Hum. Reprod. Advance Access originally published online on June 13, 2006
Human Reproduction 2006 21(9):2384-2389; doi:10.1093/humrep/del149
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A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers
1Service d’Histologie-Embryologie-Cytogénétique, Laboratoire de Biologie de la Reproduction 2Service de Médecine de la Reproduction, Hôpital Jean Verdier, Assistance Publique – Hôpitaux de Paris, Bondy and 3Service de Biostatistique et d’Informatique Médicale, CHU Saint-Louis, Assistance Publique – Hôpitaux de Paris, 75475 Paris cedex 10, France
4 To whom correspondence should be addressed at: Service d’Histologie-Embryologie-Cytogénétique-Laboratoire de Biologie de la Reproduction, Hôpital Jean Verdier, 93140 Bondy cedex, France. E-mail: jean-philippe.wolf{at}jvr.ap-hop-paris.fr
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Abstract |
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BACKGROUND: Assisted hatching (AH) in fresh embryo transfer (ET) could be associated with increased implantation rates. However, very few prospective randomized studies have specifically addressed the issue of AH during frozen-thawed embryo transfers (FET) cycles, those that have reported controversial results. The aim of this study was to evaluate the benefit of an enzymatic zona pellucida treatment of frozen-thawed embryos before transfer. METHODS: This was a prospective study including 125 non-donor FET cycles from 125 infertile couples. FETs were randomly allocated into AH group (n = 61, embryos pretreated with pronase 5 IU/ml for 1 min at 37°C) or control group (n = 64, untreated embryos). Zona pellucida thickness was measured for each transferred embryo. The main outcome parameters were clinical pregnancy and implantation rates. RESULTS: The two groups were comparable regarding mean women’s age, duration and indications of infertility, IVF outcome after fresh ETs, numbers and quality of fresh and frozen embryos, frozen-thawed embryo survival rates and blastomeres survival indexes. Despite a statistically significant decrease of zona pellucida thickness after pronase treatment [(mean ± SD) 18.5 ± 2.25 versus 14.5 ± 2.75 µm; P < 0.0001], implantation (9.6 versus 9.2%) and clinical pregnancy rates (18.0 versus 17.2%) were not statistically different after FETs, with a similar mean number of embryos transferred between AH and control groups, respectively. CONCLUSION: Within the constraints of our protocol, partial enzymatic digestion of zona pellucida by pronase was not related with any benefit of the FET outcome especially concerning the implantation ability of frozen-thawed embryos.
Key words: frozen-thawed embryo transfers outcome/partial digestion/zona pellucida
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Introduction |
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The zona pellucida is the extracellular matrix that surrounds the oocyte. It has multiple functions. Before fertilization, it presents a species-specific sperm barrier. Immediately following fertilization, it acts as a major block to polyspermy (Wassarman, 1992
). The zona pellucida protects also the embryo against the infiltration by leukocytes (Modlinski, 1970) and infection by bacterial and fungal agents (Chen and Wrathall, 1989) in the reproductive tract. Another function of the zona pellucida is to prevent the separation of blastomeres from cleaving embryos (Bronson and McLaren, 1970) and to maintain the cell arrangement of early embryos to ensure their successful development (Suzuki et al., 1995). However, hatching of the blastocyst from the zona pellucida is a prerequisite for implantation in the uterus. The mechanism of physiological hatching is not well characterized, but, in animal models, it has been shown to involve a lysis of the zona pellucida by enzymes, secreted either by the embryo or by the female reproductive tract. In vitro experiments have shown that the supplementation of embryo culture medium with protease has been associated with an improvement of the hatching rate in mice (Lee et al., 1997). Furthermore, hydrostatic pressure exerted on the zona pellucida by expansion of the blastocyst could play an associated role for hatching (Perona and Wassarman, 1986; Sawada et al., 1990; Gordon and Dapunt, 1993; Confino et al., 1997).
