[Nursing cooperation in surgical collection of testis tissues from prepubertal male patients for cryopreservation: A study of 4 cases].

OBJECTIVE: To explore nursing cooperation in surgical collection of the testis tissue from prepubertal male patients for cryopreservation. METHODS: We retrospectively analyzed the methods and effects of perioperative nursing in surgical collection of the testis tissue from 4 prepubertal male patients for cryopreservation in our Center of Reproductive Medicine. Before, during and after operation, we took strict measures in making sterilized ice containers, intraoperative nursing cooperation, protection of the isolated testis tissues and transferring of the samples. RESULTS: Testis tissues were successfully collected from all the 4 prepubertal males, 31, 31, 20 and 34 samples from each case respectively, well protected and subjected to slow cryopreservation after standard processing in the embryo laboratory. CONCLUSION: In surgical collection of the testis tissue for cryopreservation, preparation of sterilized ice containers, intraoperative nursing cooperation and protection and transferring of the samples are essential for standard processing and cryopreservation of the testis tissue in the embryo laboratory.

Progress and expectation of stem cell therapy for diabetic wound healing.

Impaired wound healing presents great health risks to diabetics. Encouragingly, the current clinical successfully found out meaningful method to repair wound tissue, and stem cell therapy could be an effective method for diabetic wound healing with its ability to accelerate wound closure and avoid amputation. This minireview aims at introducing stem cell therapy for facilitating tissue repair in diabetic wounds, discussing the possible therapeutic mechanism and clinical application status and problems.

Transcutaneous electrical acupoint stimulation based on electro-oculogram signal regulation for children with mental retardation: a randomized controlled trial / 中国针灸

OBJECTIVE@#To observe the clinical efficacy of transcutaneous electrical acupoint stimulation (TEAS) at Changqiang (GV 1) based on the modulation of electro-oculogram (EOG) signal for children with mental retardation, and explore the evaluation effect of the goal attainment scale (GAS) in children with mental retardation.@*METHODS@#Sixty children with mental retardation were randomly divided into a treatment group and a control group, with 30 cases in each one. The children in the control group were treated with conventional rehabilitation, 5 times a week. On the basis of the control group, TEAS at Changqiang (GV 1) under the modulation of EOG signal was adopted in the treatment group. When the similarity between the collected EOG signal and the template was within the range of EOG threshold, one electric stimulation was triggered at Changqiang (GV 1) for 20 s (continuous wave, 70-100 Hz in frequency, 0.1-0.2 ms in pulse width), lasting 30 min in each treatment, the intervention was given twice a week. One course of treatment was composed of 4 weeks, and 3 courses were required in total in the two groups. The infant-junior high school student's social living ability scale (S-M) and GAS were scored and compared before and after treatment in the two groups.@*RESULTS@#After treatment, the scores of self-living ability in the treatment group and communication ability in the control group were higher than those before treatment (P<0.01, P<0.05). The scores of collective activity and motor ability in the treatment group were higher than those in the control group (P<0.05). After treatment, GAS scores were higher than before treatment in both groups (P<0.001), and the score in the treatment group was higher than the control group (P<0.05).@*CONCLUSION@#TEAS under the modulation of EOG signal is conductive to improving the collective, motor and self-living abilities of the children with mental retardation and promoting children's individual goals. Compared with the standard score of S-M, the T value of GAS can better reflect the subtle progress of individual.

[The application study of Electrophysiological appropriate technology in perioperative nursing of patients undergoing microdissection testicular sperm extraction].

Obstract: To explore the application of electrophysiological appropriate technology in perioperative nursing of patients undergoing microdissection testicular sperm extraction. METHODS: A retrospective analysis was conducted on the medical records of 108 patients who underwent testicular incision and sperm extraction under a microscope at our center from May 2022 to June 2023. Among them, 51 patients received routine care and 57 patients received electrophysiological treatment. Evaluate the perioperative nursing effects of appropriate electrophysiological techniques through VAS pain score, Self Rating Anxiety Scale (SAS) score, Pittsburgh Sleep Quality Score, and Kolcaba Comfort Scale. RESULT: Patients who received appropriate electrophysiological interventions had lower VAS pain scores (2.36 ± 1.37 vs 4.16 ± 1.38, P<0.001) than the control group, and higher KOLCABA comfort scale scores than the control group (70.73 ± 19.46 vs 52.06 ± 17.50, P<0.001); There was no statistically significant difference in the Self Rating Anxiety Scale (SAS) score and Pittsburgh Sleep Quality Score. CONCLUSION: Electrophysiological techniques can effectively improve postoperative pain and comfort in patients undergoing testicular incision and sperm extraction under a microscope, and have clinical application value.

