A comparison between quarter, partial and total laser assisted hatching in selected infertility patients

BACKGROUND: The object of this study was to evaluate the efficacy of laser assisted hatching (LAH) of embryos on implantation and pregnancy rates of a selected group of infertility patients. METHODS: A total of 322 cycles using LAH was undertaken in our Centre between June 1998 and September 1999. Patients were offered LAH if they fell in either one or more of the following categories: (i) Patients over 37 years of age undergoing either IVF or intracytoplasmic sperm injection (ICSI) treatment cycles; (ii) patients with more than 2 previous treatment cycle failures; (iii) patients undergoing frozen embryo replacement cycles and (iv) women who were considered to be poor responders. The initial results of totally breaching the zona pellucida (total LAH; group 1) did not meet with our expectations. We subsequently modified the technique to thinning one area of the zona pellucida (partial LAH; group 2) and this thinned area was then extended to a quarter segment (quarter LAH; group 3). RESULTS: In group 1, the pregnancy rate was 14.6% with a clinical pregnancy rate of 5.2%. In group 2 the pregnancy rate was 20.9% with a clinical pregnancy rate of 18% and for patients in group 3 the pregnancy rate was 29.0% with a clinical pregnancy rate of 22.1%. CONCLUSIONS: Overall there was firm statistical evidence that the pregnancy and clinical pregnancy rates arising from quarter LAH were higher in comparison with partial and total LAH.     

Impact of assisted hatching on ART outcome in women with endometriosis

BACKGROUND: Assisted hatching can improve the implantation rate in cycles with poor outcome. The impact of assisted hatching in embryos from women with endometriosis is not known. Therefore, the hypothesis that the implantation potential of embryos obtained from women with endometriosis can be improved with assisted hatching was tested. METHODS: In a prospective randomized study, transfer embryos obtained from 60 women with endometriosis were hatched using a laser system and compared to embryos obtained from patients with the same diagnosis which were left intact (n = 30). RESULTS: The characteristics of cycles were similar between groups. The pregnancy (40% zona intact, 28.3% assisted hatching), and implantation rates (19.4% zona intact, 17.8% assisted hatching) did not differ in endometriosis cycles regardless of assisted hatching. CONCLUSION: Assisted hatching does not improve outcome in women with endometriosis undergoing assisted reproduction.     

Infertility: Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in-vitro fertilization patients

A study was conducted on patients who had attempted and failedprevious in-vitro fertilization (IVF) procedures an averageof 3.8 times following the application of assisted hatchingwith conventional culture systems. The aim of this investigationwas to determine if addition of co-culture methodologies couldreduce embryonic abnormalities and thus improve the prognosisfor pregnancy. The study population consisted of 123 patients,subdivided into three patient categories. Previous IVF resultsfrom conventional culture were used to evaluate any potentialbenefits derived from the present co-culture application. Followingco-culture, the rate of blastomere development was increasedand the rate of fragmentation decreased. An increased rate ofblastomere development was most noticeable in the patients aged39 years with no male factor as well as the intracytoplasmicsperm injection (ICSI) subgroup. Similarly, the rate of fragmentationwas significantly reduced in the aforementioned subgroups. Themost pronounced impact of co-culture was on pregnancy and implantationrates. The overall clinical and ongoing pregnancy rates were38% (47/123) and 36% (44/123) respectively. The correspondingimplantation rate was 17% (72/412) as shown by embryonic cardiacactivity. The ongoing pregnancy rates in the 39 years no malefactor, 40 years no male factor and ICSI no age limit patientsubgroups were 41% (21/51), 30% (8/27) and 33% (15/45) respectively.The results indicate that addition of co-culture to the IVFprocedure for poor-prognosis patients may be advisable.     

