USA), which contained the following solutions: phosphate buffered saline (PBS), 1.5 mol/l propanediol (PROH) and 1.5 mol/l PROH plus 0.1 mol/l sucrose. The freezing programme for embryos in the study unit was as follows: starting temperature 20°C; rate of cooling 2°C/min from 20°C to –7°C; soak at –7°C for 5 min; manual seeding; hold the temperature at –7°C for 10 min; rate of cooling 0.3°C/min from –7°C to –30°C; rate of cooling 3°C/min from –30°C to –120°C. The frozen straw was quickly transferred from the freezing chamber to a reservoir of liquid nitrogen. Frozen embryos were thawed on the day of embryo transfer at room temperature for 30 s and then at 30°C in a water bath for 30 s. The retrieved embryos were then transferred to a plastic five-well dish where they were washed from the cryoprotectant at room temperature by stepwise dilutions (Quinn’s). Embryos were classified as fully intact (100% cells survived), partially damaged (≥50% cells survived) or degenerated (<50% cells survived). Frozen–thawed embryos were considered to have survived if ≥50% of the blastomeres were intact. Embryos were cultured in the CO2 incubator for a short period before transfer (about 30–60 min).
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Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
Assignment On the day of embryo transfer, just before transfer, eligible patients were randomized to either the treatment group (assisted hatching) or the control group (no assisted hatching) according to a randomization list based on sequential numbers in sealed envelopes. To eliminate potential bias, group allocation was performed just before embryo transfer. By that time, the work of embryo selection had been done and it had already been decided which embryos would be transferred, cryopreserved or discarded. For frozen–thawed cycles, group allocation was randomized after embryos were thawed among those patients who met the study’s criterion. To avoid bias from different skills among the operators, the procedures of zona thinning by laser, embryo thawing and embryo transfer, were performed by specific individuals. Both patients and the clinician were blinded to the group allocation.
Assisted hatching by laser thinning of the zona pellucida On the day of embryo transfer, immediately prior to transfer, embryos undergoing laser-assisted hatching received the following treatment. The ZP was thinned using a 1.48 μm wavelength diode laser (OCTAX EyeWareTM, Germany). A few milliseconds of laser irradiation achieved a ZP thinning to about 50% of the initial ZP thickness. This irradiation was initiated at one point and continued until 25% of the ZP was thinned. That is, laser drilling was initiated at the 9 o’clock position and consecutive irradiations were generated until the 12 o’clock position was reached.
Statistical analysis Clinical pregnancies were defined by the presence of one or more gestational sacs. The implantation rate was the proportion of embryos transferred, which resulted in an intrauterine gestational sac. The primary outcome measure was implantation rate. Secondary outcome measures included pregnancy rates (rates of positive HCG, clinical pregnancy and multiple pregnancies) and live birth rate. Results were analysed using Student’s t-test for numerical variables and chi-squared test for categorical variables. A difference of P < 0.05 was considered as significant.
Results Fresh embryo transfer cycles A total of 831 IVF/ICSI cycles were performed during the study period. Of these, 772 met the inclusion criteria, but 12 patients abandoned embryo transfer for various reasons,
such as avoiding potential risks of ovarian hyperstimulation syndrome. The remaining 760 fresh cycles were finally randomly allocated into either the assisted hatching group or the control group (no assisted hatching). Patient characteristics in the assisted hatching and control groups are summarized in Table 1. The mean age, duration of infertility, causes of infertility, number of pervious IVF/ ICSI cycles, rate of ICSI performed and number of previous failed cycles were similar in both the groups. Embryology data and outcomes are summarized in Table 2. The number of oocytes retrieved, number of oocytes fertilized, number of good quality embryos and number of embryos transferred were all comparable between the two groups. No significant difference in the rates of positive HCG tests (47.5 versus 48.8%), clinical pregnancy (42.4 versus 42.6%), multiple pregnancies (40.2 versus 35.8%), implantation (26.3 versus 25.2%) and live birth (34.9 versus 35.4%) were found between the assisted hatching and control groups. Patients in each group were further subdivided into two groups according to the women’s ages (<35 years versus ≥35 years). No significant differences in pregnancy rates (rates of positive HCG tests, clinical pregnancy and multiple pregnancies), implantation or live birth rates were detected between these two groups of women (Table 3).
