Effects of crocin supplementation during in vitro culture of intact and half-destroyed four-cell mouse embryos.

OBJECTIVE: We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. METHODS: We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 µg/ml of crocin supplementation (Groups 2 and 4). RESULTS: Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. CONCLUSIONS: Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

The relationship between reactive oxygen species, DNA fragmentation, and sperm parameters in human sperm using simplified sucrose vitrification with or without triple antioxidant supplementation.

OBJECTIVE: This study examined whether the addition of triple antioxidants (3A)-10 µM acetyl-L-carnitine, 10 µM N-acetyl-L-cysteine, and 5 µM α-lipoic acid-in freezing-thawing medium during human sperm cryopreservation using the sucrose vitrification (SuV) and liquid nitrogen vapor (Vapor) techniques could improve post-thaw survival of spermatozoa. METHODS: We analyzed 30 samples from healthy human sperm donors. Each sample was allocated into one of five groups: fresh control, SuV, SuV+3A, Vapor, and Vapor+3A. The sperm motility, morphology, viability, intracellular and extracellular reactive oxygen species (ROS) levels, and sperm DNA fragmentation (SDF) were evaluated. RESULTS: The cryopreserved spermatozoa had significantly reduced percentages of motility (p<0.05) and viability (p<0.05). Antioxidant supplementation non-significantly improved these parameters (p>0.05). No significant differences were found in sperm morphology between the fresh and frozen-thawed groups (p>0.05). After freezing, the extracellular ROS levels in the frozen-thawed groups were significantly higher (p<0.05) than in the fresh group. However, we did not find any differences in intracellular ROS parameters among these groups (p>0.05). The SDF was higher in the SuV and Vapor groups than in the fresh group, but without statistical significance (p=0.075 and p=0.077, respectively). CONCLUSION: Cryopreservation had detrimental effects on sperm motility, viability, and extracellular ROS levels, without changing the morphology or intracellular ROS levels. Antioxidant supplementation was slightly effective in preventing SDF in frozen-thawed spermatozoa.

Vitrification of mouse two-cell and blastocyst stage embryos in simplified closed system using either a hemi-straw or a hollow fiber device.

Two-cell stage and blastocyst stage mouse embryos were equilibrated in a medium containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 8-15 min. Vitrification was performed in a medium containing 0.5 M sucrose and either 15% EG + 15% DMSO, 17.5% EG + 17.5% DMSO, or 20% EG + 20% DMSO for 30 s. They were then placed either on a hemi-straw (HS) or a hollow fiber vitrification (HFV) device and vitrified by cooled air inside a 0.5-ml straw. In two-cell embryos, a 100% survival rate was obtained from all groups except the 20% HS group (P > .05). All vitrified two-cell groups showed similar rates of blastocyst development to that of fresh control group (P > .05), except 17.5% and 20% HFV groups, which were significantly lower than the other groups (P < .05). In the blastocyst embryos, the HFV groups were divided into two subgroups (non-collapsed; HFV-NC and collapsed; HFV-C blastocyst). Re-expansion rate in 15% HFV-NC, 17.5% HFV-NC, and 15% HFV-C groups was reduced (P < .05), whereas the rest were similar to control. In conclusion, we established a simplified, reliable, and closed system for HFV vitrification applying hemi-straw, which does not require skilled practitioners.

Developmental competence of in vitro-matured human oocytes obtained from pregnant and non-pregnant women.

OBJECTIVE: The aim of this study was to compare the rate of maturation, fertilization, and embryo development of in vitro-matured human oocytes derived from pregnant and non-pregnant women. METHODS: Immature oocytes were obtained by needle aspiration from 49 pregnant women (group 1) who underwent a cesarean section at term and 77 non-pregnant women (group 2) who underwent a gynecological operation during the same period (8 months). Healthy immature oocytes (530 in group 1 and 539 in group 2) were cultured and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. RESULTS: The percentage of degenerated oocytes was significantly higher (12.1% vs. 6.3%; p<0.001) in group 1 than in group 2. There was no significant difference in the maturation rate (66.8% vs. 68.1%; p=0.698), fertilization rate (66.7% vs. 67.6%; p=0.857), or the rate of formation of good-quality blastocysts (46.2% vs. 47.2%; p=0.898) in oocytes obtained from pregnant and non-pregnant women. CONCLUSION: The developmental competence of immature oocytes did not differ between pregnant and non-pregnant women.