The impact of assisted hatching on live birth rates and outcomes of assisted conception: a systematic review

BACKGROUND: During the past decade in the UK, only one in six cycles of assisted conception has resulted successfully in a live birth. Assisted hatching (AH) has been proposed to improve outcome. This systematic review of randomized controlled trials addresses primary outcomes of live birth, clinical pregnancy and embryo implantation. METHODS: Trials on post-fertilization disruption of the zona pellucida were identified from the Cochrane Controlled Trials Register, MEDLINE, EMBASE and published bibliographies. Outcomes were analysed using random effects meta-analysis, sensitivity analysis, sub-grouping and meta-regression. RESULTS: Of 23 included trials recruiting 2572 women, only six reported live birth data. AH had no significant effect on live birth (OR 1.21, 95% CI 0.82-1.78). There was a significant benefit of AH on clinical pregnancy (OR 1.63, 95% CI 1.27-2.09), especially in the sub-group of women with previous failure of assisted conception (OR 2.33, 95% CI 1.63-3.34). Meta-regression suggested that AH might be more useful in older women. Implantation data were not considered valid for statistical analysis. The methodological quality of included trials was sub-optimal. CONCLUSIONS: AH probably enhances clinical pregnancy, especially in women with previous failure of assisted conception treatment and in older women; however, trials were of poor quality and so may be biased. Better quality trials reporting live birth are required to confirm any positive effects on the 'take-home-baby rate'.     

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching

Immature oocyte recovery followed by in-vitro oocyte maturationand in-vitro fertilization is a promising new technology forthe treatment of human infertility. The technology is attractiveto potential oocyte donors and infertile couples because ofits reduced treatment intervention. Immature oocytes were recoveredby ultrasoundguided transvaginal follicular aspiration. Oocyteswere matured in vitro for 36–48 h followed by intracytoplasmicsperm injection (ICSI). Embryos were cultured in vitro for 3or 5 days before replacement. Assisted hatching was performedon a day 5 blastocyst stage embryo. Embryo and uterine synchronywere potentially enhanced by luteinization of the dominant follicleat the time of immature oocyte recovery. Mature oocyte and embryoproduction from immature oocyte recovery were similar to theprevious IVF results of the patients. A blastocyst stage embryo,produced as a result of in-vitro maturation, ICSI, in-vitroculture and assisted hatching, resulted in the birth of a healthybaby girl at 39 weeks of gestation.     

Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in-vitro fertilization patients

Older patients and those who consistently return for embryotransfer but without implnatation were studied to see if a combinationof day 3 assisted hatching and co-culture (AH +CC) might bebeneficial compared to assisted hatching alone (AH-alone). Femalepatients of 38 years and couples who had previously failedto implant embryos three times or more were prospectively andrandomly assigned to either an experimental or a control group.In the experimental group all embryos were co-cultured on partialmonolayers of bovine oviductal epithelial cells for 2 days followedby assisted hatching by zona drilling (AH +CC). All controlembryos were cultured by standard procedures until day 3 whenthey also underwent zona drilling prior to uterine transfer(AH -alone). With 50 cycles in each group there was unfortunatelya marginal bias against the AH + CC group in that these patientshad undergone a higher number of previous transfer cycles. Therewas a marginally lower percentage of fragmentation and a signficantlyhigher degree of zona thickness variablity in the AH + CC embryogroup. Embryonic implantation was significantly increased (P< 0.05) in the AH xCC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantlyhigher (P < 0.05) initial pregnancy rate (52 versus 32%)in the AH +CC group, and a higher (not significant) viable pregnancyrate (38 versus 22%).     

An improved mechanical technique for assisted hatching

BACKGROUND: Varied clinical outcomes of assisted hatching (AH) have been reported. We attempt to investigate whether the size of the zona opening created by AH is adequate for blastocyst hatching, and, if not, set up a new method to improve it. METHODS: A new AH technique, long zona dissection (LZD), was established, and experiments were performed to compare the effects of different sizes of zona opening on complete hatching of blastocysts in mouse and human embryos in vitro. RESULTS: The LZD technique can create a long zona slit on early embryos, even blastocysts, with the slit size beyond two-thirds of zona diameter. Compared with three-dimensional partial zona dissection, LZD can significantly enhance the hatching speed and the rate of complete hatching of mouse blastocysts (93.9%). All (100%) human blastocysts completely hatched following LZD; however, when the slit size after AH was about two-fifths of zona diameter, more of the larger inner cell masses (ICM) became trapped by the zona opening during hatching than the smaller ICM (53.3 versus 12.5%, P = 0.01). CONCLUSIONS: Zona opening of moderate size following AH is inadequate for the completion of blastocyst hatching in vitro; in some cases, however, it can be significantly improved by LZD.     