Sex differences in clinical outcomes of extremely preterm infants/extremely low birth weight infants: a propensity score matching study / 中国当代儿科杂志

OBJECTIVES@#To study the effect of sex on the clinical outcome of extremely preterm infants (EPIs)/extremely low birth weight infants (ELBWIs) by propensity score matching.@*METHODS@#A retrospective analysis was performed for the medical data of 731 EPIs or ELBWIs who were admitted from January 1, 2011 to December 31, 2020. These infants were divided into two groups: male and female. A propensity score matching analysis was performed at a ratio of 1:1. The matching variables included gestational age, birth weight, percentage of withdrawal from active treatment, percentage of small-for-gestational-age infant, percentage of use of pulmonary surfactant, percentage of 1-minute Apgar score ≤3, percentage of mechanical ventilation, duration of mechanical ventilation, percentage of antenatal use of inadequate glucocorticoids, and percentage of hypertensive disorders in pregnancy. The two groups were compared in the incidence rate of main complications during hospitalization and the rate of survival at discharge.@*RESULTS@#Before matching, compared with the female group, the male group had significantly higher incidence rates of neonatal respiratory distress syndrome, bronchopulmonary dysplasia (BPD), severe intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, and patent ductus arteriosus (P<0.05), while after matching, the male group only had a significantly higher incidence rate of BPD than the female group (P<0.05). There was no significant difference in the rate of survival at discharge between the two groups before and after matching (P>0.05).@*CONCLUSIONS@#Male EPIs/ELBWIs have a higher risk of BPD than female EPIs/ELBWIs, but male and female EPIs/ELBWIs tend to have similar outcomes.

Metabolomics Study on Susceptibility of Pneumonia in Children / 分析化学

The changes of endogenous metabolites in urine samples that come from pneumonia patients of 6 months to 6 years old children were analyzed by metabolomics methods based on gas chromatography and mass spectrometry (GC-MS).The aim of this study was to analyze and study the pathogenesis of endogenous metabolites in children with pneumonia and the pathogenesis of pneumonia susceptibility.The urine samples were collected and divided into normal children group (NC group,n=29),first infection with pneumonia group (FIP group,n=35),and repeated infection with pneumonia group (RIP group,n=31).The urine metabolic profile of pneumonia was obtained by GC-MS.Principal component analysis(PCA) and partial least squares-discriminant analysis (PLS-DA) were used to analyze the data.The results were analyzed by one-way analysis of variance and Fold change.Finally,there was significant difference between the normal group and the pneumonia group,the significant metabolites were serine,histidine,proline,norleucine,glutamine,stearic acid,valine,isoleucine with p value<0.05 and Fold change>5,and indole-3-acetic acid,creatine,ethanolamine,mannosylglycerol and fructose were significant between the two pneumonia groups with p value<0.05.The urinary metabolites demonstrated that amino acid metabolism and glucose metabolism were the main metabolic pathways and responsible for the susceptibility to pneumonia.

Metabolomics analysis of Tripterygium wilfordii formulation based on theory of detoxicity compatibility / 中国中药杂志