Cohen et al. have demonstrated in human that a reduced implantation ability observed following IVF embryo transfers (ET) may be because of the inability of the embryo to hatch out of the zona pellucida (Cohen et al., 1990). Indeed, an extended culture period of such embryos in an artificial environment could induce a zona pellucida hardening, as evaluated on the basis of zona pellucida dissolution times (Manna et al., 2001). Zona pellucida hardening of cryopreserved oocytes was also shown to be increased by the freeze-thaw process (Manna et al., 2001). This suggests that it could be the same for frozen-thawed embryos, although this was not specifically demonstrated.
Several techniques for assisted hatching (AH) in IVF programmes have been introduced over the years to breach or to thin the zona pellucida and promote the natural process of hatching. It has been shown that AH could be associated with increased implantation rates after IVF ET (Cohen et al., 1990) or frozen-thawed embryo transfers (FET) (Tucker et al., 1991; Check et al., 1996). AH can be performed by mechanical (Cohen et al., 1990), chemical (Cohen et al., 1992), enzymatic (Fong et al., 1997) or laser manipulation (Antinori et al., 1996). A study comparing these four different techniques showed similar implantation and pregnancy rates whatever the AH procedure was (Balaban et al., 2002). Furthermore, a previous study has recently stressed that laser zona pellucida thinning rather than zona pellucida breaching could lead to better implantation rates, suggesting that the breaching of the inner zona layer could be detrimental to the implantation potential of the human embryo (Mantoudis et al., 2001). This confirmed a previous study which showed a higher hatching rate at the blastocyst stage when AH was attempted by zona thinning rather than complete zona drilling (Blake et al., 2001). These studies suggest that opening the zona pellucida may have adverse effects such as abnormal blastocyst expansion, loss of blastomeres through the breached zona and disappearance of the protective effect of the zona pellucida against infectious or immunologic aggressions (Blake et al., 2001; Mantoudis et al., 2001).
A number of studies have focused on the benefit of such AH following fresh ET, but their results have remained controversial (Edi-Osagie et al., 2003; Sallam et al., 2003). Two recent meta-analyses showed that women undergoing AH were significantly more likely to achieve a clinical pregnancy, but in comparing heterogeneous data, they failed to highlight which IVF indications clearly benefit from AH (Edi-Osagie et al., 2003; Sallam et al., 2003). Furthermore, very few prospective randomized studies have specifically addressed the issue of AH performed on frozen-thawed embryos during FET cycles. Very recent studies reported controversial results with decreased (Primi et al., 2004), similar (Ng et al., 2005) or increased (Gabrielsen et al., 2004) implantation rates in the AH group compared to the control group.
On the basis of the discrepant results in the literature, the aim of this randomized prospective study was to assess whether AH, performed using a partial enzymatic digestion of the zona pellucida from surnumerary frozen-thawed embryos, without breaching of the zona pellucida, could enhance their implantation ability.
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Materials and methods |
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Study period and patients
This randomized prospective study included 125 FETs from 125 infertile patients, undertaken between 1 January 2004 and 18 December 2004 at the assisted reproductive technologies center of Jean Verdier hospital. The study was confined to non-donor and first FET cycles. Fresh IVF attempts were performed between 1999 and 2004. Embryos were generated by either ICSI or by conventional IVF (cIVF), because it has been previously shown that no adverse effect was demonstrated concerning the implantation ability of such frozen-thawed embryos derived from either of these two techniques (Kowalik et al., 1998
; Aytoz et al., 1999; Hu et al., 1999). The indications for IVF included tubal, endometriosis, male and others (mixed factors or unexplained infertilities). On the day of FET, patients were randomized by a laboratory technician according to a computer-generated randomization list in sealed enveloppes into the AH group or the control group. This study was approved by the Regional Ethical Committee. Furthermore, each couple included in this study was asked to sign an approval consent form before enrolling in the study.
