Freezing effect on post-thawed sperm characteristics especially sperm DNA integrity comparing between liquid nitrogen vapour and computerized program freezer.

The aim of the study was to evaluate the cryodamage effects on human sperm characteristics, especially on sperm DNA integrity, after 6 months of freezing comparing between liquid nitrogen vapour (LNV) and computerized program freezer (CPF). Forty normal semen samples were collected for semen analysis. Each sample was mixed with cryoprotective media and devided into 2 straws. The first straw was frozen with LNV and the second one with CPF. After 6 months of cryostorage, semen samples were thawed, and sperm chromatin integrity as well as sperm motility, morphology, vitality and cryosurvival rate were determined. Percentages of DNA damage were higher (p<0.01) following freezing with LNV than with CPF. Sperm vitality was greater (p<0.05) after CPF than after LNV, as well as cryosurvival rate (p<0.001). Post-thawed sperm motility was greater after CPF than after LNV, either in grade A (p<0.001) or in grade B (p<0.05). No significant difference was observed in the percentage of normal sperm morphology comparing the two freezing methods. The current study demonstrated post-thawed decrease in sperm DNA integrity as well as other sperm characteristics after freezing in both methods. The CPF significantly provided superior results in post-thawed sperm DNA integrity, sperm motility and vitality than LNV did. In case of 6 months of cryostorage, therefore, we recommend the computerized program freezer as a preference for sperm cryopreservation.

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.

Hypoosmotic swelling test of sperm supplemented with pentoxifylline after preparation by two-layer percoll gradient method.

The aim of this study was to compare the percentage of sperm tail membrane swelling under hypoosmotic conditions with those with and without pentoxifylline supplements in sperm prepared by the two-layer Percoll gradient method. Twenty five normal semen samples were collected from male partners of infertile couples attending the Infretility Clinic at Siriraj Hospital. After the process of sperm preparation by the two-layer Percoll gradient method, the final samples were divided into 2 tubes, 0.5 ml was added into one tube and another tube was kept as control. The hypoosmotic swelling test was performed on both specimens. The percentage of swollen spermatozoa in the pentoxifylline supplement group was significantly higher than the control group (82.8 + 7.7 vs 70.8 + 12.7; p < 0.00). It was concluded that the addition of pentoxifylline to the sperm prepared by the two- layer Percoll gradient method can enhance the sperm membrane integrity, and it may be beneficial to add pentoxifylline to sperm preparation for use in IUI or IVF.

Induction of acrosome reaction by calcium ionophore A23187 in sperm separated by two-layer percoll gradient method.

The aim of this study was to compare thepercentages of sperm with an acrosome reaction between those with and without calcium ionophore A23187 induction after two-layer Percoll gradient separation. Thirty normal semen samples were obtained from the male partners of infertile couples attending the Infertility Clinic at Siriraj Hospital. After the process of sperm separation by two-layer Percoll gradient technique, the final samples samples were divided into 2 portions. An aliquot of 10 ตM of calcium ionophore A23187 was added to one portion to induce an acrosome reaction, while the other portion was used as a control. Fluorescein isothiocyanate-conjugated Pisum sativam agglutinin (FITC-PSA) staining was performed on both specimens and the acrosome reated-sperm were evaluated. The percentage of acrosome-reated sperm in the calcium ionophore A23187 induced group was significantly higher than those of the control group (24.8+6.6vs 15.4+6.0;p < 0.001). It is concluded that calcium ionophore can significantly induce an acrosome reaction on sperm separated by two-layer Percoll gradient technique, and it may be beneficial to add calcium ionophore A23187 to sperm preparation for use in IUI or IVF.

Sperm motility and morphology after preparation using the percoll gradient method compared with the IxaPrep gradient method.