Laser zona pellucida thinning--an alternative approach to assisted hatching

BACKGROUND: The purpose of this study was to assess the efficacy and hatching characteristics of in-vitro cultured human embryos subjected to laser zona pellucida thinning. METHOD: Zona thinning was performed on 110 embryos using a non-contact 1.48 microm diode laser and the hatch rate in vitro was compared with 42 control embryos. Variation of zona thickness and degree of zona expansion was assessed. Scanning electron microscopy was performed on embryos entrapped during hatching to identify the site of hatching. RESULTS: The rate of hatching was significantly higher in laser thinned blastocysts compared with control embryos (68 versus 33% respectively, P < 0.01). Laser thinning increased the variation of zona thickness in embryos from 11.6-27.3%. Natural zona thinning occurred in 92% of laser thinned hatching blastocysts and 100% of control embryos. CONCLUSION: These results suggest that laser zona thinning is effective and may provide significant advantages over conventional assisted hatching techniques, which create holes.     

Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing

BACKGROUND: The present study was undertaken to examine the usefulness of both vitrification and assisted hatching (AH) on blastocysts that originate from embryos showing different qualities during their cleavage stage. METHODS: A total of 281 blastocysts were vitrified (93 vitrification-warming cycles) in a mixture of ethylene glycol-dimethylsulphoxide-Ficoll and sucrose using the Hemi-Straw (HS) carrier system. After warming, AH using the partial dissection technique was performed in 36 cycles. RESULTS: After warming and culture for 24 h, a total of 168 blastocysts (60%) was suitable for embryo transfers and a total of 25 ongoing pregnancies (27%) was obtained. Forty-nine transfers of 96 no-AH blastocysts and 36 transfers of 72 AH blastocysts resulted in an implantation rate of 13 and 22% respectively (P < 0.05). The percentage of transfers with at least one hatching blastocyst was significantly higher after application of AH (69 versus 33%) (P < 0.001). In all, 73 and 38% of blastocysts showing respectively optimal and non-optimal embryo development during the early stage were available for transfer (P < 0.001). Consequently, implantation rates of 19 and 6% were obtained after transfers of blastocysts showing respectively optimal and poor embryo development. CONCLUSIONS: Artificial opening of the zona pellucida after warming of vitrified blastocysts significantly improved the rate of transfers with hatched blastocysts and the implantation and pregnancy rates. The percentage of blastocysts that survived the HS vitrification procedure and were available for embryo transfer is related to their previous developmental quality.     

Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study

BACKGROUND: Focus on the hatching process has so far been in the field of fresh embryos. Cryopreserved-thawed embryos have a lower rate of pregnancy than fresh embryos, which might be due to hardening of the zona pellucida. METHODS: During a 2 year period, a prospective randomized study enrolling 253 cryopreserved-thawed cycles was performed on day 2 embryos. Pseudorandomization to assisted hatching or a control group was done on the basis of even and odd dates for thawing. One hour before embryo transfer, hatching was carried out using acidic Tyrode's solution. RESULTS: Among 136 embryos exposed to assisted hatching, 11.4% (30) were implanted compared with only 5.8% (13) of 117 embryos not exposed to assisted hatching (P<0.05, chi(2) test). No difference in the rate of clinical pregnancy and positive serum HCG was observed between the two groups. Very few women >38 years old were included in the study, and no significant difference according to age could be found between the groups. CONCLUSIONS: These results show that assisted hatching using acidic Tyrode's solution increases the implantation rate of cryopreserved-thawed embryos (P<0.05).     

Simplifying assisted conception techniques to make them universally available--a view from India.

In-vitro fertilization and related assisted conception techniques are beyond the reach of most infertile couples in India because they are so expensive. Recent innovations such as the use of the natural cycle, the techniques of gamete intra-Fallopian transfer, intravaginal culture and transcervical oocyte--sperm transfer, vaginal ultrasound-guided oocyte retrieval and the availability of ready-to-use culture medium have helped to simplify assisted conception. Research today should focus on further developing these techniques so that better pregnancy rates can be achieved with them. These simplified inexpensive assisted conception techniques can then be adapted for conditions in the developing world, so that infertile patients the world over can benefit from them.     

