Waraporn Piromlertamorn

Rapid freezing versus slow programmable freezing of human spermatozoa

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.

Clinical, endocrine and ultrasonographic features of polycystic ovary syndrome in Thai women

Aim:  To study the prevalence, reproductive hormone profiles and ovarian sonographic appearance of Thai women with polycystic ovary syndrome (PCOS).

Effect of embryo density and microdrop volume on the blastocyst development of mouse two-cell embryos

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.

Effects of ovarian endometriotic fluid exposure on fertilization rate of mouse oocytes and subsequent embryo development

BackgroundAccidental 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.MethodsMouse 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.ResultsThe 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.ConclusionsExposure 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.

Immature Oocytes in “Apparent Empty Follicle Syndrome”: A Case Report

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.

Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing.

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.

Slow programmable and ultra‐rapid freezing of human embryos

Aim:  To compare the outcomes of slow freezing with ultra‐rapid freezing (URF) of cleavage‐stage human embryos on aluminum foil.

Follicle-stimulating hormone receptor gene polymorphism in chronic anovulatory women, with or without polycystic ovary syndrome: a cross-sectional study

BackgroundPolymorphisms 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.MethodsDNA samples from peripheral blood lymphocytes were extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism.ResultsThe 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.ConclusionsIn Thai women, there was no association between the FSHR gene polymorphism at codons 307 and 680 and chronic anovulation.

Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers

BackgroundIt 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.MethodsIn 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.ResultsNo 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.ConclusionsFull-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased.

Assisted reproductive technologies in Thailand: 2001-2007 results generated from the ART Registry, Royal Thai College of Obstetricians and Gynaecologists

Aim:  To present the results of assisted reproductive technology (ART) performed in Thailand during 2001–2007.