IVF procedures
The ovarian stimulation and oocyte collection regimens remained unchanged over the studied period and have been described previously (Cedrin-Durnerin et al., 2003). Controlled ovarian stimulation was done using a classical long protocol with a pituitary down-regulation, achieved after i.m. administration of GnRH agonist (triptorelin, Ipsen-Biotech, Paris, France), and subcutaneous administration of recombinant human FSH (Gonal F®, Serono, Boulogne, France). hCG (10 000 IU, Organon, Saint Denis, France) was administered i.m. when a consistent rise in serum estradiol concentration was associated with the presence of 
4 follicles 16 mm in diameter. Transvaginal oocyte retrieval was scheduled 36 h after hCG administration under ultrasound guidance. The luteal support was initiated on day 1 after oocyte retrieval with 400 mg/day of progesterone (Utrogestan®, Besins Iscovesco, Paris, France), administered until measurement of serum beta-hCG at day 12 and prolonged until 5–6 weeks in case of pregnancy. In the absence of ovarian hyperstimulation syndrome (OHSS) risk, 2500 and 1500 units of hCG were administrated on day 1 and 3–6 after oocyte retrieval, respectively.
The culture media used for oocytes and embryos were (i) ISM1TM from day 0 to day 2 and (ii) ISM2TM from day 2 to day 3 (Medicult, Lyon, France). Semen samples were prepared with a discontinuous gradient method using PurSperm® (Nidacon International, Gothenburg, Sweden). ICSI or cIVF were standardly carried out 3–6 h after oocytes retrieval. Assessment of fertilization was performed at 16–20 h. The embryos were cultured in a humidified 5% CO2 atmosphere maintained at 37°C for a subsequent 24 or 48 h period. On day 2 or day 3, embryos were evaluated for fresh ET and cryopreservation using routine examination of (i) the number of blastomeres, (ii) the degree of fragmentation and (iii) the uniformity of blastomeres. Embryo morphology was scored as follows: grade A, <10% fragmentation and equal blastomeres; grade B, 10–30% fragmentation and equal blastomeres; grade C: fragmentation >30–50% and/or unequal blastomeres and grade D: fragmentation >50%. Our strategy was to freeze the surnumerary embryos on days 2 or 3 only if they exhibited a favourable grading with <30% fragmentation and regular blastomeres (grades A and B).
Embryo freezing
The freezing procedure was performed using a programmable biological freezer (Planer Kryo 560–16, CryoBioSystem, Paris, France), following a slow-freeze protocol using 1,2-propanediol (PROH) as cryoprotectant (Lassalle et al., 1985). Commercial media were used following the manufacturer’s instructions (Embryo Freezing Pack, Medicult). Thereafter, one to three embryos were loaded into cryostraws (CryoBioSystem) and cooled at a rate of 2°C/min from room temperature to –7°C. Manual seeding was then performed, and freezing was resumed first at a rate of 0.3°/min to –30°C and secondly at a rate of 35°C/min to –150°C before being plunged into liquid nitrogen. Finally, the straws were stored in liquid nitrogen, until thaw.
Embryo thawing and FET
The embryos were rapidly thawed and sequentially rehydrated at room temperature in commercial thawing solutions, following the manufacturer’s instructions (Embryo Thawing Pack, Medicult). Thawed embryos were placed in ISM2TM at 37°C and evaluated under x200 magnifications for (i) blastomere survival and (ii) scoring of the embryo morphology. Embryos were classified as fully intact (100% cells survived), partially damaged (50% cells survived) or degenerated (<50% cells survived). The survival rate was defined as the number of thawed embryos with 50% cells survived over the total number of thawed embryos x100. FETs were performed using only embryos with 50% intact blastomeres. The blastomere survival index was calculated for all thawed embryos as the average post-thaw, over the average prefreeze blastomere count, x100. Day 2 thawed embryos were transferred after a 20 h culture period under IVF conditions. Zona pellucida thickness was always measured on day 3 cleaved-stage embryos.
Thawed embryos were transferred during the course of stimulated cycles (Guerif et al., 2002). Briefly, rFSH was administered (75 IU daily) from day 6 to day 12. At day 13, hCG (5 000 IU) was administered according to follicle growth >17 mm after ultrasound evaluation, serum estradiol and progesterone levels and LH surge. FET was performed on day 5 after hCG administration depending on the progesterone level (>3 ng/ml measured at day 3 after hCG). Luteal support was performed similarly to fresh ET.