Frozen–thawed embryo transfer cycles A total of 245 frozen–thawed cycles were also performed, of which 45 were excluded either because they didn’t meet the criteria of the study or embryo transfer was abandoned. Therefore, a total of 200 patients were recruited and were also randomly allocated into either the assisted hatching group or the control group (no assisted hatching). No significant differences were detected in the mean patient age at embryo thawing, duration of infertility, causes of infertility, scheme of transfer and number of previous fresh IVF/ICSI cycles between the assisted hatching and control groups (Table 4). There were also no significant differences in the number of embryos thawed, embryo survival rate, number of embryos fully intact and number of embryos transferred between two groups (Table 5). The rates of positive HCG tests (32.0 versus 17.0%), clinical pregnancy (27.0 versus 15.0%) and implantation (16.7 versus 7.3%) were significantly greater in the assisted hatching group than in the control group (P < 0.004; P < 0.04; P < 0.001, respectively) (Table 5). Although the multiple pregnancy rate (44.0 versus 28.6%) and the live birth rate (21.0 versus 12.0%) of the assisted hatching group appeared to be higher than those of the control group, the difference was not statistically significant.
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Table 1. Comparison of the characteristics of patients undergoing fresh embryo transfer cycles, with either assisted hatching or no assisted hatching. Characteristic
Assisted hatching
No assisted hatching
No. of cycles Age (years) Duration of infertility (years) Cause of infertility Male factor Tubal Mixed Unexplained ICSI performed Previous failed IVF/ICSI cycles
387 31.08 ± 4.68 4.6 ± 2.5
373 30.44 ± 4.15 4.2 ± 2.9
92 (23.8) 240 (62.0) 42 (10.9) 13 (3.4) 134 (34.6) 1.39 ± 0.82
79 (21.2) 234 (62.7) 44 (11.8) 16 (4.3) 145 (38.9) 1.34 ± 0.75
Values are either mean ± SD, or number (%). ICSI = intracytoplasmic sperm injection. There were no statistically significant differences between the two groups.
Table 2. Comparison of the outcomes of fresh embryo transfer cycles between the assisted hatching and no assisted hatching groups. Outcome
Assisted hatching
No assisted hatching
No. of cycles Oocytes retrieved Fertilized oocytes Good quality embryos Embryos transferred Positive HCG tests Clinical pregnancies Multiple pregnancies Implantations Live births
387 5194 (13.42 ± 7.39) 3399 (8.78 ± 5.71) 1685 (4.35 ± 3.72) 870 (2.25 ± 0.57) 184 (47.5) 164 (42.4) 66 (40.2) 229 (26.3) 135 (34.9)
373 5398 (14.47 ± 7.92) 3521 (9.44 ± 5.37) 1791 (4.80 ± 3.77) 846 (2.26 ± 0.53) 182 (48.8) 159 (42.6) 57 (35.8) 213 (25.2) 132 (35.4)
Values are either number (mean ± SD) or number (%). HCG = human chorionic gonadotrophin. There were no statistically significant differences between the two groups.
Table 3. Comparison of outcomes in fresh embryo transfer cycles by age between the assisted hatching and no assisted hatching groups. Outcome
Embryos transferred Positive HCG test Clinical pregnancies Implantations Live births
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<35 years of age Assisted No assisted hatching hatching (n = 297) (n = 317)
Assisted hatching (n = 90)
≥35 years of age No assisted hatching (n = 56)
641 153 (51.5) 138 (46.5) 192 (30.0) 119 (40.1)
226 31 (34.4) 26 (28.9) 37 (16.4) 20 (22.2)
140 17 (30.4) 13 (23.2) 19 (13.6) 10 (17.8)
703 165 (52.0) 148 (46.7) 194 (27.6) 122 (38.5)
Values are number, or number (%). HCG = human chorionic gonadotrophin. There were no statistically significant differences between outcome measures with or without assisted hatching for either age group.
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Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
Table 4. Comparison of the characteristics of patients undergoing frozen–thawed embryo transfer cycles, with either assisted hatching or no assisted hatching. Characteristic
Assisted hatching
No assisted hatching
No. of cycles Age at embryo thawing (years) Duration of infertility (years) Cause of infertility Male factor Tubal Mixed Unexplained Scheme of transfer Natural HRT Previous failed IVF/ICSI cycles
100 31.84 ± 3.85 4.7 ± 3.4
100 30.66 ± 4.42 4.3 ± 2.7
32 (32.0) 48 (48.0) 11 (11.0) 9 (9.0)
25 (25.0) 52 (52.0) 16 (16.0) 7 (7.0)
60 (60.0) 40 (40.0) 1.28 ± 0.82
59 (59.0) 41 (41.0) 1.33 ± 0.82
Values are either mean ± SD or number (%). HRT = hormone replacement therapy; ICSI = intracytoplasmic sperm injection. There were no statistically significant differences between the two groups.
