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OBJECTIVE: We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. METHODS: We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 µg/ml of crocin supplementation (Groups 2 and 4). RESULTS: Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. CONCLUSIONS: Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

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OBJECTIVE: To determine whether human hydrosalpinx fluid might have a deleterious effect on the fertilization rate and embryonic development of the exposed mouse oocytes. METHODS: Mouse cumulus-oocyte complexes (COCs) were randomly allocated for exposure to pure hydrosalpinx fluid (100% HSF group, n=400), EBSS containing 50% of hydrosalpinx fluid (50% HSF group, n=320) and pure EBSS (control group, n=300). RESULTS: The results showed that the fertilization rate in the 100% HSF group was significantly lower than the control group (64.0% versus 73.0%, p=0.031). The blastocyst formation rate was also lower in the 100% HSF group than 50% HSF and the control group (51.5% versus 56.9% versus 56.3%, respectively), but not statistically significant (p=0.275). There was no significant difference in the mean numbers of cells in the ICM, TE, and total cell number in blastocysts from the control group and two hydrosalpinx fluid exposure groups. CONCLUSIONS: Human hydrosalpinx fluid has a negative effect on the fertilization rate of the exposed mouse oocytes. However, this effect was found only in undiluted concentration and does not affect the subsequence of embryonic development and blastocyst cell number.

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OBJECTIVE: We investigated the effect of supplementing fertilization medium and/or culture medium with astaxanthin (AST) on the two phases of in vitro fertilization: gamete fertilization and embryo development. METHODS: Mouse cumulus-oocyte complexes were divided into four groups with 5 µM AST added to the fertilization medium (group 3, n=300), culture medium (group 2, n=300), or both media (group 4, n=290). No AST was added to the control group (group 1, n=300). RESULTS: The fertilization rate was significantly higher (p<0.001) in the groups using AST supplemented fertilization medium (group 3, 79.0%; group 4, 81.4%) than those without AST (group 1, 56.3%; group 2, 52.3%). The blastocyst rate calculated from the two-cell stage was significantly lower (p<0.001) in the groups using AST-supplemented embryo culture medium (group 2, 58.0%; group 4, 62.3%) than in those without AST (group 1, 82.8%; group 3, 79.8%). The blastocyst rate calculated from the number of inseminated oocytes was highest in group 3 (189/300, 63.0%) and lowest in group 2 (91/300, 30.3%) with statistical significance compared to other groups (p<0.001). There were significantly higher numbers of cells in the inner cell mass and trophectoderm, as well as significantly higher total blastocyst cell counts, in group 3 than in the control group. CONCLUSION: An increased blastocyst formation rate and high-quality blastocysts were found only in the fertilization medium that had been supplemented with AST. In contrast, AST supplementation of the embryo culture medium was found to impair embryo development.

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OBJECTIVE: To compare our in-house method of embryo freezing with Cryotop vitrification in terms of immediate survival, subsequent cleavage and blastocyst formation, and cell numbers in blastocysts. METHODS: Two-cell mouse embryos were randomly allocated into three groups: a non-frozen control group (group 1, n=300), a group that underwent Cryotop vitrification (group 2, n=300), and a group that underwent our in-house freezing method (group 3, n=300). RESULTS: There were no significant differences between groups 2 and 3 in the immediate survival rate (96.3% vs. 98.6%, respectively; p=0.085), the further cleavage rate (91.7% vs. 95.0%, respectively; p=0.099), or the blastocyst formation rate (80.7% vs. 78.6%, respectively; p=0.437). The cell numbers in the blastocysts from groups 1, 2, and 3 were comparable (88.99±10.44, 88.29±14.79, and 86.42±15.23, respectively; p=0.228). However, the percentage of good-quality blastocysts in the Cryotop vitrification group was significantly higher than in the group in which our in-house method was performed, but was lower than in the control group (58.0%, 37.0%, and 82.7%, respectively; p<0.001). CONCLUSION: At present, our method is inferior to the commercial Cryotop vitrification system. However, with further improvements, it has the potential to be useful in routine practice, as it is easier to perform than the current vitrification system.

