History and prospects of intracytoplasmic sperm injection (ICSI) and the development of golden hamster ICSI embryos.

BACKGROUND: Golden (Syrian) hamsters have many advantages for the study of reproductive biology and developmental biology, including a consistent estrous cycle, a stable superovulation response, and a short gestation period. However, there are serious difficulties in doing in vitro manipulations of hamster embryos, because they are very sensitive to various environmental factors. Thus, biotechnological researches of hamster embryos should be performed with high-level skills of embryo manipulations. METHODS: The authors summarized the history of hamster intracytoplasmic sperm injection (ICSI) and introduced key points for hamster ICSI, which were found in our previous studies on the production of embryos by ICSI and offspring by embryo transfer. MAIN FINDINGS: The key points for hamster ICSI were in vitro manipulations under the light-controlled environment, injection of acrosome-less sperm heads into oocytes as soon as possible before spontaneous oocyte activation occurs, and determination of the optimal culture conditions. CONCLUSION: To our knowledge, there are no available reports on production of offspring from ICSI embryos in hamsters except our reports. Moreover, success rates of hamster ICSI remain very low. For the purpose of spreading hamster ICSI, it is necessary to make further researches to improve manipulation techniques and to resolve experimental problems.

Improved developmental potential of mouse vitrified-warmed oocytes achieved by culturing in recovery medium with glutathione ethyl ester (GSH-OEt).

PURPOSE: The aim of the present study was to investigate the effect of glutathione ethyl ester (GSH-OEt) in the recovery medium on the developmental competence of mouse vitrified-warmed MII oocytes. METHODS: Vitrified-warmed oocytes were incubated for 1 h in recovery medium in the presence or absence of 0.5 mM GSH-OEt. The authors examined the effects of GSH-OEt, first on the levels of glutathione (GSH) and reactive oxygen species (ROS) in vitrified-warmed oocytes, and second, on in vitro blastocyst development, division speed to blastocysts, and total cell numbers of blastocysts from vitrified-warmed oocytes fertilized by Intracytoplasmic sperm injection (ICSI). RESULTS: Adding GSH-OEt to the recovery medium significantly (p < 0.05) increased GSH content and decreased ROS levels in vitrified-warmed oocytes. The blastocyst rate did not differ significantly between the two groups, but the speed of development to blastocysts in the GSH-OEt (+) group was significantly more rapid. In addition, the total blastocyst cell number was significantly higher in the GSH-OEt (+) group than in the GSH-OEt (-) group (92.8 ± 5.1 vs. 71.4 ± 3.5, p < 0.01). CONCLUSION: Adding GSH-OEt to the recovery medium of vitrified-warmed mouse oocytes enhances the development potential of oocytes and improves the quality of blastocysts.

Time-lapse monitoring of mouse embryos produced by injecting sonicated, frozen-thawed sperm heads with high or low chromosomal integrity.

PURPOSE: To investigate the first-division kinetics and in vitro development of embryos produced by injecting sonicated sperm heads with high or low chromosomal integrity into oocytes. METHODS: Mouse spermatozoa were frozen after separating the sperm heads from the tails by sonication in an EGTA solution (EGTA group) or M2 medium (M2 group). The chromosomal integrity of sonicated mouse spermatozoa was analyzed by injecting the sperm heads into fresh mouse oocytes. The developmental potential of spermatozoa was examined by injecting the sperm heads into vitrified-warming mouse oocytes. We used a time-lapse monitoring system to compare the first-division kinetics. RESULTS: Chromosomal integrity was preserved significantly more frequently in the EGTA group (90.6%) than in the M2 group (32.7%). Blastocysts developed significantly more often in the EGTA group (80.8%) than in the M2 group (39.6%). In the M2 group, with frequent chromosome aberrations, the time between the sperm injection and first cleavage was delayed (18.4 hours), compared to the EGTA group (16.5 hours). All results of the EGTA group were similar to that of fresh epididymal spermatozoa. CONCLUSION: The EGTA solution for sonication maintained the integrity of sperm chromosomes. Our results revealed a relationship between sperm chromosome integrity and first-division kinetics.