In order to compare the Percell gradient and IxaPrep gradient methods, the percentage of progressively motile sperm and the percentage of sperm with normal morphology were examined. Thirty five normal semen sample were collected from the male partners of infertile couples attending the Infertility Clinic. The samples were divided into two fractions of 1 ml each. Both fractions were processed using the Percoll gradient and IxaPrep gradient methods, and sperm motility and normal morphology were evaluated. The percentages of spermatozoa that showed progressive motility and normal morphology from the sperm preparation using the IxaPrep gradient method were significantly higher than those prepared by the Percoll gradient method (77.7 + 12.0 vs 73.7 + 12.3 and 64.4 + 15.1 vs 61.1 + 13.3 respectively ; p<0.05). It was concluded that sperm preparation using IxaPrep gradient method showed better sperm quality with respect to progressive motility and normal morphology than those using the Percoll gradient method, The IxaPrep gradient method can be considered as an alternative to the Percoll gradient for sperm preparation.

Comparison of EffectiveNESS between Recombinant Follicle Stimulating Hormone and Human Menopausal Gonadotrophin Administration in those with Diminished Ovarian Reserve.

The study was carried out to compare the effectiveness of recombinant follicle stimulating hormone (recombinant FSH) and human menopausal gonadotrophin (HMG) in those with diminished ovarian reserve. A total of 106 ovarian stimulation cycles from 88 poor responders were included in this study. Either recombinant FHS or HMG was administered in order to stimulate the ovary for each cycle. The pregnancy rate of the recombinant FSH group (22.5%) was higher than that of the HMG group (9.1%). The cancellation rate of the recombinant FSH group (10.0%) was lower than that of the HMG group (19.7%). In in vitro fertilisation-embryo transfer (IVF-ET) cycles, the fertilisation rate of the recombinant FSH group (62.8%) was higher than that of the HMG group (51.8%). The pregnancy rate and the implantation rate of the recombinant FSH group (23.0 and 9.1%, respectively) were higher than those of the HMG group (13.6 and 5.9%, respectively). Although this did not achieve statistical significance, only the recombinant FSH group achieved pregnancies using gamete intrafallopian transfer (GIFT). In conclusion, recombinant FSH is probably more effective than HMG in improving the IVF and pregnancy rate in poor responders.

The attitude of chronically ill patients and personnel of Siriraj Hospital to a recreational therapy service.

A study of the attitudes of 507 patients with chronic illnesses and 961 personnel working with these patients towards a recreational activity service was performed using a questionnaire. Most of them agreed that the hospital should arrange recreational activity for patients with chronic illnesses in addition to physical treatment (95.6, 99.0 percent of patient and personnel groups respectively). Both groups evaluated the benefit of recreational activity with a score of 7.93 ฑ 2.09 and 8.71 ฑ 1.42. Activities ranked first were reading books, watching television or a video, gardening and singing/listening to music. Concerning health professionals willingness to join these activities, 80% of the nurse group (registered nurses and practical nurses) were willing to do so whereas only 51.4 percent of the doctor group were available due to different working styles. Most of the personnel accepted that the recreational therapist should be the one who arranges this service. Both groups agreed that a recreational activity service should be free of charge (45.2-51.4 percent). From the results of this study, we can conclude that patients and personnel appreciate a recreational activity service for chronically ill patients, so we should develop and distribute this service to patients in order to improve their quality of life and the quality of our service.

Comparison of sperm motility between the swim-up and swim-down methods.

The objective of this study was to compare two methods for the extraction of high motility sperm; the swim-up and swim-down techniques. The result showed that both methods produced greater sperm motility than the original semen. The motility of the sperm was lower in the swim-down method compared with the swim-up method (65.5+11.2% vs 76.5+10.1%; p<0.01).

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.

Comparison between hysterosalpingo-contrast sonography ( HyCoSy ) using echovist® and hysterosalpingography ( HSG ) in uterine cavity and tubal patency assessment.