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OBJECTIVE: We studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts. METHODS: Mouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125). RESULTS: The rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group (69±19.3, 74±15.7, and 71±16.8, respectively; p=0.680). CONCLUSION: These findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers.

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BACKGROUND: Polymorphisms at codons 307 and 680 are the most commonly encountered allelic variants of the follicle-stimulating hormone receptor (FSHR) gene. Studies in Caucasians suggest that certain FSHR variants are more common in women with polycystic ovary syndrome (PCOS) than normal women. The objective of this study was to determine the distribution of FSHR gene polymorphisms at codons 307 and 680 in Thai women with chronic anovulation, without (121 women) and with PCOS (133 women), using 132 known fertile women as controls. METHODS: DNA samples from peripheral blood lymphocytes were extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The prevalence of Threonine307Threonine (TT), Threonine307Alanine (TA), and Alanine307Alanine (AA) genotypes at codon 307 was 53.0% (95% CI = 44.2-61.7%), 42.4% (95% CI = 34-51.3%), and 4.5% (95% CI = 1.9-10.1%) in controls; 52.6% (95% CI = 43.8-61.3%), 39.8% (95% CI = 31.6-48.7%), and 7.5% (95% CI = 3.9-13.7%) in PCOS women; and 50.4% (95% CI = 42.8-61.2%), 45.4% (95% CI = 34.9-53.1%), and 4.5% (95% CI = 1.5-9.6%) in anovulatory women without PCOS, respectively. The prevalence of Asparagine680Asparagine (NN), Asparagine680Serine (NS), and Serine680Serine (SS) genotypes at codon 680 was 54.5% (95% CI = 45.7-63.2%), 40.9% (95% CI = 32.5-49.8%), and 4.5% (95% CI = 1.9-10.1%) in controls; 51.9% (95% CI = 43.1-60.6%), 44.4% (95% CI = 35.8-53.2%), and 3.8% (95% CI = 1.4-9.0%) in PCOS women; and 47.9% (95% CI = 40.4-58.8%), 47.1% (95% CI = 36.5-54.7%), and 5.0% (95% CI = 2-10.9%) in anovulatory women without PCOS, respectively. The prevalence of FSHR gene polymorphisms at both codons were not statistically different among the three groups. CONCLUSIONS: In Thai women, there was no association between the FSHR gene polymorphism at codons 307 and 680 and chronic anovulation.

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BACKGROUND: It is still debatable whether a full-thickness assisted hatching (AH) is better than the partial zona thinning. In this research, we used a mouse model to study the effect of partial and complete laser-AH on the rate of completely hatched blastocyst and their cell numbers. METHODS: In experiment 1, mouse morulae had 0, 1, 2 or 3 full-thickness openings of 10 microns created in the zona pellucida with an infrared laser beam. In the second experiment, 0, 1 and 2 openings of 20 microns were studied. In the third experiment, a full-thickness opening of 20 microns or quarter-thinning of the zonal circumference to a depth of 90% was compared with non-AH controls. RESULTS: No difference in blastocyst formation was found in laser-treated groups and in the controls. In experiment 1, the rate of completely hatched blastocysts was significantly lower than the controls. In experiment 2 when the size of the opening was increased, blastocysts completely hatched at a significantly higher rate than that in the controls. In experiment 3, the rate of completely hatched blastocysts was the highest in the full-thickness group. Cell numbers in completely hatched blastocysts from both AH groups were significantly fewer than those in the controls. CONCLUSIONS: Full-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased.