AH by pronase thinning of the zona pellucida
This was adapted from a protocol previously described (Fong et al., 1998). Culture dishes, with both 20 µl droplets of the pronase solution (5 IU/ml) (Protease, Sigma P-8811, Sigma-Aldrich, St Quentin Fallavier, France) and 20 µl droplets of ISM2TM medium under mineral oil, were prepared 24 h before FETs and maintained at 37°C in humidified 5% CO2 atmosphere. This pronase work solution was obtained after dilution (1 : 20 v/v) of an aliquot (100 IU/ml in ISM2TM medium) stored at –20°C until use. Briefly, cleaved embryos were transferred 15 min before FETs from embryo culture dishes to 20 µl droplets of the pronase solution for 1 min at 37°C in humidified 5% CO2 atmosphere. The aim was to thin the zona pellucida without complete removal. The embryos were then put in fresh ISM2TM medium, gently washed and placed into the incubator until transfer. Zona pellucida thickness was evaluated at day 3 by two independent operators after examination of all transferred embryos from the study and the control groups, on the heated stage of an inverted microscope using a WHK 10X/20 l occulometer (Olympus, Rungis, France). The accuracy of the measure assessed by the occulometer was controlled and validated using videocinematographic software (Lucia®, Nikon, France), following the manufacturer’s instructions. This measure was repeated four times on different places to limit a potential effect of zona pellucida thickness variation, and a mean was calculated for each embryo. All measures were done (i) at the time of FET (control group) and (ii) before and after pronase hatching (AH group).
IVF and FET cycles outcome
In both fresh ET and FET cycle outcomes, a clinical pregnancy was assessed by a positive fetal heartbeat on transvaginal ultrasound at 5–6 weeks of pregnancy. The implantation rate was calculated as the number of gestational sacs identified on transvaginal ultrasound per number of (i) fresh or (ii) frozen transferred embryos.
Statistical analysis
Assuming that (i) the average implantation rate of frozen-thawed and fresh embryos in our laboratory was 8.0 and 20.0% (when fresh ETs are associated with freezing), respectively and (ii) at least a two-fold increase in the implantation rate is expected in the AH group compared to the control group (Gabrielsen et al., 2004), the sample size required would be 120 patients to achieve statistical significance (P < 0.05) with a power of 0.8. Categorical data are presented as counts (percent) and quantitative data as mean (SD) or median (range) when the distribution was skewed. Groups were compared using chi-square test or Fisher’s exact test if more appropriate for categorical variables and Student’s t-test with unequal variances or Wilcoxon rank-sum test for quantitative variables. Differences before and after treatment were tested using paired Student’s t-tests.
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One hundred and thirty-two couples were asked to participate in the study, but seven declined (5.3%). In all, 61 and 64 FETs, resulting from 125 patients (ICSI: n = 68; cIVF: n = 57), were included in the AH group and the control group, respectively (Figure 1). Data from ICSI or cIVF were pooled, because no difference was observed concerning the mean (±SD) of zona pellucida thickness from embryos generated by ICSI (18.4 ± 2.7 µm) or cIVF (18.7 ± 2.7 µm).
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Table I summarizes the demographic data and some parameters related to the IVF outcome. No statistically significant difference was observed comparing the (i) mean women age at the time of oocyte retrieval (32.3 ± 4.0 versus 32.0 ± 4.4 years), (ii) duration of infertility (4.1 ± 2.5 versus 3.6 ± 2.2 years), (iii) percentages of primary/secondary infertility, (iv) infertility indications, (iv) numbers and percentages of good-quality frozen embryos [5.0 ± 3.4; (76.4%) versus 5.1 ± 3.0; (71.6%)] and (v) implantation, clinical pregnancy and live birth rates after fresh ET (15.2/28.0/27.0 versus 17.8/31.0/30.0%) in the AH and control groups, respectively.