Selection of high-quality and viable blastocysts based on timing of morula compaction and blastocyst formation.

PURPOSE: The time-lapse system is a device that allows continuous monitoring without removing embryos from the incubator. Using a time-lapse system, we retrospectively investigated cleavage speed time points as potential indicators for selecting high-quality viable blastocysts. METHODS: This study included 963 zygotes of two pronuclei retrieved from 196 patients between January 2015 and December 2016. All embryos in culture were monitored by time-lapse after intracytoplasmic sperm injection. Of 492 blastocysts developed in vitro, 128 vitrified-warmed single blastocyst transfers were classified into pregnancy and non-pregnancy groups, and the parameters were compared. RESULTS: In the pregnancy group, timing of both morula compaction and regular blastocyst formation was significantly faster than in the non-pregnancy group. Furthermore, the optimal cutoff values for compacted morula (94.9 hours) and regular blastocyst (113.9 hours) were determined using the receiver operator characteristic curve analysis. Embryos that formed compacted morulae within 94.9 hours and developed into regular blastocysts within 113.9 hours were associated with a significantly higher pregnancy rate than those that did not (44.4% vs 16.0%). CONCLUSION: The timing of morula compaction and regular blastocyst formation is important as an indicator of high-quality blastocysts to increase odds for pregnancy after embryo transfer.

Development of golden hamster embryos effectively produced by injection of sperm heads sonicated in Tris-HCl buffer with EGTA.

PURPOSE: To investigate the effects of sperm treatment medium-TCM199 or EGTA in Tris-HCl buffer (TBS + EGTA)-for sonication of frozen-thawed hamster spermatozoa in terms of sperm chromosome integrity and development of hamster oocytes injected with the sperm heads (ICSI). METHODS: Frozen-thawed hamster spermatozoa were separated into heads and tails by sonication in TCM199 or TBS + EGTA. Sperm heads were injected into mouse oocytes to assess hamster sperm chromosomes. We further compared the development of hamster ICSI embryos produced by injecting sonicated sperm heads in TCM199 vs TBS + EGTA. RESULTS: Sperm chromosome integrity was greater following sonication of frozen-thawed hamster spermatozoa in TBS + EGTA than in TCM199 (89.7% vs 69.0%). Embryonic development was improved following hamster oocyte injection with sperm heads sonicated in TBS + EGTA compared to in TCM199 (8-cell: 84.1% vs 65.4%; morula: 78.4% vs 43.2%; blastocyst: 42.0% vs 17.3%). Gene expression of zygotic genome activation in 2-cell embryos was significantly higher with TBS + EGTA than with TCM199. We transferred 43 morulae/blastocysts from the TBS + EGTA group to foster mothers, and 4 (9.3%) developed into live offspring. CONCLUSION: These results showed that the rapid injection of hamster sperm heads separated by sonication in TBS + EGTA effectively produced more ICSI embryos during a short time.

Meiotic spindle size is a strong indicator of human oocyte quality.

PURPOSE: To investigate the relationship between the meiotic spindle size in human metaphase II oocytes and embryo developmental potential after intracytoplasmic sperm injection (ICSI). METHODS: Analyzed were 1302 oocytes with a visible meiotic spindle from 281 patients aged under 40 years undergoing ICSI cycles. The meiotic spindle was imaged by using PolScope before ICSI. The oocytes were classified into three groups, according to spindle size: group A (<90 µm2), group B (90-120 µm2), and group C (>120 µm2). RESULTS: Overall, 389 (29.9%) oocytes were classified into group A, 662 (50.8%) into group B, and 251 (19.3%) into group C. The fertilization rate of the group B oocytes was significantly higher than for the A and C oocytes. The blastocyst formation rate in group B was significantly higher than in group A. In addition, the pregnancy rate in group B was significantly higher than in the other two groups. CONCLUSION: The oocytes with a spindle size of 90-120 µm2 showed higher fertilization, blastocyst formation, and clinical pregnancy rates than those with larger or smaller spindles. The measurement of the meiotic spindle size thus has a positive predictive value for identifying human embryo developmental potential clinically.

