The successful pregnancy and birth of a healthy baby after human blastocyst vitrification using Cryo-E, first case in Siriraj Hospital.

This case report illustrates a successful pregnancy and birth after vitrification of a human blastocyst using modified cryostorage- Cryo E. A 31-year-old woman underwent short protocol for ovarian hyperstimulation in an IVF treatment program. After administration of intramuscular hCG, all oocytes were recovered transvaginally with ultrasound guidance. Twelve mature oocytes were obtained and eight were fertilized Three out of eight embryos became blastocyst, and two blastocysts were transferred on day 5, but no implantation occurred The other was cryopreserved by vitrification technique using a cryo E. Three months after freezing, the cryopreserved blastocyst was thawed and survived. It was transferred to the patient's uterus. Blastocyst implantation resulted in a healthy single pregnancy. After continuing pregnancy until term, she delivered by cesarean section due to unfavorable cervical conditions. A male baby with Apgar score 9/10, weighing 3500 g was born. Her child showed normal growth.

Evaluation of cryo-injury of sperm chromatin according to computer controlled rate freezing method part 2.

OBJECTIVE: The present study was designed to determine the effect of the freeze-thawing procedure, computer controlled rate freezing and duration for six months, on human sperm chromatin (assessed by acridine orange test), vitality, motility, and morphology. DESIGN: Experimental study MATERIAL AND METHOD: Twenty semen samples were obtained from patients attending the infertility unit. The semen analysis was measured according to WHO criteria. Sperm morphology was evaluated by strict Kruger criteria and sperm chromatin were detected by acridine orange test. After semen analysis, each sample was mixed with cryoprotectant and divided into straw. The straw was frozen with computer controlled rate freezing method After 6 months of cryostorage, semen samples were thawed and then the semen was analyed, and sperm chromatin and morphology were determined. RESULTS: After six months of cryostorage, the mean percentage of normal sperm chromatin decreased significantly (87.3 +/- 9.0 vs. 51.9 +/- 27.4, p < 0.001). Vitality, motility, and normal morphology of sperm decreased significantly (78.7 +/- 1.9 vs. 32.8 + 10.8, 52.6 + 1.9 vs. 24.1 +/- 10.9 and 21.4 +/- 4.3 vs. 18.0 +/- 4.4 respectively, p < 0.001). CONCLUSIONS: The computer controlled rate freezing of sperm for six months and thawing process significantly decreased normal sperm chromatin, vitality, motility, and normal morphology.

Comparison between sperm treated with pentoxifylline and 2-deoxyadenosine using hypo-osmotic swelling test.

OBJECTIVE: To compare the percentage of sperm tail membrane swelling under hypo-osmotic conditions between sperm treated with pentoxifylline and 2-deoxyadenosine. DESIGN: Experimental in vitro study. MATERIAL AND METHOD: Thirty normal semen samples from male partners of infertile couples were collected. After sperm preparation by two-layer Percoll gradient method, each sperm sample was divided into three specimens. Pentoxifylline and 2-deoxyadenosine were separately added into two specimens, while the third specimen was used as a control. Hypo-osmotic swelling test was performed in all specimens. Percentage of swollen spermatozoa in each specimen was evaluated. RESULTS: The mean percentage of swollen spermatozoa in the semen samples supplemented with pentoxifylline and 2-deoxyadenosine were both significantly higher than those in the control (82.8 +/- 7.7 and 83.0 +/- 9.5 vs 70.8 +/- 12.7; p < 0.001). There was no significant differences of swollen spermatozoa between pentoxifylline and 2-deoxyadenosine (p = 0.898). CONCLUSION: Addition of pentoxifylline and 2-deoxyadenosine to the sperm prepared by the two-layer Percoll gradient method can almost equally enhance the sperm membrane integrity. Therefore, it may be beneficial to add these compounds to sperm preparation for use in assisted reproduction.

Evaluation of cryoinjury of sperm chromatin according to liquid nitrogen vapour method (I).