Fertilization and early embryology: Use of lasers in assisted fertilization and hatching

The erbium-yttrium-aluminium-garnet (Er: YAG) laser has beenapplied to micromanipulation in humans. It was used in the fertilizationprocess for both subzonal insemination (SUZI) and for partialzona dissection (PZD). Laser-assisted micromanipulation achievedsignificantly higher fertilization rates (34.8%) when comparedto mechanical SUZI (16.1%), but use of the laser did not improvethe PZD results (laser 14.8% versus mechanical 14%). The Er:YAG laser was used to assist hatching. In the mouse it significantlyimproved the hatching rate (80 versus 29.3%) 110 h after administrationof human chorionic gonadotrophin. This technique was appliedin two different centres to patients with previous in-vitrofertilization (IVF) failures. The implantation rate per embryo(14.4% laser-assisted hatching versus 6% control group) andthe pregnancy rate per transfer (40 versus 16.2%) were improved.     

Zona thinning with the use of laser: a new approach to assisted hatching in humans

From 1993 to 1994, in our centre, laser-assisted hatching wasperformed on 2- to 4-cell stage embryos obtained from in-vitrofertilization (TVF) patients. We treated 376 embryos from 96patients with repeated IVF failures (two to four attempts) (groupA) and 397 embryos from 111 patients undergoing IVF for thefirst time (group B). Embryos were transferred immediately afterthe laser treatment. Both groups were compared to control groups(A' and B') whose embryos were transferred with intact zonapellucida (ZP). The resulting clinical pregnancies were 41 inA and 44 in B versus 24 hi A' and 23 in B' respectively. Thepregnancy rates per patient were 42.7 and 39.6% versus 23.1and 19% in the control groups (P < 0.05), while the implantationrates per embryo were 12.2% in A and 11.8% in B versus 13 and7.1%. These results show that laser zona thinning of human embryosat 48 h after egg retrieval significantly increases the implantationand pregnancy rate (P < 0.05).     

Possibilities and limitations of techniques of assisted reproduction for the treatment of male infertility

Since relatively few spermatozoa are needed for oocyte fertilization during gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization (IVF), these methods have been applied in couples with infertility due to male causes. Forty-six couples with male factor infertility were enrolled in this study and results were compared with those attained in 48 couples treated with the same techniques for other than male causes. Overall, GIFT resulted in 26% ongoing pregnancies. GIFT seems to be particularly successful when the sperm concentration is 20 x 10(6)/ml or more, but sperm motility and/or morphology are poor. Nine pregnancies occurred out of 26 GIFT cycles in 18 cases selected on this basis. The ongoing pregnancy rate after IVF was 16% per patient. The latter treatment should be attempted in male immune infertility and in cases with a low sperm concentration, with or without abnormal sperm motility and/or morphology. In these circumstances, five pregnancies were attained out of 28 cycles in 14 cases. For similar sperm concentrations, the conception rate per cycle attained with techniques of assisted reproduction was more than twice that attained with conventional treatment of male infertility.     

A formal comparison of the practice of assisted reproductive technologies between Europe and the USA

In this study, we compared pregnancy and delivery outcomes after the utilization of assisted reproductive technologies (ART) in Europe and the United States (US). ART outcomes were compared between Europe and the US for the year 2001, based on formal reports published by the European Society for Human Reproduction and Embryology (ESHRE) and the Center for Disease Control and Prevention (CDC) in collaboration with the American Society for Reproductive Medicine (ASRM) and Society for ART (SART). Europe utilizes ART at approximately twice the rate of the US (P < 0.001). United States patients showed a significantly decreased likelihood of reaching oocyte retrieval (P < 0.001) and embryo transfer (P < 0.001). Despite this lower chance of reaching oocyte retrievals and embryo transfers, US patients experienced significantly higher clinical pregnancy rates (P < 0.001) and delivery rates per started cycle (P < 0.001) than European patients. Amongst patients reaching oocyte retrieval, the difference in clinical pregnancy rates and live birth rates was even more pronounced in favour of the US. However, US patients received significantly more embryos per embryo transfer (P < 0.001) and experienced a significantly higher multiple pregnancy rate (P < 0.001). Significant differences in favour of US patients in pregnancy rates and live birth rates were also observed for frozen embryo cycles and oocyte donation cycles, where the difference was most pronounced. The better pregnancy and live birth outcomes in the US are not explainable by the transfer of larger embryo numbers alone.     