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We compared AH performed on 125 frozen-thawed embryos at the time of FET with a control group of 130 embryos. There was no difference comparing the (i) frozen-thawed embryo survival rates (79.1 ± 22.5 versus 74.3 ± 26.2%), (ii) blastomeres survival indexes (84.9 ± 14.9 versus 83.9 ± 13.9%) and (iii) zona pellucida thickness of transferred embryos (18.5 ± 2.2 versus 18.5 ± 3.0 µm) between the AH group and the control group, respectively (Table II). Despite a statistically significant decrease of zona pellucida thickness after pronase treatment (14.5 ± 2.75 versus 18.5 ± 2.25 µm; P < 0.0001), implantation (9.6 versus 9.2%) and clinical pregnancy rates (18.0 versus 18.7%) were not statistically different after FETs in the AH group compared to the control group, with a similar number of frozen-thawed and good-quality embryos transferred (Table II). According to the median, the implantation rates were not statistically different when the zona pellucida thickness was <18.5 µm (8.6 versus 10.0%) or
18.5 µm (10.9 versus 8.3%) between the AH group and the control group, respectively. Twin pregnancy and miscarriage rates per clinical pregnancy were also similar in the two groups (Table II).
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Discussion |
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AH in fresh ET could be associated with increased implantation rates (Edi-Osagie et al., 2003
; Sallam et al., 2003). Then, AH was proposed to rescue the implantation ability of surnumerary frozen-thawed embryos using mechanical (Tucker et al., 1991), chemical (Check et al., 1996; Gabrielsen et al., 2004) or laser manipulation (Primi et al., 2004; Ng et al., 2005). Three very recent, well-designed prospective studies reported controversial issues on frozen-thawed embryos from AH. Results from these studies are summarized in Table III. Gabrielsen et al. reported that zona drilling (opening) using Tyrode’s acid led to a two-fold increase of the implantation rate in the AH group compared to the control group (Gabrielsen et al., 2004), confirming a previous retrospective study (Check et al., 1996). The two other studies showed that AH, performed using laser manipulation (one opening and one thinning), did not lead to higher implantation rate in patients initiating an FET (Primi et al., 2004; Ng et al., 2005) (Table III). We reported in this prospective randomized study our experience of AH from cryopreserved embryos which failed to demonstrate any benefit of an enzymatic zona pellucida thinning before FET when compared to a control, and this is the limit of our protocol.
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AH can be performed using several different techniques, which have been reported to lead to similar implantation rate results (Balaban et al., 2002). However, the benefit of AH either opening or thinning the zona pellucida is still debated. Indeed, a previous study reported a failure of chemical zona thinning to enhance human embryo implantation significantly, contrary to AH by complete zona drilling (Tucker et al., 1993
). The authors suggested that the bilayered human zona pellucida needed to be fully breached to enhance implantation ability of such embryos. However, other studies reported that zona opening of mouse embryos might have adverse effects as (i) the possibility of loss of blastomeres or of the whole embryo during contractions of the female reproductive tract (Nichols and Gardner, 1989) or (ii) the inhibition of natural expansion of blastocyst (Malter and Cohen, 1989). To avoid these drawbacks, cruciate thinning of the mouse zona pellucida was attempted, obtaining a significant enhancement of blastocyst hatching (Khalifa et al., 1992). In this way, thinning of the human zona pellucida rather then a complete zona drilling was also shown to increase (i) blastocyst hatching (Blake et al., 2001) and (ii) implantation rates (Antinori et al., 1996; Mantoudis et al., 2001). We used an enzymatic procedure to thin the zona pellucida, because this method has been previously and successfully used on blastocysts (Fong et al., 1998). Furthermore, the safety of the method has been previously described where the ultrastructure of the embryos subjected to pronase solution was examined (Fong et al., 2001). This work determined the optimal time of zona pellucida treatment by the pronase solution for softening or complete removal of the zona pellucida to be safe, without any change in the fine structure of blastocysts, compared with controls. Analysis of the human zona pellucida by polarized light microscopy quantitatively distinguishes its multi-laminar structure. Relative to oocytes, filaments of the inner zona layer are oriented radially (layer 1), whereas filaments of the outer zona layer (layer 3) are oriented tangentially. These are separated by a middle layer (layer 2) exhibiting a minimal birefringence which suggests a random orientation of filaments (Pelletier et al., 2004). Zona thinning with pronase presents two theorical advantages: the outer layer is dissolved, whereas inner layers are weakened by the enzyme. However, no data are available to assess the safety of the other AH techniques, especially concerning chemical or laser manipulations. The latter technique seemed suitable, allowing non-contact, microscope objective-delivered accessibility of laser light to the target with minimal absorption by the culture dish and the medium (Schopper et al., 1999) but with a high cost. However, mechanical AH with partial zona dissection, previously used in our laboratory, is thought to be limited by the difficulty to create a hole of consistent size and appeared to generate a risk of lysis of a blastomere during the procedure. This could be potentially associated with toxic influences by damaged blastomeres on sibling cells in the embryo (Edgar et al., 2000). The use of pronase solution to circumferentially thin the zona pellucida without a hole created in the zona pellucida could also protect the hatched embryos from external aggressions. Indeed, Primi et al. reported that AH by laser zona pellucida breaching could have a negative effect when not supported by an immunosuppressive and antibiotic (IA) treatment, with an implantation rate of 1.6% in the group without any treatment compared to 6.0% in the group with IA (Primi et al., 2004). The authors hypothesized that IA treatment could prevent some undefined immune reactions localized near the hole in the zona pellucida of hatched embryos which could destroy them. In conclusion, despite the efficacy of this easy-to-perform and low-cost method to thin significantly the zona pellucida thickness, AH by pronase did not improve the implantation rate of FET cycles and should not be routinely performed. Further studies are needed to assess whether AH performed with pronase zona pellucida thinning may be helpful in classical AH indications.