assessment of uterine cavity and tubal patency compare with hysterosalpingography ( HSG ). Thirty four of infertile women undergoing investigation for infertility were enrolled from July 1997 until November 1998 for the current study. HyCoSy using Echovistฎ and HSG were successful carried out during the first half of each patient’s menstrual cycle. The flow of contrast medium through the uterine cavity and each fallopian tube was visualised in real time by transvaginal ultrasonography for HyCoSy. Meanwhile, HSG was performed and reported by consultant radiologists. Regarding the uterine cavity evaluation, 32 of 34 cases ( 94.1 % ) was reported to be normal by both procedures. Only 2 cases were noted with different results. From the 68 fallopian tubes studied, though 47 of 68 tubes were showed patent by the two procedures, HyCoSy could detect occlusion in only 5 of 10 left fallopian tubes and in 5 of 9 right fallopian tubes which were reported to be occluded by HSG. Mean length of time for HyCoSy was 8.7 minutes (range 2-30 minutes, Std. Dev. 5.4) and mild pelvic pain was the only complication incurred. In conclusion, HyCoSy using Echovistฎ is a reliable and safe procedure for outpatient uterine cavity evaluation. However, it does not substitute to HSG for tubal patency assessment in basic infertility investigation.

Semen parameters retrieved by electroejaculation in spinal cord injured Thai men: Preliminary report.

Semen characteristics from seven spinal injured Thai men retrieved by electroejaculation were studied at Spinal cord injury unit and Infertility unit, Siriraj hospital. The mean age of the subjects was 24.7 years. Three cases had sperm count within normal limit ( > 20x106/c.c. ) and oligospermia in three cases. One case had azoospermia. All had low motility or no motility at all. All had leukospermia ( > 1x106/c.c. ) except one case. This study shows the change in semen parameters and may help for planning the management of spinal cord injured men.

Tubal patency assessment: A comparison of hysterosalpingo contrast sonography (HyCoSy) and chromolaparoscopy.

Twenty nine fallopian tubes from 15 patients were examined for tubal patency using HyCoSy followed by chromolaparoscopy. The mean volume of contrast medium used was 14.3 ml and the duration of HyCoSy was 8.5 minutes. Concordance, sensitivity, specificity, positive predictive value and negative predictive value were 75.9, 44.4, 90.0, 66.7 and 78.3% respectively. The only adverse effect was mild pelvic pain and this was found in 60%. These results suggest that HyCoSy may become an established method for assessing fallopian tubal patency in the future.

Hypo-osmotic swelling test of washing and swim-up sperm treated with pentoxifylline.

An experimental study was carried out in order to evaluate the effect to of pentoxifylline on the membrane function of washing and swim-up sperm in the process of sperm preparation for assisted reproductive techniques. Normal semen samples were taken according to World Health Organization criterias, from 30 male partners who attended the infertility clinic from November 1996 to January 1997, and divided into two aliquots. In the process of sperm preparation by the washing and swim-up method, pentoxifylline was added in only one aliquot. The hypo-osmotic swelling test was used to evaluate the outcome of both aliquots. From the study, the percentage of sperm that showed swollen tails in the hypo-osmotic solution prepared by the washing and swim-up method and treated by pentoxifylline was higher than that in the group which did not have pentoxifylline added in the process of sperm preparation. However, the difference was not atatistically significant (54.9 and 49.2 percent; P>0.01). It was therefore concluded that pentoxifylline added to washing and swim-up sperm in the process of sperm preparation dose not improve membrane function of spermatozoa. Thus, it may not be necessary to use pentoxifylline in the treatment of male infertility.

Fertilization rate during intracytoplasmic sperm injection procedure comparing 6 and 12 O’clock polar body position.

A Prospective study was designed to compare fertilization rate between the . and 12 o’clock position of the polar body during the intracytoplasmic sperm injection (ICSI) procedure. A total of 128 oocytes were injected by placing the first polar body at 6 o’clock and 12 o’clock alternately. Normal fertilization rates were 70.31 and 71.87 percent respectively. There was no statistical significance between the two groups.

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.

Basal body temperature chart as a guide for donor insemination.

The use of BBT in a single donor insemination and the conception rate according to cycle day was studied. For 107 such cycles, inseminations were performed on day 0 in 21.5% of cycles, on days-1,+1 and –2 in 17.8%, 16.8% and 13.1% respectively. The overall pregnancy rate achieved was 44%. The success rate per cycle was 15%, and the highest success rates were obtained on day 0 (43.7%) and day –1 (31.2%). No pregnancies occurred when insemination was done before day -3 and beyond day +1. In conclusion BBT provides a reasonable guide to the 2-to 3-day period on either side of the nadir in donor insemination.