Rapid freezing versus Cryotop vitrification of mouse two-cell embryos.

OBJECTIVE: To compare our in-house method of embryo freezing with Cryotop vitrification in terms of immediate survival, subsequent cleavage and blastocyst formation, and cell numbers in blastocysts. METHODS: Two-cell mouse embryos were randomly allocated into three groups: a non-frozen control group (group 1, n=300), a group that underwent Cryotop vitrification (group 2, n=300), and a group that underwent our in-house freezing method (group 3, n=300). RESULTS: There were no significant differences between groups 2 and 3 in the immediate survival rate (96.3% vs. 98.6%, respectively; p=0.085), the further cleavage rate (91.7% vs. 95.0%, respectively; p=0.099), or the blastocyst formation rate (80.7% vs. 78.6%, respectively; p=0.437). The cell numbers in the blastocysts from groups 1, 2, and 3 were comparable (88.99±10.44, 88.29±14.79, and 86.42±15.23, respectively; p=0.228). However, the percentage of good-quality blastocysts in the Cryotop vitrification group was significantly higher than in the group in which our in-house method was performed, but was lower than in the control group (58.0%, 37.0%, and 82.7%, respectively; p<0.001). CONCLUSION: At present, our method is inferior to the commercial Cryotop vitrification system. However, with further improvements, it has the potential to be useful in routine practice, as it is easier to perform than the current vitrification system.

The effect of the site of laser zona opening on the complete hatching of mouse blastocysts and their cell numbers.

OBJECTIVE: We studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts. METHODS: Mouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125). RESULTS: The rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group (69±19.3, 74±15.7, and 71±16.8, respectively; p=0.680). CONCLUSION: These findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers.

Comparison of blastocyst and Sage media for in vitro maturation of human immature oocytes.

In vitro maturation (IVM) of human oocytes is an attractive alternative to conventional assisted reproductive technology (ART) treatment, as it involves no or minimal ovarian stimulation. Currently, commercialized media specifically designed for IVM are often used. These media are expensive, have limited shelf life, and must be ordered in advance. If standard culture media can be used in place of the specialized IVM media, it would simplify management and make IVM more feasible and more widely employed in ART centers around the world, especially in developing countries where resources are scarce. This study was, therefore, conducted to test the hypothesis that blastocyst medium was as good as commercial IVM medium to support maturation and developmental competence of human immature oocytes as previously shown in the mouse system. Immature oocytes were obtained by needle aspiration from 89 pregnant women during cesarean deliveries between April 2012 and February 2013. Sibling oocytes were allocated to Sage IVM media (512 oocytes) or blastocyst medium (520 oocytes) and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. There was no difference in maturation rate (65.0% vs 68.7%; P = .218) or fertilization rate (66.9% vs 66.4%; P = .872) of oocytes matured in vitro in both media. There was also no difference in the formation of good-quality blastocysts (46.6% vs 45.9%; P = .889) in the 2 groups. Further study should be done to ascertain implantation and pregnancy potential of these embryos.

Impact of hemostasis methods, electrocoagulation versus suture, in laparoscopic endometriotic cystectomy on the ovarian reserve: a randomized controlled trial.