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Acknowledgements |
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The authors thank Mrs Samia Kanafani for reviewing the English translation.
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References |
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Antinori S, Panci C, Selman HA, Caffa B, Dani G, Versaci C. (1996) Zona thinning with the use of laser: a new approach to assisted hatching in humans. Hum Reprod 11:590–594.
Aytoz A, Van den Abbeel E, Bonduelle M, Camus M, Joris H, Van Steirteghem A, Devroey P. (1999) Obstetric outcome of pregnancies after the transfer of cryopreserved and fresh embryos obtained by conventional in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1999:2619–2624.
Balaban B, Urman B, Alatas C, Mercan R, Mumcu A, Isiklar A. (2002) A comparison of four different techniques of assisted hatching. Hum Reprod 17:1239–1243.
Blake DA, Forsberg AS, Johansson BR, Wikland M. (2001) Laser zona pellucida thinning – an alternative approach to assisted hatching. Hum Reprod 16:1959–1964.
Bronson RA and McLaren A. (1970) Transfer to the mouse oviduct of eggs with and without the zona pellucida. J Reprod Fertil 22:129–137.
Cedrin-Durnerin I, Bstandig B, Galey J, Bry-Gauillard H, Massin N, Hugues JN. (2003) Beneficial effects of GnRH agonist administration prior to ovarian stimulation for patients with a short follicular phase. Reprod Biomed Online 7:179–184.[Medline]
Check JH, Hoover L, Nazari A, O’Shaughnessy A, Summers D. (1996) The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril 65:254–257.[Web of Science][Medline]
Chen SS and Wrathall AE. (1989) The importance of the zona pellucida for disease control in livestock by embryo transfer. Br Vet J 145:129–140.[Web of Science][Medline]
Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, Wiemer K. (1990) Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 5:7–13.
Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. (1992) Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 7:685–691.
Confino E, Rawlins R, Binor Z, Radwanska E. (1997) The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo. Fertil Steril 68:164–167.[CrossRef][Web of Science][Medline]
Edgar DH, Bourne H, Speirs AL, McBain JC. (2000) A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 15:175–179.
Edi-Osagie EC, Hooper L, McGinlay P, Seif MW. (2003) Effect(s) of assisted hatching on assisted conception (IVF & ICSI). Cochrane Database Syst Rev 4,CD001894.
Fong CY, Bongso A, Ng SC, Anandakumar C, Trounson A, Ratnam S. (1997) Ongoing normal pregnancy after transfer of zona-free blastocysts: implications for embryo transfer in the human. Hum Reprod 12:557–560.
Fong CY, Bongso A, Ng SC, Kumar J, Trounson A, Ratnam S. (1998) Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation. Hum Reprod 13:2926–2932.
Fong CY, Bongso A, Sathananthan H, Ho J, Ng SC. (2001) Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties. Hum Reprod 16:540–546.
Gabrielsen A, Agerholm I, Toft B, Hald F, Petersen K, Aagaard J, Feldinger B, Lindenberg S, Fedder J. (2004) Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study. Hum Reprod 19:2258–2262.