Tripterygium wilfordii Hook. f. induced-hepatotoxicity was the main limitation for its usage in clinic. Qingluo Tongbi formulation showed obvious attenuation for hepatotoxicity in clinic and fundamental research in vivo. To explore the potential mechanism of the attenuation, we conducted a study on the plasma metabolomic profiles of T. wilfordii and Qingluo Tongbi formulation in rats by a sensitive gas chromatography-mass spectrometry (GC-MS/MS) method. In plasma samples, a total of 72 compounds were analyzed by EI source MS, and were successfully identified by matching NIST database. The semi-quantification results were then calculated by OPLS-DA model with SIMCA-P 13.0 software. The three groups were clearly distinguished in OPLS-DA score plot. In addition, the observation values of Qingluo Tongbi formulation showed the obvious trend towards the control levels, suggesting the detoxicity effect of the formulation. Variation metabolites were further analyzed by VIP and One Way ANOVAs, and the results showed a significant increase in compounds of glycogenic amino acids, such as alanine, proline, serine and glutamine after the administration of T. wilfordii, indicated that the tissue proteins were decomposed and amino acids were leakage into blood. Qingluo Tongbi formulation could reverse the amino acids into normal level. On the contrary, the levels of glucose, lactic acid and hydroxy butyrate decrease, and the formulation can relieve the disorder in the levels of lactic acid, suggesting the regulation of the energy metabolism. Additionally, the level of branched chain amino acid was decreased, suggested the toxicity was induced, but the formulation cannot increase it into the normal levels. Nevertheless, all the above results suggested that the classical Qingluo Tongbi formulation displayed the liver protection effect by adjusting the amino acid levels and regulating the energy metabolism. Qingluo Tongbi formulation was developed based on traditional Chinese medicine theory "detoxicity compatibility", and contained Panax notoginseng (Burk.) F. H. Chen to nourish blood and absorb clots. Modern pharmacology suggested that its liver protection effect was correlated with the promotion of protein synthesis. Another important herb is Rehmannia glutinosa Libosch., which can regulate the energy metabolism. Both were consistent with the metabolomic results in this study, which explained the potential mechanism of "detoxicity compatibility" theory. Therefore, the currently developed metabolomic approach and the obtained results would be highly useful for the comprehensive toxicity studies for other herbal medicines and various complex deoxicity formulations.

Effect of ginkgolide B on high glucose -induced human lens epithelial cell apoptosis / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To study the effect of ginkgolide B on high glucose-induced human lens epithelial cell (HLEC) apoptosis. METHODS: The high glucose-induced HLEC model was established. Different concentrations of ginkgolide B were intervened. Cell viability was assayed by MTT assay, morphology of cell apoptosis was observed by hochest33258 staining. Cell ultrastructural changes were detected by transmission electron microscopy. Expressions of apoptosis - related factors caspses - 3 and caspase-9 were tested by colorimetric detection. RESULTS: High glucose inhibited the activity of HLECs, induced apoptosis reaction of HLECs, caused high expression of apoptosis factors in cells; while ginkgolide B inhibited the decrease of cell viability induced by high glucose, decreased the HLEC apoptosis and reduced the expression of apoptosis factors Caspase-3 and Caspase-9.CONCLUSION: Ginkgolide B may inhibit the expression of caspses-3 and caspase-9 then effectively inhibited high glucose-induced apoptosis in HLECs.

Reduced and delayed expression of GDF9 and BMP15 in ovarian tissues from women with polycystic ovary syndrome.

PURPOSE: Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play crucial roles in follicular development and oocyte maturation. This study aimed to investigate and compare the expression of these proteins in ovarian tissues of women with and without polycystic ovary syndrome (PCOS). METHODS: Ovarian tissues from 28 patients with PCOS and 26 normal ovulatory women were collected, and the expression of GDF9 and BMP15 in oocytes and granulosa cells was evaluated via immunohistochemical staining. RESULTS: GDF9 and BMP15 were first expressed in primordial follicles at very low levels, and their expression increased gradually with follicular development, reaching the highest levels in Graafian follicles. However, less GDF9 and BMP15 expression was observed in primordial, primary, and secondary follicles in ovarian tissues of PCOS patients compared with levels in the control tissues (P < 0.05). In Graafian follicles, GDF9 and BMP15 expression reached comparable levels in the PCOS and control groups (P > 0.05). CONCLUSIONS: The expression of GDF9 and BMP15 in ovarian tissues varies among the developmental stages in both oocytes and granulosa cells in human ovarian tissues. The expression of these proteins is reduced and delayed in the early follicular stage in PCOS ovarian tissues, and these differences in expression may be associated with aberrant follicular development in patients with PCOS.

Inhibitory effects of controlled ovarian stimulation on the expression of GDF9 and BMP15 in oocytes from women with PCOS.