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BACKGROUND: Accidental exposure of oocyte/cumulus complex to endometriotic fluid is not uncommon during oocyte retrieval. Only two studies were available on this subject and they gave conflicting results. In this study, we used a mouse model to evaluate the effect of controlled exposure of oocytes to ovarian endometriotic fluid. METHODS: Mouse oocytes/cumulus complexes (n = 862) were divided into 4 groups, and were exposed to endometriotic fluid (group 1), pooled sera from subjects without endometrioma (group 2), phosphate-buffered saline (group 3), and fertilization medium (controls). After five minutes, oocytes were washed and inseminated. Embryo development was observed daily. The quality of hatching blastocysts was assessed by counting the number of inner cell mass (ICM) and trophectoderm (TE) cells. RESULTS: The fertilization, cleavage and blastocyst formation rates in the four groups were not statistically different. The proportions of hatching/hatched blastocysts from fertilized oocytes in groups 1 and 2 were significantly lower than those in group 3 and controls (P = 0.015). Hatching blastocysts from all groups showed no significant difference in the number of ICM and TE cells. CONCLUSIONS: Exposure of mouse oocytes/cumulus complexes to endometriotic fluid had subtle detrimental effects on subsequent blastocyst development. However, one should be cautious in projecting the results of this study to contaminated human oocytes in a clinical setting.

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In this study, we compared the effects of repeated freezing/thawing of human sperm by our in-house method of rapid freezing with slow programmable freezing. Sperm samples from 11 normozoospermic subjects were processed through density gradients and divided into three aliquots: non-frozen, rapid freezing and slow programmable freezing. Sperm in the rapid freezing group had better motility and viability than those in the slow freezing group (P<0.01) after the first, second and third cycles of freezing/thawing, but there was no difference in morphology. In the second experiment, rapid freezing was repeated three times in 20 subjects. The samples from each thawing cycle were evaluated for DNA fragmentation using the alkaline comet assay. DNA fragmentation began to increase considerably after the second cycle of freezing/thawing, but to a level that was not clinically important. In the third experiment, rapid freezing was done repeatedly in 10 subjects, until no motile sperm were observed after thawing. The median number of repeated freezing/thawing that yielded no motile sperm was seven (range: 5-8, mean: 6.8). In conclusion, we demonstrated that repeated freezing/thawing of processed semen using our rapid freezing method gave better results than standard slow programmable freezing. This method can help maximize the usage of precious cryopreserved sperm samples in assisted reproduction technology.

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AIM: To present the results of assisted reproductive technology (ART) performed in Thailand during 2001-2007. METHODS: All licensed ART centers are obliged to submit annual reports on the number of patients, cycles, ART techniques and treatment outcomes to the Reproductive Medicine Subcommittee of the Royal Thai College of Obstetricians and Gynaecologists. Data from all centers were aggregated and analyzed retrospectively. RESULTS: Cycles were categorized into fresh and frozen/thawed embryo transfer (FET) cycles. Initiated cycles in the first category for 2001 to 2007 were 2183, 2112, 2780, 2717, 3458, 3579 and 4288, respectively. FET cycles during the same period were 467, 558, 733, 768, 1136, 1210 and 1473, respectively. The average pregnancy rate for in vitro fertilization (IVF) was 28.9% per retrieval (range, 26-32.3%) or 33.8% per transfer (range, 30.7-38.6%). Multiple pregnancies (of which 89.3% were twins) from all treatment procedures during this period were 11.4% (range, 9.2-14.5%). A congenital abnormality was reported in 0.56% of live births. The number of embryos per transfer in IVF decreased from 4.1 to 2.9, with no drop in pregnancy rates. Oocyte insemination by intracytoplasmic sperm injection (ICSI) was utilized more often than standard IVF, while gamete intrafallopian transfer and zygote intrafallopian transfer were almost completely replaced by IVF/ICSI. There was a significant difference in pregnancy rates (P < 0.01) when clinics were classified by cycle volumes (<100, 100-400 and >400 cycles/year). CONCLUSIONS: Despite many limitations, the data provided in this report will help patients, clinicians and policy makers understand the current situation of ART practice in Thailand.

