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.
ObjectivesThis study explored the association between a history of oligomenorrhea and onset of menopause and metabolic parameters.MethodsThe study population was 605 postmenopausal women who were patients at the Menopause Clinic, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand between February 2015 and December 2015. A questionnaire was used to ask all women about their history of oligomenorrhea. The study also collected medical data, including weight, height, waist circumference, blood pressure, and blood glucose and lipid profile.ResultsOf the 231 postmenopausal women with a complete data record, 31 had a history of oligomenorrhea and 200 did not. The age of onset of menopause was around 48 years in both groups. Prevalence of metabolic syndrome was 12.1%. More women with a waist circumference larger than 80 cm had a history of oligomenorrhea at the interview than women who had not, but a history of oligomenorrhea did not relate to other metabolic parameters. The adjusted odds ratio of a history of oligomenorrhea to waist circumference was 3.69 (95% confidence interval, 1.17–11.64).ConclusionsA history of oligomenorrhea did not affect the age at menopause, but was associated with waist circumference during menopause.
Objectives: Hemoglobin E disease, c.26G>A variant of beta-globin gene, is the most common hemoglobinopathy in Asia. Compound heterozygotes inheriting Hb E disease and beta-thalassemia generate beta-thalassemia-Hb E disease with severe anemia. This study aimed to develop a pre-implantation genetic testing for monogenic disorders (PGT-M) protocol for beta–thalassemia (c.17A>T mutation)-Hb E disease (c.26G>A mutation) using multiplex fluorescent polymerase chain reaction (PCR) and mini-sequencing. Materials and Methods: bthalw1 primers were used to amplify a beta-globin gene fragment covering both mutations, i.e. beta– thalassemia (c.17A>T) and Hb E disease. D21S11 microsatellite marker was included for contamination detection. Novel mini-sequencing primers were designed and tested for detection of both mutations. Results: Pre-clinical work up of the optimized PGT-M protocol using 20 single buccal cells of a heterozygous subject showed 100% amplification efficiency and 0% allele drop out (ADO) rate for both primers. In clinical PGT-M cycle, 15 embryos were subjected to biopsy. The results showed two normal, one heterozygous for beta-thalassemia, six heterozygous for Hb E disease, one affected for beta-thalassemia-Hb E disease and five with ambiguous results. Two normally diagnosed embryos were chosen for transfer, one singleton pregnancy was obtained. A healthy baby boy was resulted. Postnatal testing confirmed PGT results. Conclusions: Novel PGT-M protocols for beta-thalassemia-Hb E disease using multiplex fluorescent PCR and mini-sequencing were developed and described here. The protocol was applied in a clinical PGT-M cycle and gave rise to one successful pregnancy and consequently a healthy baby boy. Mini-sequencing was proved to be rapid, accurate and cost-effective protocol for PGT-M.
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