PURPOSE: To explore the effects of controlled ovarian stimulation (COS) on the expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes and granulosa cells from patients with or without polycystic ovary syndrome (PCOS). METHODS: This case-control study was conducted in the university affiliated hospital. The study comprised four groups of patients: eighteen PCOS patients with COS (stimulated-PCOS) and twenty-two PCOS patients without COS (unstimulated-PCOS), twenty-nine normal ovulatory women with COS (stimulated-control) and twenty-eight normal ovulatory women without COS (unstimulated-control). The oocytes and granulosa cells were collected and the abundance of GDF9 and BMP15 mRNA in the cells were detected by nested quantitative real-time PCR. RESULTS: The abundance of GDF9 and BMP15 mRNA was significantly higher both in oocytes (P < 0.01, P < 0.001, respectively) and GCs (P < 0.01, P < 0.05, respectively) from stimulated-control group than in unstimulated-control group. However, there was no significant difference for GDF9 or BMP15 mRNA in oocytes from stimulated-PCOS goup compared with unstimulated-PCOS group (P > 0.05, P > 0.05, respectively). The abundance of GDF9 mRNA was significantly lower (P < 0.01) while the abundance of BMP15 mRNA was significantly higher (P < 0.001) in GCs from stimulated-PCOS group than in unstimulated-PCOS group. CONCLUSIONS: The controlled ovarian stimulation can promote the expression of GDF9 and BMP15 both in oocytes and GCs from normal ovulatory women. However, the stimulating effects may be inhibited in oocytes from PCOS patients, which subsequently impair cytoplasm maturation and lead to poor oocyte quality.

Comparison of amino acid metabolism in frozen-thawed and fresh early-stage human embryos.

AIMS: To compare the amino acid differences of changes of frozen-thawed early-stage human embryos and fresh cultured early-stage human embryos. MATERIAL AND METHODS: Discarded embryos and their in vitro culture medium of patients who underwent in vitro fertilization-embryo transfer (IVF-ET) at the Research Center for Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University, from September 2010 to April 2011 were collected. Amino acid levels were determined by high performance liquid chromatography. RESULTS: The amino acid differences of changes in the culture medium of fresh embryos (661.50 µmol/L) were significantly higher than in the medium of post-thawed embryos (232.00 µmol/L) at 0.5 h (P < 0.001). At 1 and 2 h, no significant difference of change was found in all amino acids. Differences in the concentration of amino acids between post-thawed embryos and blank control medium were already present beginning at 1 h. CONCLUSIONS: The level of amino acid metabolism of frozen-thawed early-stage human embryos has already recovered from the state of metabolic stagnation during cryopreservation at 1 h of incubation after thawing, and the amino acid metabolism level at that time approximates that in fresh embryos before freezing. This may be established as the optimal embryo transfer time in IVF-ET.

Day-2 and day-3 sequential transfer improves pregnancy rate in patients with repeated IVF-embryo transfer failure: a retrospective case-control study.

The purpose of this study was to evaluate the effect of sequential embryo transfer in patients with repeated IVF failure. A retrospective matched case-control study was conducted and the outcomes of 213 patients with a history of repeated IVF-embryo transfer failure were analysed, of which 33 women underwent sequential embryo transfer on day 2 and day 3 (D2/D3 group), 66 women on day 3 and day 5 (D3/D5 group), 85 women underwent day-3 embryo transfer only (D3 control group) and 29 women underwent day-5 embryo transfer only (D5 control group) in the assisted reproduction centre of the Sixth Affiliated Hospital of Sun Yat-sen University from August 2010 to December 2011. The results showed that the clinical pregnancy rate of the D2/D3 group was higher than that of the D3 group (48.5% versus 22.4%, P=0.006) while the clinical pregnancy rates of the D3/D5 and D5 groups were not significantly different (50.9% versus 45.8%). Day-2 and day-3 sequential embryo transfer may improve the clinical outcomes for patients with repeated IVF-embryo transfer failures. The purpose of this study was to evaluate the effect of sequential embryo transfer in patients with repeated IVF failure. A retrospective matched case-control study was conducted and the outcomes of 213 patients with a history of repeated IVF-embryo transfer failure were analysed, of which 33 women underwent sequential embryo transfer on day 2 and day 3 (D2/D3 group), 66 women on day 3 and day 5 (D3/D5 group), 85 women underwent day-3 embryo transfer only (D3 control group) and 29 women underwent day-5 embryo transfer only (D5 control group) in the assisted reproduction centre of the Sixth Affiliated Hospital of Sun Yat-sen University from August 2010 to December 2011. The results showed that the clinical pregnancy rate of the D2/D3 group was higher than that of the D3 group (48.5% versus 22.4%, P=0.006) while the clinical pregnancy rates of the D3/D5 and D5 groups were not significantly different (50.9% versus 45.8%). Day-2 and day-3 sequential embryo transfer may improve the clinical outcomes for patients with repeated IVF-embryo transfer failures.