OBJECTIVE: To evaluate the impact on ovarian reserve between two different methods ofhemostasis after laparoscopic ovarian endometrioma excision. MATERIAL AND METHOD: A randomized controlled study was conducted from January to December 2013 in Thammasat University Hospital, Thailand. Reproductive women, age 18-45years who underwent laparoscopic ovarian cystectomy were randomized in electrocoagulation and suture groups. Clinical baseline data and ovarian reserve outcome (anti-Mullerian hormone (AMH)) were evaluated. RESULTS: Fifty participants were recruited and randomized in two groups. Electrocoagulation and suture groups consisted of 25 participants. Baseline characteristics between 2 groups (age, weight, BMI, height, cyst diameter, duration and estimated blood loss) were not statistically different. There were no significant difference of AMIH between electrocoagulation and suture group atpre-operative (2.90±2.26 vs. 2.52±2.37 ng/ml), 1 week (1.78±1.51 vs. 1.99±1.71 ng/ml), 1 month (1.76±1.50 vs. 2.09±1.62 ng/ml), 3 months (2.09±1.66 vs. 1.96±1.68 ng/ml) and 6 months (2.11±1.84 vs 1.72±1.68 ng/ml), respectively. However mean AMH ofboth groups significantly decreased since the first week of operation. Effect oflaparoscopic ovarian surgery had significantly declined and sustained AMH level until 6 months. CONCLUSION: Laparoscopic cystectomy of ovarian endometrioma has negative impact to ovarian reserve. Either electroco- agulation or suture method had no different effects.

Follicle-stimulating hormone receptor gene polymorphism in chronic anovulatory women, with or without polycystic ovary syndrome: a cross-sectional study.

BACKGROUND: Polymorphisms at codons 307 and 680 are the most commonly encountered allelic variants of the follicle-stimulating hormone receptor (FSHR) gene. Studies in Caucasians suggest that certain FSHR variants are more common in women with polycystic ovary syndrome (PCOS) than normal women. The objective of this study was to determine the distribution of FSHR gene polymorphisms at codons 307 and 680 in Thai women with chronic anovulation, without (121 women) and with PCOS (133 women), using 132 known fertile women as controls. METHODS: DNA samples from peripheral blood lymphocytes were extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The prevalence of Threonine307Threonine (TT), Threonine307Alanine (TA), and Alanine307Alanine (AA) genotypes at codon 307 was 53.0% (95% CI = 44.2-61.7%), 42.4% (95% CI = 34-51.3%), and 4.5% (95% CI = 1.9-10.1%) in controls; 52.6% (95% CI = 43.8-61.3%), 39.8% (95% CI = 31.6-48.7%), and 7.5% (95% CI = 3.9-13.7%) in PCOS women; and 50.4% (95% CI = 42.8-61.2%), 45.4% (95% CI = 34.9-53.1%), and 4.5% (95% CI = 1.5-9.6%) in anovulatory women without PCOS, respectively. The prevalence of Asparagine680Asparagine (NN), Asparagine680Serine (NS), and Serine680Serine (SS) genotypes at codon 680 was 54.5% (95% CI = 45.7-63.2%), 40.9% (95% CI = 32.5-49.8%), and 4.5% (95% CI = 1.9-10.1%) in controls; 51.9% (95% CI = 43.1-60.6%), 44.4% (95% CI = 35.8-53.2%), and 3.8% (95% CI = 1.4-9.0%) in PCOS women; and 47.9% (95% CI = 40.4-58.8%), 47.1% (95% CI = 36.5-54.7%), and 5.0% (95% CI = 2-10.9%) in anovulatory women without PCOS, respectively. The prevalence of FSHR gene polymorphisms at both codons were not statistically different among the three groups. CONCLUSIONS: In Thai women, there was no association between the FSHR gene polymorphism at codons 307 and 680 and chronic anovulation.

Relaxation for perimenopausal and postmenopausal symptoms.