Variations in bovine embryo production between individual donors for OPU-IVF are closely related to glutathione concentrations in oocytes during in vitro maturation.

Variations in embryo production between individual oocyte donors represent a serious problem in cattle production, when implementing ovum pick-up (OPU) and in vitro maturation (IVM) of oocytes. However, the precise cause of this problem is unknown. Here, we aimed to investigate the relationship between the glutathione (GSH) concentration in IVM oocytes and embryo development to explore a potential cause of the variations between individual donors. First, we found a high positive correlation (r = 0.80) between the GSH concentration in IVM oocytes and the blastocyst development rate of oocytes collected in the same OPU session for each donor (N = 11). Second, we selected two donors with significantly different blastocyst development rates. In samples from these donors, we examined the dynamics of oocytes GSH concentrations, and the gene expression of the glutathione synthetase (GSS) and glutathione peroxidase (GPX) genes in cumulus-oocyte complexes (COCs) during IVM. At 0 and 24 h after IVM, oocytes from the donor with the highest blastocyst development rate (high donor) exhibited significantly higher oocyte GSH concentrations than oocytes from the donor with the lowest blastocyst development rate (low donor, P < 0.05). At 4 and 9 h after IVM, GSH concentrations gradually decreased in oocytes from both donors. The GSS expression levels at 0, 4, and 9 h after IVM were significantly higher in COCs from the high donor than in COCs from the low donor (P < 0.05). The expression levels of GPX, a marker of oxidative stress, at 4 and 9 h after IVM were significantly higher in COCs from the low donor than in COCs from the high donor. Finally, adding cysteine into the IVM medium of oocytes collected in the same OPU session from the low donor significantly increased oocytes GSH concentrations and blastocyst development rates (P < 0.05). In conclusion, we showed that oocyte GSH concentrations were related to the differences in embryo development between individual donors. Our results suggested that increasing the GSH concentration in IVM oocytes could reduce the individual differences in embryo production between donors.

Estradiol benzoate treatment before ovum pick-up increases the number of good quality oocytes retrieved and improves the production of transferable embryos in Japanese Black cattle.

The aim of this study was to evaluate the efficacy of treatment with estradiol benzoate (EB) at luteal phase prior to the ovum pick-up (OPU) during in vitro production of transferable embryos in Japanese Black cattle. A total of 15 cows were used as oocyte donors for OPU. Of those, four donors were randomly allocated (three times) into each of two treatment groups as a crossover study, and OPU session was carried out six times per one donor. Another eleven donors were used in a paired difference test by one crossover trial. Donors in the control group received no hormonal treatment; whereas, donors in the EB group received 1 mg of EB as a single injection. First, we observed dynamics of ovarian follicles and emergence of follicular wave after EB injection using transrectal ultrasonography. The number and proportion of medium-sized follicles with 4 to 6 mm in diameter increased gradually and achieved a peak at 72 and 96 hours after EB injection. The OPU was performed 88 hours after EB injection. The EB-treated donors had a higher proportion of follicles with 4 to 6 mm in diameters at the time of OPU. The stimulation with EB significantly increased the numbers of follicles aspirated, and the good quality cumulus-oocyte complexes per OPU. Furthermore, in the EB group, the percentage of transferable blastocysts was significantly greater than that in the control group (P<0.05). In conclusion, a single EB injection before OPU increases the number of medium-sized follicles and can produce more transferable embryos.

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro.

Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter.