Gordon JW and Dapunt U. (1993) A new mouse model for embryos with a hatching deficiency and its use to elucidate the mechanism of blastocyst hatching. Fertil Steril 59:1296–1301.[Web of Science][Medline]
Guerif F, Bidault R, Cadoret V, Couet ML, Lansac J, Royere D. (2002) Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal. Hum Reprod 17:1321–1326.
Hu Y, Maxson WS, Hoffman DI, Ory SJ, Eager S. (1999) A comparison of post-thaw results between cryopreserved embryos derived from intracytoplasmic sperm injection and those from conventional IVF. Fertil Steril 72:1045–1048.[CrossRef][Web of Science][Medline]
Khalifa EA, Tucker MJ, Hunt P. (1992) Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse. Hum Reprod 7:532–536.
Kowalik A, Palermo GD, Barmat L, Veeck L, Rimarachin J, Rosenwaks Z. (1998) Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization. Hum Reprod 10:2848–2851.
Lassalle B, Testart J, Renard JP. (1985) Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 44:645–651.[Web of Science][Medline]
Lee DR, Lee JE, Yoon HS, Lee HJ, Kim MK, Roh SI. (1997) The supplementation of culture medium with protease improves the hatching rate of mouse embryos. Hum Reprod 12:2493–2498.
Malter HE and Cohen J. (1989) Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 24:67–80.[CrossRef][Web of Science][Medline]
Manna C, Rienzi L, Greco E, Sbracia M, Rahman A, Poverini R, Siracusa G, De Felici M. (2001) Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques. Zygote 9:201–210.[CrossRef][Web of Science][Medline]
Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. (2001) A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 16:2182–2186.
Modlinski JA. (1970) The role of the zona pellucida in the development of mouse eggs in vivo. J Embryol Exp Morphol 23:539–547.[Web of Science][Medline]
Ng EH, Naveed F, Lau EY, Yeung WS, Chan CC, Tang OS, Ho PC. (2005) A randomized double-blind controlled study of the efficacy of laser-assisted hatching on implantation and pregnancy rates of frozen-thawed embryo transfer at the cleavage stage. Hum Reprod 20:979–985.
Nichols J and Gardner RL. (1989) Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse. Hum Reprod 4:180–187.
Pelletier C, Keefe DL, Trimarchi JR. (2004) Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos. Fertil Steril 81:Suppl. 1, 850–856.
Perona RM and Wassarman PM. (1986) Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm. Dev Biol 114:42–52.[CrossRef][Web of Science][Medline]
Primi MP, Senn A, Montag M, Van der Ven H, Mandelbaum J, Veiga A, Barri P, Germond M. (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. Hum Reprod 19:2325–2333.
Sallam HN, Sadek SS, Agameya AF. (2003) Assisted hatching – a meta-analysis of randomized controlled trials. J Assist Reprod Genet 20:332–242.[CrossRef][Web of Science][Medline]
Sawada H, Yamazaki K, Hoshi M. (1990) Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties. J Exp Zool 254:83–87.[CrossRef][Web of Science][Medline]
Schopper B, Ludwig M, Edenfeld J, Al-Hasani S, Diedrich K. (1999) Possible applications of lasers in assisted reproductive technologies. Hum Reprod 14:Suppl. 1, 186–193.
Suzuki H, Togashi M, Adachi J, Toyoda Y. (1995) Developmental ability of zona-free mouse embryos is influenced by cell association at the 4-cell stage. Biol Reprod 53:78–83.[Abstract]
Tucker MJ, Cohen J, Massey JB, Mayer MP, Wiker SR, Wright G. (1991) Partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol 165:341–345.[Web of Science][Medline]
Tucker MJ, Luecke NM, Wiker SR, Wright G. (1993) Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate. J Assist Reprod Genet 10:187–191.[CrossRef][Web of Science][Medline]
Wassarman PM. (1992) Mouse gamete adhesion molecules. Biol Reprod 46:186–191.[Abstract]
Submitted on July 28, 2005; resubmitted on November 22, 2005; resubmitted on April 3, 2006; accepted on April 7, 2006.
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