Anti-apoptotic effects of glycine against neuron injury induced by oxygen glucose deprivation / 第二军医大学学报

To investigate the effects of glycine on neuron injury induced by oxygen glucose deprivation (OGD) and the underlying mechanisms. Methods In vitro OGD model was established with cultured rat primary neurons. The study was randomly divided into normal control group, OGD group and glycine treatment group. The influences of glycine on cell activity and survival rate were detected by MTT, and cell apoptosis was detected by flow cytometry. Western blotting analysis was used to examine the effect of glycine on expression of apoptotic proteins Bcl-2, Bcl-xL, Bad, and Bax. Results Glycine could improve the activity and survival of primary neurons of rats and decrease neuron apoptosis. Glycine also significantly increased anti-apoptotic proteins Bcl-2 expression and significantly decreased pro-apoptotic proteins Bax expression (P<0. 05 or P<0. 01). Conclusion Glycine can notably alleviate neuron injury induced by OGD, which is probably through up-regulation of anti-apoptotic factors and down-regulation of pro-apoptotic factors.

[Change and significance of growth differentiation factor 9 and bone morphogenetic protein expression during oocyte maturation in polycystic ovary syndrome patients with ovarian stimulation].

OBJECTIVE: To investigate the expression pattern and significance of two important oocyte-secreted factors: growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) during oocyte maturation in women with polycystic ovary syndrome (PCOS) and infertile women due to husband factors. METHODS: Total of 25 oocytes [9 at germinal vesicle GV stage, 9 at MI stage and 7 at MII stage] were obtained from 12 patients with PCOS and 82 oocytes (29 at GV stage, 26 at MI stage and 27 at MIIstage) were from 56 controls. The nested quantitative real time (RT) PCR was used to detect the abundance of GDF9 and BMP15 mRNA in each oocyte. RESULTS: (1) The expression level of GDF9 mRNA at the GV stage, MI stage and MII stage in PCOS group were 44.8 (4.2 - 529.0), 27.6 (9.8 - 172.7) and 49.0 (0.2 - 65.9) respectively, the expression in were 149.9 (55.4 - 387.9), 29.9 (2.5 - 205.8) and 657.8 (149.4 - 1376.2) in control group, respectively. The expression of GDF9 mRNA at MII stage was significantly lower in PCOS group than in controls (P < 0.01), however, the differences didn't reach statistical significant at GV or MI stage between the two groups (P > 0.05). The expression of GDF9 mRNA displayed some changes at different maturation stage in controls (P < 0.05, P < 0.01), however, the expression didn't demonstrate any dynamic changes in PCOS group (P > 0.05). (2) The expression level BMP15 mRNA at the GV stage, MI stage and MII stage in PCOS group were 0.1 (0.1 - 22.0), 3.2 (0.6 - 55.0) and 6.4 (3.2 - 8.5), respectively, the expression were 41.6 (6.5 - 96.1), 4.0 (2.0 - 10.4) and 49.7 (2.3 - 139.5) in control group, respectively. The expression of BMP15 mRNA at GV stage was significantly lower in PCOS group than in controls (P < 0.01), however, the differences were not significant at MI or MII stage between the two groups (P > 0.05). The expression of BMP15 mRNA also displayed some changes at different maturation stage in controls (P < 0.05), however, the level didn't demonstrate any dynamic changes in PCOS group (P > 0.05). CONCLUSION: It was suggested that the low expression of oocyte secreted factors in mature oocytes from PCOS patients might be associated with impaired oocyte quality and developmental competence in PCOS.