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OBJECTIVE: To study the effect of embryo density and microdrop volume on mouse two-cell embryo development. DESIGN: Experimental study. SETTING: Assisted conception laboratory. ANIMAL(S): Two-cell mouse embryos (n = 1511). INTERVENTION(S): One, five, 10, or 15 embryos were cultured in 10-µL drops of cleavage medium. In the second study, embryos were cultured singly in 0.5-, 1-, 2-, 5-, and 10-µL drops. Finally, they were cultured in pair in 0.5-, 1-, and 2-µL drops. After 48 hours, embryos were transferred into blastocyst medium for an additional 24 hours. MAIN OUTCOME MEASURE(S): Cleavage and blastocyst formation and inner cell mass (ICM) and trophectoderm (TE) cell numbers. RESULT(S): No differences in cleavage or blastocyst formation were found in different groups in experiment 1, 2, or 3. Embryos cultured singly had fewer ICM and TE cells than those cultured in groups. Embryos cultured singly in 0.5 µL had fewer TE cells than those in 10 µL, but had insignificant difference in the ICM. Duo culture in 0.5-2 µL appeared to give the same results as group culture in 10-µL drops. CONCLUSION(S): Group culture is preferred when using sequential media. Beneficial effects cannot be mimicked by volume reduction in single-embryo culture.

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OBJECTIVE: To determine the prevalence of metabolic syndrome and insulin resistance in Thai women with polycystic ovary syndrome (PCOS). MATERIAL AND METHOD: Oral glucose tolerance tests were performed in 70 PCOS women. Blood was taken for the measurement of fasting insulin, 2-hr postprandial insulin, lipid profiles, testosterone and sex hormone binding globulin RESULTS: The prevalence of metabolic syndrome and insulin resistance was 24.3% and 30.7%, respectively. Diabetes mellitus and impaired glucose tolerance was detected in 11.4% and 31.4%, respectively Homeostatic model assessment (HOMA) was the most sensitive screening test for insulin resistance. PCOS women with metabolic syndrome had significantly higher body mass index, free testosterone index and insulin levels than those without the syndrome. CONCLUSION: There was a high prevalence of metabolic syndrome, insulin resistance, diabetes mellitus and impaired glucose tolerance in Thai PCOS women. A combination of fasting glucose, a 2-hr glucose tolerance test and fasting insulin level effectively identified PCOS women who were at high risk of metabolic syndrome.

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Empty follicle syndrome (EFS) is a condition in which no oocytes are obtained after an apparently successful ovarian stimulation. Genuine EFS (GEFS) is differentiated from false EFS by an optimal level of human chorionic gonadotropin on the day of oocyte retrieval. Some believe that GEFS does not exist and that it is only a reflection of the margin of error attendant upon the procedure of oocyte aspiration. Others believe that GEFS is caused by dysfunctional folliculogenesis, resulting in early atresia of oocytes. In this report, we present a case of apparent GEFS, in which immature oocytes were identified after filtration of follicular aspirates. Our findings suggest that delayed maturation of oocyte cumulus complexes in response to HCG might be an etiologic mechanism in some cases of GEFS. This creates a situation similar to the aspiration of immature follicles, where germinal vesicle-stage oocytes with dense scanty cumulus cells are often difficult to identify under a dissecting microscope.

