A comparison of swim-up and simple swim-up methods for the extraction of high motility sperm.

The objective of this study was to compare two methods for the extraction of high motility sperm; swim-up and simple swim-up technique. The results were comparable in concentration, motility and morphology.

Premature Ovarian Failure (POF): Cytogenetic and SRY analyses.

Premature ovarian failure (POF) is a condition causing amenorrhea, hypoestrogenism and elevated gonadotropins and can be found in 1% of women before the age of 40. There are many causes of POF. One of these is a chromosome abnormality especially of the sex chromosome, the most common of which is 45,X. Translocation between regions on the X and Y chromosome and X chromosome deletions have also been reported. The objectives of this study were to perform chromosome analysis and to look for the SRY gene in the patient with POF. 25 patients with POF attending the Gynecologic Endocrinology Clinic at the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, were included in this study. Peripheral blood was collected from the patients for cytogenetic and SRY analyses. There were 4 patients (16%) with identifiable abnormal X chromosomes, which were 45,X/46,XX ; 46,X,i(Xq) ; 46,XX/47XXX and 47,XXX. All patients were SRY negative. POF may be caused by other causes such as auto-immunity, mumps oophoritis etc. Interestingly, this study revealed much higher chromosome abnormalities rate among Thais with POF than has been previously reported elsewhere although those chromosome anomalies are very similar. Chromosome study should be routinely performed as a part of basic laboratory evaluation for all patients with POF. The information obtained will be useful for genetic counseling, planning patient management and family members' decisions in the future.

Long-term compliance with hormonal replacement therapy: Experience of Siriraj menopause clinic.

A retrospective analysis of women who registered for treatment at Siriraj Menopause Clinic in the year 1995 was performed to assess long-term compliance with hormonal replacement therapy (HRT). There were 217 women who registered that year and all were followed up for the following four years. Of these 217 women, 195 commenced HRT and were divided into two groups. The first group of 1,105 women (natural menopause, N) comprised 25 menopause transition and 80 postmenopause cases. The second one of 91 women (surgical menopause, S) was the group who previously underwent total hysterectomy with bilateral salpingooophorectomy. The average age at first consultation in the N group was 52.1 + 6.5 year which was significantly higher than 46.7 + 7.7 year in the S group (P<0.0001). In the N group, compliance was 61.9% at 1 year and 56.1%, 50.4%, 43.8% at 2,3 and 4 years while in the S group compliance was 74.7% at 1 year and 64.8%, 58.2%, 47.2% at 4 years respectively. Compliance with HRT was not statistically different between both groups (P>0.05). Drop out cases were maximum in the first years in both groups and total drop out cases increased slowly and steadily every year for the next four years. Ages at first consultation of compliant and non compliant women in each group were also not statistically different (P>0.05). Long-term compliance with HRT in this study was comparable with previously reported data. Main reasons for non compliance at 4 years were breast symptoms including breast masses and mastalgia, abnormal vaginal bleeding or irregular menses, weight gain and skin problems including acne, melasma and hair loss. These problems are different from other reports. Factors affecting long-term compliance with HRT should be further studied especially in those women who have undergone a surgical menopause.

Comparison of three methods for DNA extraction from amniotic fluid for fetal sex determination by multiplex polymerase chain reaction (PCR).

The objective of this study was to compare three methods used for amniotic fluid DNA extraction. These methods were: Proteinase K/Phenol-Chloroform, Proteinase K/7.5M Guanidine-HCl and DNAzolา BD Reagent. Ten samples of uncontaminated amniotic obtained by amniocentesis performed in mothers with advanced maternal age for detection of fetal chromosome abnormality were studied. Each sample was divided into three tubes, 1 ml placed in each, and DNA extraction was performed by all three methods. The quality and quantity of DNA extracted by each method were compared by electrophoresis on 3% agarose gel and spectrophotometric study at 260 & 280 nm. The DNA obtained was subsequently used for fetal sex determination by multiplex PCR method. The primers used for multiplex PCR were specific for X and Y chromosomes. Accuracy of fetal sex determination was compared with the results from amniocyte culture and the sex at birth. The results showed that DNA extracted by DNAzolาBD Reagent was 100% accurate when used to determine fetal sex by PCR; eight samples on 1st PCR and the other two on repeat PCR. DNA extracted by Proteinase K/7.5M Guanidine-HCl also yielded 100% accuracy in fetal sex determination; seven samples on 1st PCR, two samples on 2nd PCR and one sample needed 3rd PCR. The Proteinase K/Phenol-Choroform method yielded only 90% accuracy and one sample failed to determine fetal sex after having repeated PCR three times. The extraction method which gave the maximum amount of DNA was the Proteinase K/Phenol-Chloroform one but the failed to give 100% accuracy in determining fetal sex. The method which produced the least protein contamination was DNAzolฎBD Reagent and gave 100% accuracy in determining fetal sex with smallest number of PCR reactions. In conclusion, DNAzolฎBD reagent method is relatively rapid for amniotic fluid DNA extraction with high accuracy for fetal sex determination but when large amount of DNA is also needed for other purposes, Proteinase K/Phenol-Chlorofrom is recommended as the method of choice.

Cryopreservation of human spermatozoa: The effect of cryostorage time on the post – thaw sperm outcome.

A prospective study was reported comparing the effect of short-term (1 week) and long-term (6 months) cryostorage time on the post-thaw sperm outcome in four different freezing techniques. Eighty five semen samples were included in the study. Each sample was divided and subjected to four cryopreservative techniques: CEG-NCR, CEG-CR, TEST-EYG-NCR, and TEST-EYC-CR. The cryopreservation of each technique was performed in similar way by mixing the semen with the cryopreservative media (CEG or TEST-EYG) in ratio 1:1, then the mixture was aspirated into two straws. The temperature was then reduced (by NCR or CR freezing method) to -80 C, then the straws were transferred into liquid nitrogen. One straw of each technique was thawed after 1 week and the other straw after 6 months of cryostorage time for assessing the post-thaw sperm outcome. As the results, the post-thaw sperm motility and survival rate of the 1 week cryostorage group was slightly higher than the 6 months group in the CEG-NCR and the CER-CR freezing technique, but these did not have clinical significance because the differences were into small. There were no differences of post-thaw sperm outcome between 1 week and 6 months group in the TEST-EYG-NCR and TEST-EYG-CR freezing techniques. We concluded that the duration of cryostorage did not have clinical effect on the post-thaw sperm outcome.