Clinical observation on treatment of primary dysmenorrhea with acupuncture and massage / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the clinical therapeutic effect of acupuncture massage for primary dysmenorrhea and its influence on prostaglandin F2alpha level in menstrual fluid.</p><p><b>METHODS</b>Ninety patients with primary dysmenorrhea were assigned equally to the acupuncture group, the massage group and the control group (oral administrated with Somiton tablet). The treatment course was 3 menstrual cycles. The effect in relieving menalgia was evaluated using numerical rating scale (NRS) before and after treatment and change of PGF2alpha content in menstrual fluid was tested as well.</p><p><b>RESULTS</b>The NRS score was 0.60 +/- 0.67 in the acupuncture group and 0.53 +/- 0.63 in the massage group respectively, significantly lower than that in the control group, which was 2.30 +/- 1.73 (P < 0.01). The PGF2alpha level after treatment was significantly lower in the acupuncture group (176.58 +/- 17.85 ng/L) and the massage group (171.32 +/- 17.15 ng/L) than that in the control group (220.56 +/- 54.47 ng/L), respectively (P <0.01). There showed no difference in the two aspects between the acupuncture group and the massage group (P >0.05).</p><p><b>CONCLUSION</b>Acupuncture and massage has a reliable therapeutic effect on primary dysmenorrhea, which is superior to that of Somiton tablet, and its mechanism might be exerted by regulating PGF2alpha level in menstrual fluid.</p>

[Effects of three-stage rehabilitation treatment on acute cerebrovascular diseases: a prospective, randomized, controlled, multicenter study].

OBJECTIVE: To evaluate the effect of tertiary rehabilitation treatment on acute cerebrovascular diseases. METHODS: Fifteen tertiary rehabilitation networks were set up throughout the country. 1078 patients with acute cerebrovascular diseases were randomly divided into 2 groups: rehabilitation group and control group, out of which 19 patients died, 157 dropped out, and 7 successive evaluations were completed in 902 patients that. 439 of the remaining 902 patients in the rehabilitation group, 266 males and 173 females, aged 61 +/- 11, 278 cases with cerebral infarction and 161 with cerebral hemorrhage, received routine treatment and early rehabilitation for 28 days in the ward of neurology, and then went home and received community rehabilitation for 6 months or underwent specialized reinforcement training for 2 months and after that went home and received community rehabilitation for 4 months. The 463 patients in the control group, 281 males and 182 females, aged 60 +/- 11, 291 of which with cerebral infarction and 172 with cerebral hemorrhage, received only routine treatment and early rehabilitation for 28 days in the ward of neurology, and then went home to conduct rehabilitation training by themselves or their family members for 6 months. Evaluation was conducted 7 times, with National Institutes of Health Stroke Scale (NIHSS), Fugl-Meyer motor function scale, Barthel index, SF-36 scale, Lowenstein occupational therapy cognitive assessment (LOTCA), Westen aphasia battery, Hamilton depression scale, and modified Ashworth spasm scale, one week after the onset and by the ends of 1, 2, 3, 4, 5, and 6 months after the onset respectively. RESULTS: The scores of clinical neurological impairment, Fugl-Meyer scores, SF-36 scores, incidence of PSD, and modified Ashworth scores (for upper and lower limbs) were lower, and LOTCA scores and Barthel indexes were higher at different time points in the rehabilitation group than in the control group; and the differences were statistically significant since the 2nd month after the onset. By the end of the 6th month, the patients of the rehabilitation group basically re-achieved the ability of self-care in daily activities with a Barthel index of 84 +/- 33. The patients of the control group also recovered to a certain degree, however, to a smaller extent in comparison with the rehabilitation group. CONCLUSION: Tertiary rehabilitation treatment of cerebrovascular diseases is effective in improving motor function, ability of daily living activities, and quality of life and reducing the incidence rates of secondary complications.

[Sex determination of human preimplantation embryo using nested polymerase chain reaction].

OBJECTIVE: Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis. METHODS: One (or two) lymphocyte and blastomere (n=50/group) were collected and prepared under the following conditions: (1) water only (H(2)O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65 degree centigrade, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene. RESULTS: The amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method. CONCLUSION: The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.