Fucci technology makes possible the distinction between live cells in the G(1) and S/G(2)/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G(1) and S/G(2)/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G(1) and S/G(2)/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

Androstenedione induces abnormalities in morphology and function of developing oocytes, which impairs oocyte meiotic competence.

OBJECTIVE: To obtain insight into the effects of androstenedione on ovarian folliculogenesis and oogenesis. DESIGN: Experimental study. SETTING: St. Marianna University School of Medicine. ANIMAL(S): Prepubertal (14-day-old) BDF1 female mice. INTERVENTION(S): Early secondary follicles were isolated from the ovaries and were cultured individually in vitro with or without androstenedione (10(-11) to 10(-5) M) for 12 days. Thereafter, the follicles were treated with hCG and epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Diameters and morphology of follicles and oocytes; E(2) and P secretion; and chromatin configuration and expression of growth differentiation factor 9 (GDF9) in oocytes were examined. RESULT(S): Early secondary follicles developed to the preovulatory stage. Androstenedione treatments increased the follicle diameters, reduced survival rates of follicles, and promoted the formation of follicles with abnormal morphology, including misshapen oocyte. The secretion of E(2) and P was significantly higher in androstenedione-exposed follicles. Androstenedione prevented the alteration in chromatin configuration and reduced oocyte GDF9 expression. When follicles cultured with androstenedione were treated with hCG and EGF, the first polar body exclusion, chromosome alignment on metaphase plate, and spindle assembly were inhibited in the oocytes. CONCLUSION(S): These results demonstrate that excess androgen induces abnormalities in the morphology and function of developing oocytes, which impairs oocyte meiotic competence.

Relationship between meiotic spindle characteristics in human oocytes and the timing of the first zygotic cleavage after intracytoplasmic sperm injection.

PURPOSE: To investigate the relationship between meiotic spindle characteristics in human oocytes and the timing of the first zygotic cleavage after intracytoplasmic sperm injection (ICSI). METHODS: Zygotes that had cleaved to two-cell stage by 27 h post-ICSI were classified as early cleaving and the remainder as late cleaving. Meiotic spindle parameters previously imaged using the PolScope were compared between the two groups. RESULTS: Of 384 embryos, 163 were classed as early cleaving and 221 as late cleaving. The rate of blastocyst formation or pregnancy by Day 2 embryo transfer was significantly higher following early cleavage than after late cleavage (52.4% vs. 24.4% or 32.6% vs. 11.4%). Spindle areas (108.0 vs. 89.8 µm(2)), lengths (14.7 vs. 13.4 µm) and PolScope retardance were also significantly greater in the early cleaving group. CONCLUSIONS: Meiotic spindle parameters determine the timing of the first zygotic cleavage and are strong indicators of human embryo developmental potential.

Effect of preantral mouse follicle culture period on meiotic maturation and developmental competence of oocytes.

Purpose: The aim of this study is to determine the optimal culture period for meiotic maturation and developmental competence of in vitro-grown mouse oocytes. Methods: Early preantral follicles with diameter of 100-130 µm were collected mechanically from day 14 mouse ovaries and cultured for 8, 10, and 12 days. The diameters of follicles and oocytes were measured, and chromatin configuration in oocytes was observed. We also examined meiotic maturation by human chorionic gonadotropin (hCG)/epidermal growth factor (EGF) stimulation, developmental competence of fertilized oocytes to blastocysts, and apoptosis in blastocysts. Results: The follicular diameter increased significantly from days 4 to 10, and the diameter of day 12 oocytes was significantly larger than day 8 or earlier oocytes. Chromatin configuration around the nucleolus was transformed from "nonsurrounded (immature)" to "surrounded (mature)" after 10 days. Furthermore, MII rate of day 10 and 12 oocytes was significantly higher than that of day 8 oocytes. The blastocyst rate of day 10 oocytes was higher than that of day 8 or 12 oocytes. The blastocyst apoptotic rate of day 12 oocytes was higher than that of day 10 oocytes. Conclusions: Long culture periods of in vitro-grown oocytes affect meiotic maturation, developmental competence to blastocysts, and apoptosis.