BACKGROUND: Since the time of publication of the Women's Health Initiative (WHI) study, menopausal symptom management has become more complex because of increased awareness of the risks associated with hormone replacement therapy (HRT). Currently, a wide range of management options is available. Some women take prescription drugs, and others use self care strategies, including lifestyle modifications, over-the-counter preparations and complementary and alternative therapies, such as herbal preparations, exercise programmes and relaxation techniques. Relaxation techniques consist of a group of behavioural interventions. They are considered relatively harmless, but their effectiveness in treating vasomotor symptoms and sleep disturbances remains debatable.  OBJECTIVES: To determine the effectiveness of relaxation techniques as treatment for vasomotor symptoms and associated sleep disturbances in perimenopausal and postmenopausal women. SEARCH METHODS: Searches of the following electronic bibliographic databases were performed in February 2014 to identify randomised controlled trials (RCTs): the Cochrane Menstrual Disorders and Subfertility Group Specialised Trials Register; the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, AMED, PsycINFO, Social Science Citation Index and CINAHL. Handsearches of trial registers, relevant journals and published conference abstracts were also performed. SELECTION CRITERIA: RCTs were included if they compared any type of relaxation intervention with no treatment or other treatments (except hormones) for vasomotor symptoms in symptomatic perimenopausal/postmenopausal women. DATA COLLECTION AND ANALYSIS: Two review authors selected studies, assessed quality and extracted data. Included studies were combined, if appropriate, by using a random-effects model to calculate pooled mean differences and 95% confidence intervals. MAIN RESULTS: Four studies were eligible for inclusion (281 participants): Two studies compared relaxation with electroacupuncture or superficial needling, one study compared relaxation with paced respiration or placebo control (α-wave electroencephalographic biofeedback) and one study compared relaxation with no treatment.No evidence was found of a difference between relaxation and acupuncture or superficial needle insertion in the number of hot flushes per 24 hours (mean difference (MD) 0.05, 95% confidence interval (CI) -1.33 to 1.43, two studies, 72 participants, I(2) = 0%; very low-quality evidence). Nor did any evidence suggest a difference between the two interventions in hot flush severity, measured using the Kupperman Index (MD -1.32, 95% CI -5.06 to 2.43, two studies, 72 participants, I(2) = 0%; very low-quality evidence).The other two studies found no clear evidence of a difference in hot flush frequency between relaxation and paced respiration, placebo or no treatment. The data for these comparisons were unsuitable for analysis.None of these studies reported night sweats, sleep disturbances associated with night sweats or adverse effects as an outcome.The main limitations of identified evidence were lack of data, imprecision and failure to report study methods in adequate detail. AUTHORS' CONCLUSIONS: Evidence is insufficient to show the effectiveness of relaxation techniques as treatment for menopausal vasomotor symptoms, or to determine whether this treatment is more effective than no treatment, placebo, acupuncture, superficial needle insertion or paced respiration.

Developmental competence of human embryos derived from in vitro maturation of immature oocytes retrieved during cesarean section.

AIM: To investigate the developmental competence of human embryos that originated from in vitro matured oocytes retrieved during cesarean section. METHODS: Immature oocytes were collected from 95 pregnant women, who underwent cesarean section at Buddhachinaraj Hospital Medical School and consented to participate in the study. Retrieved oocytes were cultured in blastocyst medium supplemented with 75 IU/L of human menopausal gonadotropin. Oocyte maturation was assessed at 30 and 48 h after culture. In vitro matured oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 h. The fertilization, cleavage and blastocyst formation rates were observed. RESULTS: Maturation rate of oocytes after 30 h of culture was 67.9% compared with 13.1% at 48 h (P < 0.0001). Insemination of oocytes in both groups resulted in similar fertilization, cleavage and blastocyst formation rates. CONCLUSION: A large proportion of oocytes retrieved at the time of cesarean section exhibited the capacity to undergo maturation in vitro. They can be fertilized and developed into good-quality blastocyst stage.

Antimullerian hormone changes after laparoscopic ovarian cystectomy for endometrioma compared with the nonovarian conditions.

Laparoscopic ovarian cystectomy is recommended for surgical procedure of endometrioma. The negative impact on ovarian reserve following removal had been documented. Little evidence had been reported for nonovarian originated effects. Objective. To evaluate the impact of laparoscopic ovarian cystectomy for endometrioma on ovarian reserve, measured by serum antimullerian hormone (AMH), compared to nonovarian pelvic surgery. Materials and Methods. A prospective study was conducted. Women who underwent laparoscopic ovarian cystectomy (LOC) and laparoscopic nonovarian pelvic surgery (NOS) were recruited and followed up through 6 months. Clinical baseline data and AMH were evaluated. Results. 39 and 38 participants were enrolled in LOC and NOS groups, respectively. Baseline characteristics (age, weight, BMI, and height) and preoperative AMH level between 2 groups were not statistically different. After surgery, AMH of both groups decreased since the first week, at 1 month and at 3 months. However, as compared to the LOC group at 6 months after operation, the mean AMH of the NOS group had regained its value with a highly significant difference. Conclusion. This study demonstrated the negative impact of nonovarian or indirect effects of laparoscopic surgery to ovarian reserve. The possible mechanisms are necessary for more investigations.