Table 5. Comparison of outcomes in frozen–thawed embryo transfer cycles between the assisted hatching and no assisted hatching groups. Outcomes
Assisted hatching
No assisted hatching
P-value
No. of cycles Thawed embryos Embryo survival rate No. of fully intact embryos Embryos transferred Positive HCG tests Clinical pregnancies Multiple pregnancies Implantation Live births
100 344 (3.44 ± 1.61) 249 (72.4) 83 233 (2.33 ± 0.67) 32 (32.0) 25 (25.0) 11 (44.0) 39 (16.7) 21 (21.0)a
100 353 (3.53 ± 1.68) 265 (75.1) 75 246 (2.46 ± 0.76) 17 (17.0) 14 (14.0) 4 (28.6) 18 (7.3) 12 (12.0)
– NS NS NS NS 0.004 0.04 NS 0.001 NS
Values are either number (mean ± SD) or number (%), unless otherwise stated. HCG = human chorionic gonadotrophin; NS = not statistically significant. a One case of monozygotic twinning.
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Discussion It is well known that hatching failure is one of the important reasons for low embryo implantation rates in IVF/ICSI. It has been postulated that assisted hatching could increase embryo implantation rates. There are three possible mechanisms by which assisted hatching could improve embryo implantation. First, suboptimal culture conditions (De Felici and Siracusa, 1982) or cryopreservation (Carroll et al., 1990) procedures might make the ZP thicken and/or harden, which might cause hatching difficulty. By thinning, creating a hole or other means, assisted hatching could mechanically facilitate the hatching process. Second, studies in human (Liu et al., 1993) and animal models (Schiewe et al., 1995) found that assisted hatching resulted in earlier hatching than in non-assisted hatching embryos. Facilitation of earlier embryonic hatching might be particularly important given that the short window of endometrial receptivity appeared to be shifted 1–2 days earlier in cycles with ovarian stimulation for assisted reproduction treatment compared with natural cycles (Develioglu et al., 1999; Nikas et al., 1999). Third, the artificial gap produced by hatching may also serve as a channel for the exchange of metabolites, growth factors and messages between embryos and the endometrium (Cohen et al., 1992a). However, so far, the clinical results of performing assisted hatching are still conflicting. Several authors have reported that assisted hatching of cleaved embryos increases the implantation and pregnancy rate (Cohen et al., 1992b; Magli et al., 1998; Gabrielsen et al., 2004; Petersen et al., 2005; Ghobara et al., 2006). Other studies did not find a significant benefit from assisted hatching of embryos (Hurst et al., 1998; Rufas-Sapir et al., 2004; Ng et al., 2005; Frydman et al., 2006). However, among these studies, very few randomized studies were available and most reports were of retrospective analyses. This prospective randomized study failed to show any beneficial effect of laser ZP thinning when compared with control group in terms of implantation rates (26.3 versus 25.2%), clinical pregnancy rate (42.4 versus 42.6%) and live birth rates (34.9 versus 35.4%) in fresh IVF/ICSI cycles. This is consistent with some other studies in which assisted hatching was performed before transfer of embryos to patients with non-selective or good prognosis (Hellebaut et al., 1996; Tucker et al., 1996; Hurst et al., 1998; Sagoskin et al., 2007) Cohen and co-workers failed to demonstrate a significant benefit from non-selective assisted hatching in a randomized study of patients with normal FSH concentration (Cohen et al., 1992a). Hellebaut et al. (1996) evaluated implantation rates after assisted hatching using partial zona dissection, which included 120 randomized non-selective patients and found no increase in rates of pregnancy or implantation when compared with the control group. Tucker et al. (1996) also failed to demonstrate a significant benefit from non-selective assisted hatching in ICSI cycles.