A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation.

OBJECTIVE: To present the effectiveness of intracytoplasmic sperm injection (ICSI) using globozoospermic sperm and assisted oocyte activation by electrical stimulation. DESIGN: A case report. SETTING: A private IVF center in Japan. PATIENT(S): A man with globozoospermia. INTERVENTION(S): Acridine orange (AO) test, mouse oocyte activation test, and ICSI with electrical oocyte activation. MAIN OUTCOME MEASURE(S): Fertilization, pregnancy, and live birth. RESULT(S): In the first ICSI attempt, neither of the two injected oocytes fertilized. Staining of the patient's sperm with AO showed that only 2.9% of the sperm emitted a green fluorescence at the characteristic round head (sperm with native DNA content). The mouse oocyte activation test using the roundheaded sperm showed that the normal fertilization rate was 78.9% when SrCl(2) was used for assisted oocyte activation; however, it was 6.0% without assisted oocyte activation. We confirmed that the sperm had defective ability to activate oocytes. In the second ICSI attempt, human oocytes were activated electrically with use of a single square direct current pulse after microinjection. All the seven injected oocytes fertilized normally, and two eight-cell embryos were transferred on day 3. Clinical pregnancy was confirmed, and a healthy girl weighing 2362 g was delivered at 37 weeks of gestation by cesarean section. CONCLUSION(S): This is the first successful outcome of ICSI using globozoospermic sperm and electrical oocyte activation. The electroactivation obviates the need for the use of potentially harmful drugs for activation.

Impact of the size of zona pellucida thinning area on vitrified-warmed cleavage-stage embryo transfers: a prospective, randomized study.

PURPOSE: The aim of this study was to determine if the size of zona pellucida thinning area by laser assisted hatching could affect the rates of pregnancy and implantation for vitrified-warmed embryo transfers at the cleavage-stage. METHODS: A total of 120 vitrified-warmed cleavage-stage embryo transfers were randomly assigned to either quarter or half of zona pellucida thinning group. RESULTS: The rates of clinical pregnancy (46.7 versus 25.0%) and implantation (32.0 versus 16.2%) were significantly greater in the half thinning group than in the quarter thinning group (P = 0.0218 and P = 0.0090, respectively). CONCLUSIONS: The results of this investigation show that, in vitrified-warmed embryo transfers at the cleavage-stage, the size of zona pellucida thinning area by laser assisted hatching impacts the rate of clinical pregnancy and implantation and that half of zona pellucida thinning significantly increases both of these results compared with quarter of zona pellucida thinning.

Case report: successful delivery following the transfer of a human re-vitrified day-7 spontaneously hatched blastocyst developed from vitrified cleaved embryos.

PURPOSE: To report a successful delivery after the transfer of a re-cryopreserved day-7 hatched blastocyst. METHODS AND RESULTS: A 30-year-old woman underwent a long-treatment protocol for ovarian stimulation. Fourteen mature oocytes were obtained, and twelve were fertilized. On day 3, two cleaved embryos were transferred, but no implantation occurred. The remaining embryos were vitrified. Subsequently, two vitrified day-3 embryos were transferred. The woman became pregnant and delivered a healthy baby. Subsequently, two vitrified day-3 embryos were transferred, but no pregnancy occurred. Subsequently, all the remaining vitrified day-3 embryos were cultured. On day 5, no blastocyst was obtained. The remaining embryos were continued to be cultured. On day 7, a hatched blastocyst was obtained and re-vitrified. Subsequently, the re-vitrified day-7 hatched blastocyst was transferred. The woman became pregnant and delivered a healthy female. CONCLUSIONS: The day-7 hatched blastocyst developed from vitrified embryos can be re-vitrified and have pregnancy potential after re-warming.

