Livebirth derived from in vitro matured oocytes.

The authors presented a case report of a woman who suffered from primary infertility due to anovulation. She was diagnosed to have polycystic ovarian syndrome (PCOS) and was treated by ovarian stimulation with conventional technique, such as clomiphene citrate alone, the combination of clomiphene citrate and metformin and the combination of clomiphene citrate with gonadotropins. Nevertheless all of them resulted in no response. Therefore, in vitro maturation of oocyte (IVM) was started and followed by intracytoplasmic sperm injection (ICSI) of 22 oocytes. 14 embryos developed and three of them were transferred back to the uterine cavity. She conceived and the ultrasonography revealed twin pregnancy at the 8-weeks gestation. She gave birth at the 31 weeks’ gestation in Siriraj Hospital. The newborns were admitted in the hospital for 24 days for phototherapy in treating jaundice which was due to prematurity. Then both of them were discharged healthily.

A comparison between conventional slow freezing and vitrification of mouse blastocysts using cryo-E.

Objective: To compare the efficiency of two cryopreservations between conventional slow freezing and vitrification of mouse blastocysts using cryo-E. Methods: ICR female mice (8 weeks) were superovulated with 5 IU/ml of pregnant mare serum gonadotrophin (PMSG), the successfulness of mating with males was verified by the presence of a vaginal plug. Blastocysts were obtained between 3.5 and 4.5 days per p.c. or 96-108 hours after hCG administration by flushing the uterus. Randomly selected blastocysts were simultaneously frozen by slow-rate freezing and a vitrification method. One month later, the embryos were thawed and cultured in the blastocyst medium (COOK; Sydney IVF, Australia). Results: Based on 250 slow freezing and 310 vitrified mouse blastocysts, vitrification resulted in a slightly higher survival and hatching rates than the slow-freezing method (83.9% VS 82.0%, and 68.8% VS 66.8%, respectively). Conclusion: Both slow freezing and vitrification of mouse blastocysts are useful methods for cryopreservation. These results showed that vitrification is better than slow freezing in terms of simplicity, duration, and cost-effectiveness.

Preliminary data of preimplantation genetic diagnosis for aneuploidy screening using fluorescent in situ hybridization in Siriraj Hospital.

Objective: Preimplantation genetic diagnosis (PGD) is technique for detecting genetic diseases. Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using fluorescent in situ hybridization (FISH) has been used worldwide including at Siriraj Hospital. The objective of this study was to comparethe pregnancy rate between a PGD-AS group with standard assisted reproductive techniques (ARTs) in Siriraj Hospital. Methods: Couples who requested PGD-AS underwent a standard ARTs process followed with blastomere biopsy for FISH analysis. The pregnancy rate was compared among the PGD-AS group and the control group. The control group was divided into 2 subgroups – all patients required ARTs subgroup and age ≥ 35 yrs. subgroup. Results: 6 stimulated cycles from 4 patients were performed in the PGD-AS group. The pregnancy rate per stimulated cycle in the PGD-AS group, control group and age ≥ 35 yrs group were 33.33%, 16.20% and 12.05% respectively. Moreover, the pregnancy rate per transferred cycles in the PGD-AS group, control group and age ≥ 35 yrs group were 40.00%, 21.02% and 13.17% respectively. Conclusion: PGD is an advanced method for detecting genetic defects. PGA-AS might increase the pregnancy rate.

Correlation between hyaluronan binding assay and TUNEL assay for sperm DNA integrity identification.

Objective: To study the correlation between the percentages of HA-unbound sperm and DNA fragmented sperm by TUNEL assay. Methods: The semen residue from semen analysis was tested by HBA and TUNEL assay. Results: The mean age of patients included in the study was 34.8 years (± 3.7 years). The proportion of HA-unbound sperm ranged from 11.3% to 24.2%, with a mean of 17.08% (± 3.24%). The range of TUNEL positive in semen samples was 2% to 11.75%, with a mean of 5.78% (± 2.28%). Pearson’s correlation between two tests was 0.848 (p<0.01). Intraobserver variation of the results of HBA ranged from 3.3% to 7.6%, with a mean of 6.23% (± 1.11%). Intraobserver variation of the results of TUNEL assay ranged from 0% to 6.9%, with a mean of 1.54% (± 2.7%). Agreement measuring of each test was determined by using intraclass correlation. The intraclass correlation coefficient of HBA and TUNEL assay were 0.970 (P<0.001) and 0.997 (P<0.001) respectively. Conclusion: As several studies have found, the binding capacity of sperm to HA is correlated with several sperm parameters. In this study, the strong correlation between the percentages of HA-unbound sperm and TUNEL positive sperm implies, furthermore, that the HA-bound sperm percentage correlates with low levels of DNA fragmentation.

Preimplantation Genetic Diagnosis for alpha thalassemia: experience in Siriraj Hospital.