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Recently, one randomized study specifically addressed good prognosis patients (defined as patients <39 years of age with normal FSH and oestradiol concentrations, no more than one previous unsuccessful cycle of IVF-embryo transfer and good embryo quality on day 3) and they also found no benefit
from assisted hatching among this patient population. Rates of implantation, pregnancy, spontaneous pregnancy loss and live birth were all very similar regardless of whether or not embryos underwent assisted hatching before transfer (Sagoskin et al., 2007). Another retrospective study with good prognosis patients arrived at the same conclusion that assisted hatching did not improve clinical results (Hurst et al., 1998). In addition, the effect of assisted hatching on two age subgroups was studied to further clarify the relationship between assisted hatching and maternal age. No significant difference was detected in clinical results for either older or younger subgroups. These conclusions are in agreement with other studies that have also failed to find any beneficial effect of assisted hatching in older patients (Ali et al., 2003; Frydman et al., 2006; Makrakis et al., 2006). However, other studies showed assisted hatching could significantly improve the outcome of IVF cycles of older women (Magli et al., 1998; Meldrum et al., 1998). Two meta-analyses demonstrated that assisted hatching increased implantation and pregnancy rates in older women (Edi-Osagie et al., 2003; Sallam et al., 2003). One of the reasons that have been suggested to explain the generally lower implantation rate after transfer of frozen– thawed embryos compared with fresh embryos is the alteration of the architecture of the ZP during the freezing–thawing process. This alteration causes the embryo to have ‘exacerbated abnormal zona hardness’, which results in failure of embryonic ZP rupture (Tucker et al., 1991; Balaban et al., 2006). Six very recent, well-designed prospective studies reported controversial issues regarding frozen–thawed embryos and assisted hatching (Gabrielsen et al., 2004; Primi et al., 2004; Ng et al., 2005; Balaban et al., 2006; Petersen et al., 2006; Sifer et al., 2006). Ng et al. (2005) failed to show any improvement in implantation or pregnancy rates after the transfer of laserthinned thawed embryos (9.0% and 12.5%, respectively) compared with control embryos (6.8% and 15%, respectively). This result was very similar to the results obtained by three other randomized studies. In frozen–thawed embryo transfer cycles no benefits of ZP thinning by pronase (Sifer et al., 2006), by laser (Petersen et al., 2006) or drilling by laser (Primi et al., 2004) was demonstrated. In contrast, the present study indicated that ZP thinning using laser before frozen–thawed embryo transfer could significantly improve the rates of pregnancy and implantation. This result was consistent with two other prospective studies (Gabrielsen et al., 2004; Balaban et al., 2006). The study by Gabrielsen et al. (2004) showed that zona drilling using acid increased the implantation rate of frozen–thawed embryos. The implantation rate in the assisted hatching group was 11.4% while that in the control group was only 5.8%. Balaban et al. (2006) also showed that ZP thinning using laser significantly increased the implantation and pregnancy rates (20.1 and 40.9%, respectively) compared with control (9.9 and 27.3%, respectively). In this study, assisted hatching was performed about 20 h after thawing and only on embryos that showed evidence of cleavage. Several points may explain the difference between studies showing a positive effect of assisted hatching and those showing no effect of assisted hatching. Firstly, different methods of assisted hatching were used. Although many studies showed RBMOnline®
Article - Impact of assisted hatching on fresh and frozen–thawed embryo transfer cycles - H-S Ge et al.
that no significant difference was found between methods of assisted hatching (Balaban et al., 2002), different research groups might in fact have some discrepancies, since assisted hatching is technical work and needs operators that have some experience. Secondly, different patient characteristics and selection criteria were involved. Some centres use assisted hatching for either poor-prognosis patients, such as women with advanced age, poor-quality embryos, embryos with thick ZP or previous implantation failures, or for good prognosis patients. Others use assisted hatching non-selectively in all couples undergoing IVF. Even if patients were chosen with the same basic characteristics, diversity might still exist because centres may have different stimulation schemes and embryo culture systems, or may transfer different numbers of embryos, have different skill levels and even measure clinical outcomes differently, which all may affect the results. Thirdly, regarding freeze–thawing cycles, different embryo freezing criteria and stage are employed: some centres freeze all surplus embryos (Petersen et al., 2006) and others only freeze good quality embryos (Ng et al., 2005). Also, some freeze cleavage embryos and blastocysts (Hiraoka et al., 2007), and others freeze pronuclei stage embryos. Furthermore, whether embryos were cultured after thawing might also affect research results. Finally, the design methods were different. Thus, a prospective randomized study may result in a more credible conclusion than that of a retrospective study. In summary, two conclusions were reached in the present study. For fresh embryo transfer cycles, assisted hatching by zona thinning had no significant impact on the rates of implantation, clinical pregnancy or live birth. In contrast, in frozen–thawed embryo transfer cycles, ZP thinning could significantly increase the rates of positive HCG test (P = 0.004), clinical pregnancy (P = 0.04) and implantation (P = 0.001). ZP thinning should be performed routinely in frozen–thawed embryo transfer cycles.
Acknowledgements This study was supported by grants from Zhejiang provincial Natural Science Foundation of China (Y204272). The authors are very grateful to all personnel of the Reproductive Medical Centre, The First Affiliated Hospital of Wenzhou Medical College for their work in this programme, and to Professor Ji-Qiang Lu and Mrs Ya-Mei Xue for their review of the manuscript.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 7 August 2007; refereed 3 September; accepted 14 November 2007.
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