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OBJECTIVE: To compare the efficacy of rapid freezing (RF) and slow programmable freezing (SPF) of human spermatozoa. DESIGN: Experimental study. SETTING: University-based assisted conception laboratory. PATIENT(S): Semen from 30 normospermic men. INTERVENTION(S): Semen was processed through density gradients and divided into three groups: nonfrozen control, RF, and SPF. MAIN OUTCOME MEASURE(S): Sperm motility, kinematics, and morphology were assessed by a computer-assisted semen analyzer; viability by eosin-Y staining and the hypo-osmotic swelling test; DNA integrity by comet assay; and sperm binding by hemi-zona assay. RESULT(S): Post-thaw sperm motility (53.9%, 37.0%, and 75.5% for RF, SPF, and controls, respectively) and sperm vitality (hypo-osmotic swelling test 60.1%, 44.1%, and 77.9%; eosin-Y staining 64.8%, 50.4%, and 81.8% for RF, SPF, and controls, respectively) were higher in the RF than in the SPF group, but lower than in the nonfrozen control group. There was no significant difference in post-thawed normal sperm morphology (14.9%, 14.4%, and 16%) and sperm DNA integrity by comet assay (93.6%, 94.5%, and 94.2%) in the RF, SPF, and controls, respectively. The hemi-zona index was no different between the two cryopreserved groups. CONCLUSION(S): The RF gave superior post-thaw motility and cryosurvival than SPF.

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PURPOSE: To compare closed-system solid surface vitrification with slow freezing. METHODS: Mouse 2-cell embryos (n = 348) were divided into vitrification, slow freezing and non-frozen groups. For vitrification, embryos were exposed to 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 10 min, then transferred into 17.5% EG, 17.5% DMSO, 0.25 M trehalose and 10% FBS in PBS. They were placed on hemi-straws and inserted into 0.5 ml straws inside a previously cooled aluminum cylinder. Slow freezing was done in straws by the conventional method. RESULTS: Vitrified embryos had significantly higher survival, further cleavage and blastocyst formation rates than those in the slow freezing group (p < 0.001) and were comparable to controls. Blastocysts in the vitrification and control groups had significantly more cells than those in the slow freezing group (p < 0.05). CONCLUSIONS: Closed-system vitrification was more effective than conventional slow freezing.

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AIM: To compare the outcomes of slow freezing with ultra-rapid freezing (URF) of cleavage-stage human embryos on aluminum foil. METHODS: Two-cell mouse embryos were used to test our method of ultra-rapid freezing. The embryos were randomly allocated to a non-frozen control (208 embryos), and slow (204 embryos) or ultra-rapid freezing groups (204 embryos). Immediate survival rate, further cleavage and blastocyst formation were compared. After validating our ultra-rapid freezing method on mouse embryos, we applied a similar ultra-rapid freezing protocol to human embryos. Consecutive human frozen/thawed embryo transfer (FET) cycles from October 1998 to June 2005 were reviewed. The survival rate, further cleavage rate and the pregnancy outcomes were compared between the URF and slow programmable freezing. RESULTS: Mouse embryos in the URF group survived the freezing/thawing process better than those in the slow freezing group (93.1% vs 82.8%, P = 0.001). Blastocyst and hatching blastocyst formation of the surviving embryos were comparable in the URF and slow freezing group (59% vs 58.6%, P = 0.944 and 32.6% vs 42%, P = 0.066, respectively). There were 146 human FET cycles in the URF group and 28 cycles in the slow freezing group. The immediate survival of embryos was higher in the URF group than in the slow freezing group (87.9% and 64.3%, P < 0.001). There was no significant difference in the mean number of embryos per transfer (3.7 +/- 1.3 and 3.3 +/- 1.2, P = 0.178), clinical pregnancy rate per transfer (28.5% and 21.4%, P = 0.444) and implantation rate per embryo (10.98% and 10.9%, P = 0.974) in the URF or slow freezing groups. CONCLUSION: Our in-house URF method gave comparable results to slow programmable freezing. Although the risk of potential contamination is a major drawback of the present ultra-rapid freezing technique, future refinement will minimize or entirely eliminate this concern.