Improved hatching in mouse embryos brought about by combined partial zona dissection and co-culture

In this study 874 mouse embryos were allocated to six groupsincluding a control, co-culture, and four groups that underwentpartial zona dissection (PZD): at the 2-cell (PZD-2) and morulastages (PZD-M) both with and without co-culture. Rates of completeblastocyst hatching on day 5 increased in the following order:control, co-culture alone, PZD-2 alone, PZD-M alone, PZD-2 withco-culture and PZD-M with co-culture (P < 0.00001). PZD-Mled to significantly higher rates of complete blastocyst hatchingcompared to PZD-2 (P < 0.03). This study showed also thatco-culture apparently compensates for any minor damage incurredduring the PZD technique at the 2-cell and morula stages, (P< 0.01 and P < 0.01) respectively. Therefore PZD and co-cultureseem mutually beneficial techniques that promote early blastocysthatching in the mouse.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

Fertilization and early embryology: Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy

The aim of this study was to examine the safety and the efficiencyof a ‘non-contact’ UV laser to assist hatching throughzona opening of human embryos. Between January and November1995 we performed zona drilling for assisted hatching usinga new laser system (PALM UV Laser microbeam), operating in a‘non-contact’ mode to create a hole in the zonapellucida of human embryos. In a randomized study, laser zonaopening was applied on embryos from two groups of patients withrepeated in-vitro fertilization (IVF) failures (two to fourattempts): group A was composed of 107 patients who receivedmixed embryos (216 laser-treated and 223 not treated) and groupB of 72 patients who received 218 laser-treated embryos only.Both groups were compared with a control group of 98 patientswhose embryos were not laser treated (n = 407) (group C). Themean ages of all groups (38.1, 38.2 and 37.8 years respectively)and the number of IVF attempts (two to four attempts) were similar.The resulting clinical pregnancies were 39 (36.4%) in groupA, 32 (44.4%) in group B and 19 (19.3%) in group C. The implantationrates/embryo were 9.3% in A, 16% in B and 5.1% in the controlgroup. In total, 17 normal babies have been delivered (10 ingroup A and seven in group B). These results show that laserzona drilling increased the pregnancy and implantation ratesin all the treated patients. The increase was slight but significantin patients of group A (P < 0.01 and P < 0.02), it waseven higher in the patients of group B (P < 0.05).     

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10^‐5) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright © 2012 John Wiley & Sons, Ltd.     

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

BACKGROUND: Assisted hatching (AH) in fresh embryo transfer (ET) cycles increases the implantation and pregnancy rates, especially in women with a poor prognosis, repeated implantation failures and in older women. Little information exists in the literature regarding the role of AH in frozen-thawed embryo transfer (FET) cycles. METHODS: Embryos were cryopreserved at the cleavage stage. On the day of FET, 160 patients were randomized according to a computer-generated randomization list in sealed envelopes into the AH group and the control group. The patients and the clinicians were blinded to the group assigned. In the AH group, the outer half of the zona pellucida over a quarter of the diameter of zona was removed using a 1480 nm non-contact laser. RESULTS: The two groups were comparable in terms of demographic characteristics, ovarian response of the stimulated cycle and quality of fresh and frozen-thawed embryos. No differences in implantation, pregnancy and multiple pregnancy rates were found between the two groups. There was a non-significant trend of a higher implantation rate in the AH group when the zona thickness was > or = 16 mm. CONCLUSION: Laser AH did not improve the implantation rate of FET cycles and should not be performed routinely in all frozen-thawed embryos at the cleavage stage.