Effectiveness of a modified version of the applied relaxation technique in treatment of perimenopausal and postmenopausal symptoms.

BACKGROUND: Awareness of the risks associated with hormone therapy for menopausal symptoms has sparked a global decline in this treatment. Alternative treatments to relieve menopausal symptoms are therefore required. The applied relaxation (AR) technique has proven to be successful for symptom amelioration, but requires participation in 12 weekly classes. The purpose of this study was to determine the effectiveness of a modified relaxation version (MR) of AR for treatment of hot flashes, night sweats, and sleep disturbances. METHODS: We conducted a12-week, randomized, parallel, open-label, controlled trial in perimenopausal and postmenopausal women visiting the menopausal clinic. Participants were randomly assigned to an MR or AR group. The MR group (n=36) received a single session of (MR) training and the AR group (n=35) received conventional 12-week training. Participants were instructed to practice the techniques daily at home for 12 weeks. The main outcome was the measure on the severity scale and frequency of hot flashes, night sweats, and sleep disturbances. RESULTS: All participants completed the study. Total severity scores in both groups decreased after 12 weeks, but there was no difference between the groups (P=0.93). The severity score for hot flashes in the MR group decreased more than in the AR group (P=0.02). The severity scores for night sweats and sleep disturbances decreased in both groups. The frequency of hot flashes, night sweats, and sleep disturbances were also decreased in both groups. CONCLUSION: A shorter, modified version of the AR was equally effective or slightly better than the conventional AR for the relief of hot flashes, night sweats, and sleep disturbances in perimenopausal and postmenopausal women. Recommendations for future research include confirmatory studies and trials with larger samples.

Modified relaxation technique for treating hypertension in Thai postmenopausal women.

AIM: To examine the effectiveness of a modified relaxation (MR) technique in reducing blood pressure levels in Thai postmenopausal women with mild hypertension, compared with a control group who received health education. METHODS: This is a 16-week, randomized, parallel, open-label, controlled trial in a menopausal clinic in a tertiary health care center in Northeastern Thailand. The intervention group received a 60-minute session of MR training and were encouraged to practice 15-20 minutes a day, at least 5 days a week. The control group received lifestyle education, including diet and exercise. The primary and secondary outcomes were systolic and diastolic blood pressure (SBP and DBP). RESULTS: Of 432 participants, 215 and 217 were randomly allocated to the MR and control groups, respectively. Of those, 167 participants in the MR group and 175 participants in the control group completed the study. The SBP was significantly more reduced in the MR group, with a mean of 2.1 mmHg (P < 0.001). There was no significant difference between groups on the changed DBP. CONCLUSION: The MR technique may be effective in lowering SBP in Thai postmenopausal women visiting a menopause clinic. Its efficacy may be observed as soon as 4 weeks after start of treatment. Long-term and combined relaxation therapy and antihypertensive agents are warranted in a large cohort of this population. This trial is registered in clinicaltrials.gov (number NCT01429662).

Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers.

BACKGROUND: It is still debatable whether a full-thickness assisted hatching (AH) is better than the partial zona thinning. In this research, we used a mouse model to study the effect of partial and complete laser-AH on the rate of completely hatched blastocyst and their cell numbers. METHODS: In experiment 1, mouse morulae had 0, 1, 2 or 3 full-thickness openings of 10 microns created in the zona pellucida with an infrared laser beam. In the second experiment, 0, 1 and 2 openings of 20 microns were studied. In the third experiment, a full-thickness opening of 20 microns or quarter-thinning of the zonal circumference to a depth of 90% was compared with non-AH controls. RESULTS: No difference in blastocyst formation was found in laser-treated groups and in the controls. In experiment 1, the rate of completely hatched blastocysts was significantly lower than the controls. In experiment 2 when the size of the opening was increased, blastocysts completely hatched at a significantly higher rate than that in the controls. In experiment 3, the rate of completely hatched blastocysts was the highest in the full-thickness group. Cell numbers in completely hatched blastocysts from both AH groups were significantly fewer than those in the controls. CONCLUSIONS: Full-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased.