Perinatal outcomes following transfer of human blastocysts vitrified at day 5, 6 and 7.

Little information is available on perinatal outcome of cryopreserved day-7 blastocyst transfer. In the present report, perinatal outcomes for transfers of cryopreserved blastocysts by a vitrification method were examined with respect to the day of blastocyst expansion among on day 5, 6 or 7 before cryopreservation. We investigated 263 cycles of vitrified-warmed blastocyst stage embryo transfer performed between April 2005 and April 2009, which were reviewed retrospectively. There were 144 cycles with day-5 blastocyst, 100 cycles with day-6 blastocyst, and 19 cycles with day-7 blastocyst transfers. There were no differences among the vitrified day-5, day-6 and day-7 blastocyst transfer groups regarding mean number of embryos transferred, pregnancy rate, implantation rate and miscarriage rate. At this time, 71 deliveries have occurred with no reported abnormalities. There were 47 infants from 41 deliveries with day-5 blastocyst, 26 infants from 23 deliveries with day-6 blastocyst, and 8 infants from 7 deliveries with day-7 blastocyst. There were no differences among the three groups in the mean gestational age, preterm delivery rate and mean birth weight. These results indicated that blastocysts have similar inherent viability regardless of whether they develop by day 5, 6 or 7.

Vitrified human day-7 blastocyst transfer: 11 cases.

This case report describes successful pregnancies after vitrification of human day-7 blastocysts. A total of 16 day-7 blastocysts were vitrified and warmed. All 16 blastocysts survived after warming and were transferred to 11 patients. Six of the women (55%) became clinically pregnant and the implantation rate was 44% (7/16). Among these women, one woman delivered a healthy baby, two pregnancies ended in miscarriage, and three pregnancies are ongoing at 10, 29 and 34 weeks of gestation. This is the first report of successful pregnancies after vitrification of human day-7 blastocysts.

Case report: two successful pregnancies following the transfer of re-vitrified human day 7 blastocysts developed from vitrified cleaved embryos.

PURPOSE: To report successful pregnancies after the transfer of re-vitrified human day 7 blastocysts developed from vitrified cleaved embryos. METHODS AND RESULTS: A total of five day 7 blastocysts developed from vitrified cleaved embryos were re-vitrified and re-warmed. All of five re-vitrified day 7 blastocysts (100%) survived after warming and were transferred to three patients. Two of the women became clinically pregnant. Of these women, one woman delivered a healthy baby and the other pregnancy is ongoing at 26 weeks of gestation. CONCLUSIONS: This is the first report of successful pregnancies after the transfer of re-vitrified human day 7 blastocysts developed from vitrified cleaved embryos.

Effect of the size of zona pellucida opening by laser assisted hatching on clinical outcome of frozen cleaved embryos that were cultured to blastocyst after thawing in women with multiple implantation failures of embryo transfer: a retrospective study.

PURPOSE: To evaluate the effect of the size of zona pellucida opening by laser assisted hatching for frozen cleaved embryo that were thawed after both fresh and frozen cleaved embryo transfer cycles failed and were cultured to blastocyst after thawing in patients with multiple implantation failures. MATERIALS AND METHODS: Of 101 consecutive procedures (October 2003 to June 2006), 30 patients declined to perform assisted hatching and were selected as control group, 40 patients had 40 microm opening of the zona (October 2003 to January 2005), 31 patients had 50% of the zona opening (February 2005 to June 2006). RESULTS: The pregnancy, implantation and delivery rates were significantly higher in 50% opening group (74%, 52% and 65%) compared to control group (17%, 10% and 13%; P < 0.01) and 40 microm opening group (43%, 27% and 38%; P < 0.04). CONCLUSIONS: The size of the zona pellucida opening may affect the outcome of frozen cleaved embryo transfer.