Objective: Detection of fetal thalassemia using preimplantation genetic diagnosis (PGD) can make a diagnosis before pregnancy so termination of pregnancy in that patient is eliminated. The objective of this study was to develop a single gene polymerase chain reaction (PCR) protocol for PGD of alpha thalassemia in Siriraj Hospital. Methods: A couple with a history of repeated Bart’s hemoglobinopathy in fetus underwent an artificial reproductive technique (ART) process using a standard ovarian stimulation protocol with intracytoplasmic sperm injection (ICSI) to reduce sperm DNA contamination. On day 3 post fertilization, laser biopsy was performed on the cleavage stage embryos to obtain a blastomere for PCR analysis of alpha thalassemia 1 SEA type. Results: 11 embryos from a total of 15 oocytes were biopsied, 2 normal, 1 alpha thal 1 trait and 3 affected embryos were detected. No contamination and allele drop out were detected, but a high PCR failure rate of 5 from 11 total biopsied embryos. Conclusion: PGD for alpha thalassemia was first established in Siriraj Hospital, but the result had a high failure rate so then optimized laboratory techniques were required.

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.

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.

Efficacy of oral micronized progesterone when applied via vaginal route.

The aim of the study was to compare the efficacy of oral micronized progesterone when applied by the vaginal route. The comparative study of serum progesterone levels between oral and vaginal micronized progesterone administration was conducted in sixty female volunteers. The subjects were equally divided into two groups to receive the drug either via the oral or vaginal route. The subjects' profiles showed that there was no significant difference in general characteristics between these two groups. The blood tests for estrogen and progesterone levels were performed on all volunteers before and after the drug administration. The data collected from the experiment revealed that the serum progesterone levels achieved by oral administration (5.06 +/- 2.95 ng/ml) differed significantly (p < 0.001) from those achieved by vaginal administration (8.26 +/- 4.09 ng/ml). The data also revealed that the serum progesterone levels of the oral administration group (4.23 +/- 2.68 ng/ml) did not differ significantly (p = 0.925) from the other group (4.15 +/- 3.40 ng/ml) when the serum estrogen level was less than 30 pg/ml. On the contrary, when the serum estrogen level was at least 30 pg/ml, there was a significant (p < 0.005) difference in the serum progesterone levels between these two groups (6.32 +/- 2.99 ng/ml for the oral route and 9.76 +/- 3.23 ng/ml for the vaginal route).

Effect of pentoxifylline on human sperm preparation.

The aim of the research was to study the effects of pentoxifylline on the sperm motility. Thirty samples of semen were studied. Each sample was divided into two sub-samples swim up technique. The other sub-samples were mixed with 3.3 mmol pentoxifylline solution for thirty minutes before entering the process of the sperm swim up preparation. The data regarding sperm concentration, motility, and morphology after 5 minutes of the swim up were recorded. The study showed that the samples that were mixed with 3.3 mmol pentoxifylline had a significantly higher sperm concentration (p< 0.001), greater sperm motility (p = 0.014), and a more normal sperm morphology (p < 0.001) than those samples prepared using the conventional method. It was concluded that pentoxifylline added in the process of sperm preparation could improve the sperm quality outcome.

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.

Effect of different exposure periods to the infrared 1.48 m diode laser on the inner cell mass and trophectoderm of blastocysts.

This study was carried out to determine the effect of the duration of exposure to an infrared 1.48 mm diode laser, on the number of cells in the inner cell mass and the trophectoderm of blastocysts following laser-assisted embryo biopsy. A total of 102 mouse embryos were used in the study. The embryos were randomly divided into three groups; group A (n = 22), group B (n = 47) and group C (n = 33). The embryos in group A were biopsied using the laser with an exposure of 600 ms, whereas those in group B were biopsied using the same laser with an exposure of 5 ms. The embryos in group C were incubated in culture without any procedures, as a control group. The blastocyst formation rates of group B (46/47, 97.8%) and group C (33/33, 100%) were significantly higher than that of group A (12/22, 54.5%). The number of cells in the inner cell mass, trophectoderm and the total number of cells in the blastocyst in group A (16.1 ฑ 5.1, 35.5 ฑ 10.9, 51.6 ฑ 12.9) were similar to those in group B (14.0 ฑ 5.6, 36.0 ฑ 12.7, 50.0 ฑ 18.3). The number of cells in the inner cell mass, trophectoderm and the total number of cells in the blastocysts in group C (19.1 ฑ 6.5, 45.8 ฑ 14.0, 65.0 ฑ 18.7) was significantly higher than those of the study groups. In conclusion, the longer duration of exposure to the infrared 1.48 mm diode laser might adversely affect blastocyst formation. However, it might not affect the quality of the blastocysts with regard to the number of cells in the inner cell mass and the trophectoderm.

Disseminated tuberculosis.