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA.

In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at ×6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs. 1.7%; P=0.032), with no significant difference in aneuploidy rate (0.8% vs 0.7%; P=0.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

Effects of ovarian endometriotic fluid exposure on fertilization rate of mouse oocytes and subsequent embryo development.

BACKGROUND: Accidental exposure of oocyte/cumulus complex to endometriotic fluid is not uncommon during oocyte retrieval. Only two studies were available on this subject and they gave conflicting results. In this study, we used a mouse model to evaluate the effect of controlled exposure of oocytes to ovarian endometriotic fluid. METHODS: Mouse oocytes/cumulus complexes (n = 862) were divided into 4 groups, and were exposed to endometriotic fluid (group 1), pooled sera from subjects without endometrioma (group 2), phosphate-buffered saline (group 3), and fertilization medium (controls). After five minutes, oocytes were washed and inseminated. Embryo development was observed daily. The quality of hatching blastocysts was assessed by counting the number of inner cell mass (ICM) and trophectoderm (TE) cells. RESULTS: The fertilization, cleavage and blastocyst formation rates in the four groups were not statistically different. The proportions of hatching/hatched blastocysts from fertilized oocytes in groups 1 and 2 were significantly lower than those in group 3 and controls (P = 0.015). Hatching blastocysts from all groups showed no significant difference in the number of ICM and TE cells. CONCLUSIONS: Exposure of mouse oocytes/cumulus complexes to endometriotic fluid had subtle detrimental effects on subsequent blastocyst development. However, one should be cautious in projecting the results of this study to contaminated human oocytes in a clinical setting.

Factors affecting pregnancy rates after microsurgical reversal of tubal sterilization.

The aim of this study was to determine the prognostic factors and pregnancy rates after microsurgical reversal of tubal sterilization. Patients undergoing tubal anastomosis from 2001 to 2008 were included. Relevant data were extracted from their medical records. Pregnancy outcomes were ascertained by responses to mailed questionnaires and telephone contact. A total of 98 patients were identified. We found that the mean duration of follow-up was 67 ± 28 months. Fifty-five patients conceived (pregnancy rate 62.5%; 95% confidence interval [CI] 52 to 72.8%). Of these, 50 were intrauterine and 5 were tubal pregnancies. Life-table analysis estimated cumulative pregnancy rates to be 30.7%, 39.8%, 49%, and 53.7% at 6, 12, 18, and 24 months after reversal, respectively. Age at the time of reversal was the only significant prognostic factor multivariate model. We concluded that age of the patient at the operation is the most important prognostic factor.

Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing.

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.

Preimplantation genetic diagnosis of alpha-thalassemia-SEA using novel multiplex fluorescent PCR.

PURPOSE: Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis (PND) giving couples at risk a chance to start a pregnancy with a disease-free baby. This study aimed to develop a new PGD protocol for alpha-thalassemia(-SEA) mutation, the commonest Mendelian disorder. PATIENTS AND METHODS: Multiplex fluorescent PCR was employed for mutation, contamination and linkage analysis. A couple experienced termination of pregnancy following positive PND decided to join the project. RESULTS: Novel primers for alpha-thalassemia(-SEA) mutation amplifying 5 DNA fragments were developed. Two PGD cycles were performed, resulting in an un-affected baby. PND confirmed the heterozygous result. From 24 embryos, 87.5% of affected genotype were of best quality compared to 0% and 18.2% of those with normal and heterozygous, respectively. CONCLUSIONS: A novel PCR protocol for the common alpha-thalassemia(-SEA) mutation is reported. This test should be widely applicable. Interestingly, a potential effect of alpha-thalassemia(-SEA) mutation on preimplantation embryonic development was noticed.