OBJECTIVE: To evaluate cryodamage on human sperm motility, cryosurvival rate, and sperm chromatin assessed by acridine orange staining method (AO test) after a six-month freeze-thawing process using liquid nitrogen vapor. STUDY DESIGN: Experimental study. MATERIAL AND METHOD: Twenty normal semen samples were obtained from the male partner of infertile couples attending the infertility unit, Siriraj Hospital. After semen analysis, each semen sample was frozen with liquid nitrogen vapor. The acridine orange test was used for assessment of chromatin structures. After 6 months of cryostorage, semen samples were thawed and the effects of cryopreservation on sperm chromatin integrity, motility, morphology, vitality, and cryosurvival rate were evaluated. RESULTS: The mean percentage of normally condensed sperm chromatin in the native semen sample decreased significantly (87.3 +/- 9.1 vs 47.9 +/- 26.2; p < 0.001) after the freeze-thawing process using liquid nitrogen vapor. Furthermore, the mean percentage of sperm motility and vitality also decreased significantly after the freeze-thawing process (52.6 +/- 1.9 vs 23.2 +/- 10.6 and 78.7 +/- 5.6 vs 30.3 +/- 8.8 respectively; p < 0.001). In contrast, the numbers of sperm with normal morphology after cryopreservation were not different from those before the procedure (21.4 +/- 4.3 vs 24.2 +/- 23.9; p = 0.606). CONCLUSION: The freeze-thawing procedure using liquid nitrogen vapor had effects on chromatin, motility, and vitality of human spermatozoa. The six-month cryopreservation of semen is a good method for avoiding the window period of HIV; however, this can cause a lot of damage to spermatozoa, thus, limits their further use in the treatment of infertility.

Vitrification of mouse oocyte using open pulled straws compared with needles.

OBJECTIVE: To compare the survival rate of mouse oocytes and fertilization rate between using open pulled straws (OPS) and needles for vitrification. MATERIAL AND METHOD: Meiosis II oocytes from female C57B/6J mice aged 7-8 weeks were collected and allocated to two groups for vitrification by using OPS or needles. Vitrified oocytes were thawed, morphological survival and fertilization rate were examined. RESULTS: There was no obvious difference between the morphological survival rates of vitrified mouse oocytes using OPS and needles (66.7% vs 64.8%). Proportions Difference 1.9% (95% CI -7.1, 10.7). The vitrified oocytes from the needle had significantly higher percentages of fertilization rate than OPS (76.8% vs 62.5%). Proportions Difference -14.3% (95% CI -24.5, -3.6). CONCLUSION: Vitrification method of mouse oocytes using needles when compared to OPS provides a similar morphological survival rate and higher fertilization rate.

Analysis of aneuploidy in mini-percoll gradient centrifuged human sperm for intracytoplasmic sperm injection (ICSI) using fluorescence in situ hybridization.

AIM: To study the aneuploidy rates of chromosomes 13, 18, 21, X and Y in Percoll gradient centrifuged sperm from infertile patients with male infertility factor treated by intracytoplasmic sperm injection (CSI) compared with healthy fertile donors and infertile patients with normal semen parameters. METHODS: This case-controlled study was conducted in a university hospital. Semen samples were obtained from three healthy fertile donors, eight infertile patients with normal semen parameters, and 18 infertile patients with male infertility factor. All samples were subjected to mini-Percoll gradient centrifugation before being processed through fluorescent in situ hybridization. The incidences of aneuploidy were compared using Chi-squared test. RESULTS AND CONCLUSIONS: A total of 64949 spermatozoa were analyzed. The disomy rates for chromosomes 13, 18, 21, and X or Y of sperm from patients with male infertility factor were 0.21%, 0.37%, 0.36% and 0.63%, respectively, whereas the diploidy rate was 0.17-0.23%. These incidences were higher than those from men with normal semen parameters. The result suggested that the embryos of patients with male infertility factor treated by ICSI are at increased risk of chromosome abnormalities.