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AIM: To study the prevalence, reproductive hormone profiles and ovarian sonographic appearance of Thai women with polycystic ovary syndrome (PCOS). METHODS: One thousand and ninety-five women were screened for oligomenorrhea/amenorrhea, and the clinical symptoms of hyperandrogenism. Ovarian morphology and volume were assessed by ultrasonography in diagnosed cases. Blood was taken for the measurement of the follicle stimulating hormone, luteinizing hormone, prolactin, testosterone, androstenedione, dehydroepiandrosterone and 17-hydroxyprogesterone. RESULTS: The prevalence of PCOS was 5.7%. The mean age of women with PCOS was less than that of non-PCOS cases (27.4 +/- 6.5 and 31.1 +/- 6.4 years, respectively; P < 0.0001). Abnormal uterine bleeding and infertility were the leading presenting symptoms. The mean ovarian volume in women with PCO appearance was 9.22 +/- 4.36 mL compared to 6.53 +/- 3.31 mL in those without this appearance (P = 0.04). Hyperandrogenemia was confirmed in 23 of the 62 cases (37.1%). CONCLUSIONS: The prevalence and clinical presentations of Thai women with PCOS were similar to those in other reports. However, hirsutism, elevated testosterone level and acanthosis nigricans were uncommon in our population. Serum androstenedione was a more sensitive indicator of hyperandrogenemia than total testosterone. Further research is needed to clarify whether there is an ethnic difference in endocrine profiles and risks of metabolic syndrome.

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AIM: To investigate the possible causes of oligozoospermia and azoospermia in infertile Thai men, and to find the frequencies of Y chromosome microdeletions and cytogenetic abnormalities in this group. METHODS: From June 2003 to November 2005, 50 azoospermic and 80 oligozoospermic men were enrolled in the study. A detailed history was taken for each man, followed by general and genital examinations. Y chromosome microdeletions were detected by multiplex polymerase chain reaction (PCR) using 11 gene-specific primers that covered all three regions of the azoospermic factor (AZFa, AZFb and AZFc). Fifty men with normal semen analysis were also studied. Karyotyping was done with the standard G- and Q-banding. Serum concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) and testosterone were measured by electrochemiluminescence immunoassays (ECLIA). RESULTS: Azoospermia and oligozoospermia could be explained by previous orchitis in 22.3%, former bilateral cryptorchidism in 19.2%, abnormal karyotypes in 4.6% and Y chromosome microdeletions in 3.8% of the subjects. The most frequent deletions were in the AZFc region (50%), followed by AZFb (33%) and AZFbc (17%). No significant difference was detected in hormonal profiles of infertile men, with or without microdeletions. CONCLUSION: The frequencies of Y chromosome microdeletions and cytogenetic abnormalities in oligozoospermic and azoospermic Thai men are comparable with similarly infertile men from other Asian and Western countries.

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AIM: To study the rate of blastocyst formation in 4-cell mouse embryos after laser destruction of one blastomere, with or without microsurgical removal of the destroyed blastomere. METHODS: Mouse embryos were randomly allocated to two control and two experimented groups. Control embryos were either non-manipulated (117 embryos) or underwent laser ablation of zona only (114 embryos). Experimented embryos had laser destruction of zona and the adjacent blastomeres. Destroyed blastomeres were either left in situ (115 embryos) or were microsurgically removed (107 embryos). They were cultured in sequential media for 72 h and were assessed for cleavage/morula arrest and blastocyst formation rates. RESULTS: Embryos arrested at cleavage/morula stages were higher when destroyed blastomeres remained in situ (30.4%) than when they were immediately removed (15.0%, P < 0.05). Blastocysts in the group with immediate removal of the destroyed blastomeres (85%) were significantly higher than when destroyed blastomeres were left in situ (69.6%, P < 0.05). Blastocyst formation in the repaired embryos was significantly lower than the non-manipulated (91.5%) and the manipulated controls (94.8%, P < 0.05). Hatching blastocysts were highest in control embryos with zonal ablation (72.8%). Proportions of hatching/hatched blastocysts in embryos, with or without removal of destroyed blastomeres, were not significantly different (39.3% and 33.9%, respectively). The percentage of embryonic loss during an attempt at microsurgical repair was 6.1%. CONCLUSION: Microsurgical removal of destroyed blastomere was effective in restoring blastocyst development. It could reduce the rate of cleavage/morula arrest.

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