Tuberculosis usually causes symptoms. Many patients may first be aware of fatigue, anorexia, weight loss, irregular menses, or a low grade fever that persists over weeks to months. Symptomatic patients with disease can be characterized as having generalized or systemic signs and symptoms, pulmonary signs and symptoms, signs and symptoms related to other organs, or a combination of these features, namely miliary or disseminated tuberculosis. Acute symptoms may be superimposed on a more chronic pattern which may simulate many other diseases. Tuberculosis may mimic, or occur concurrently, with pneumonia, sarcoidosis, fungal infection, or neoplasm. However, medical attention should be sought in order that appropriate treatment for tuberculosis can be given to the patients.

Comparison of human menopausal gonadotrophin and recombinant follicle-stimulating hormone in in-vitro fertilisation and pregnancy outcome.

The study was carried out to compare the effectiveness of human menopausal gonadotrophin (HMG) and recombinant follicle-stimulating hormone (recombinant FSH) in term of in vitro fertilisation (IVF) and pregnancy outcome. A total of 238 patients who underwent IVF for infertility treatment were included in the study. The first attempt of controlled ovarian stimulation was recorded and evaluated. A long protocol of ovarian stimulation was performed with gonadotrophin releasing hormone analogue (GnRH-a) administration. Gonadotrophin, which was either HMG (group A) or recombinant FSH (group B), was administrated to each patient for ovarian stimulation. The results of this study showed no difference in the number of stimulation days, fertilised oocytes, transferred embryos and cycles with embryos available for freezing between the two groups. Although the starting doses of both gonadotrophins were similar, the total dosage of HMG was higher than that of recombinant FSH (48.8ฑ20.8 versus 42.9ฑ20.0, p = 0.03). The number of retrieved oocytes in group A was higher than that in group B (9.5ฑ4.4 versus 8.3ฑ4.3, p = 0.04). The differences in cancellation rate, fertilisation rate, pregnancy rate per cycle and per transfer, as well as implantation rate between the two groups was not statistically significant. In conclusion, patients who underwent ovarian stimulation with GnRH-a long down-regulation still benefit for HMG for their treatment. We did not find any difference in fertilisation rate or pregnancy rate as well as implantation rate between HMG and recombinant FSH. A greater number of oocytes were retrieved in patients treated with HMG. However, more ampoules of HMG were administrated to achieve ovarian stimulation, compared with recombinant FSH.

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.

Eclampsia in Siriraj Hospital: Fifteen years analysis.

The study of the epidemiological characteristics of eclamptic patients was carried out among patients admitted to Siriraj Hospital from January 1972 to December 1986. Particular attention was paid to antenatal care patterns, maternal and perinatal mortalities. Three hundred and eleven eclamptic patients were encountered among 301,533 deliveres, a prevalence of 103.1/100,000 deliveries. The majority of eclamptic patients had no antenatal care, only 14.74% of these patients had good antenatal supervision. There were 9 maternal deaths encountered during the whole period of study with no death encountered during the last 5 years. Twenty seven perinatal deaths were encountered of which intrapatum deaths were frequently found. Most of the deaths were due to prematurity. However, there has been no perinatal death among eclampic patients during the last 5 years.

Colpocytologic study by ink staining technique for assessing ovulation.

Vaginal smears were taken from women who came for artificial insemination using donor’s semen, to assess the time of ovulation. The smears were obtained from the upper 1/3 of the lateral vaginal wall, and immediately placed in a staining bottle containing sheaffer ink sulution. The slides were examined under light microscope, and the eosinophillic index (E.I.) were recorded. The result of the study of 78 cycles demonstrated that 59 in 65 cycles (90.8%) which were evaluated between 3 days before and 2 days following ovulation showed the E.I. over 50. Vaginal smears from 2 normal menstruating women were also examined daily, and the peak of E. I. Was clearly over 50 at the time of ovulation. The E.I. values of this technique were compared to the maturation index which stained by Papanicolaou method, and good correlation was recognized. We concluded that the colpocytologic study ink staining technique is simple, rapid, and inexpensive. It can be used reliably with other methods to assess the time of ovulation.

Rupture of the gravid uterus.

Rupture of the gravid uterus, the most tragic obstetric emergence, is said to occur infrequently nowadays due to efficient antenatal and intranatal intensive care. Forty-six incidents of uterine rupture were reviewed among 197, 254 deliveries during 1978-1987, that is 1 per 4288 deliveries. There were 20 cases of previous scarred rupture, 18 traumatic, and 8 cases of spontaneous ruptures. A rupture of a previously scarred uterus had an approximately equal number of classical and low transverse scars. Maternal and fetal prognoses were much better with low transverse scars. A rupture of the previously unscarred uterus was a more dramatic event. It tended to be longitudinal and complete, and fetal prognoses were relative poor. About 28% of the ruptures were diagnosed prior to delivery, 33% were noticed at operation, and 39% were observed only after delivery and diagnosed by manual uterine exploration. The majority of cases were managed by hysterectomy rather than repair. Under special circumstances, the uterus may be repaired for subsequent reproduction. Maternal mortality was 2.2% and fetal loss 42.6%, 37% suffering from asphyxia at birth. Preventative measures, early diagnosis, and prompt treatment are emphasized.