A modified hydrostatic microfluidic pumpless device for in vitro murine ovarian tissue culture as research model for fertility preservation.

This study aimed to compare the efficacies of conventional and non-conventional (modified hydrostatic microfluidic pumpless device, MHPD) systems on ovarian tissue culture and in vitro follicle growth using a mouse model. A total of 56 ovarian cortical tissues retrieved from seven wild-type mice were divided into three groups: 1) fresh control, 2) conventional culture system (control), and 3) non-conventional system with MHPD. Ovarian tissues were cultured for 96 hours and evaluated for follicle morphology, developmental stage, intact follicle density, and relative gene expression levels (proliferating cell nuclear antigen, insulin like growth factor 1, BAX, and Bcl-2). Our major data demonstrated that the mean percentage of primary follicle development was increased by the MHPD (P<0.05). In addition, this device could maintain and support follicle development better than the conventional culture systems. However, the overall outcomes were not significantly improved by our first-design prototype. Consequently, nextgeneration platforms should be developed as alternative medical tools for fertility preservation research.

Influence of hydrogel encapsulation during cryopreservation of ovarian tissues and impact of post-thawing in vitro culture systems in a research animal model.

OBJECTIVE: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality. METHODS: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. RESULTS: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2%±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls. CONCLUSION: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.

Epigenetic modification of long interspersed elements-1 in cumulus cells of mature and immature oocytes from patients with polycystic ovary syndrome.

OBJECTIVE: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. METHODS: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. RESULTS: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. CONCLUSION: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.

Concurrent bilateral pheochromocytoma and thoracic paraganglioma during pregnancy.

Although hypertension occurring during pregnancies is not uncommon and its prognosis is generally excellent, some of its unusual causes can lead to catastrophic consequences, especially in undiagnosed cases. Here, we report a pregnant woman who presented with hypertension in her early pregnancy. It was subsequently found to be caused by bilateral pheochromocytoma. After removal of both tumors, catecholamine levels unexpectedly and unexplainably remained elevated. At 23 weeks of gestation, the fetus was found dead in utero. After the fetal death, additional studies were performed and revealed a thoracic paraganglioma. To our knowledge, this is the first report of a case of three catecholamine-producing tumors occurring concurrently during a pregnancy. Genetic analysis helped identify this unprecedented condition; the patient harbored a heterozygous missense mutation c.482G>A in exon 3 of the VHL gene, indicating von Hippel-Lindau syndrome. Physicians who care for hypertensive pregnant patients should be aware of this condition as its diagnosis would probably lead to a better outcome.

The salivary ferning test and ovulation in clomiphene citrate-stimulated cycles.

OBJECTIVE: To determine the day of ovulation by the salivary ferning test in clomiphene citrate-treated women. DESIGN: A descriptive study. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. SUBJECT: Seventy-five infertile women with regular menstrual cycles. MATERIAL AND METHOD: Infertile women were given 100 mg of clomiphene citrate for five days and collected their saliva samples daily until seven days after ovulation. Transvaginal ultrasound was performed daily to detect ovulation. The salivary ferning formation was examined by a normal light microscope and graded from 1-3, according to its extent and intensity. MAIN OUTCOME MEASURE: The salivary ferning score, the peak salivary ferning day, and the day of ovulation detected by ultrasound. RESULTS: The patients' age and cycle length (mean +/- SD) were 32.9 +/- 3.7 years and 28.4 +/- 1.3 days. The peak salivary ferning day corresponded with the ultrasound ovulation day in only 7.1%. There were two peaks of median salivary ferning scores; one was two days prior ovulation and the other was five days post ovulation. There was no correlation between the peak salivary ferning day and day of ovulation detected by ultrasound (r = 0.102, p > 0.05). CONCLUSION: In clomiphene citrate-stimulated cycles, the saliva ferning test does not seem to associate with ovulation.

Comparison of two different fixed doses of follitropin-beta in controlled ovarian hyperstimulation: A prospective randomized, double blind clinical trial.

A prospective randomized, double blind, single centre study was conducted to compare the efficacy, efficiency and clinical side effects of daily fixed dose regimen of either 100 IU or 200 IU of recombinant follicle stimulating hormone(rFSH) Follitropin beta in down-regulated women undergoing controlled ovarian hyperstimulation(COH) for either conventional in vitro fertilization(IVF) or intracytoplasmic sperm injection(ICSI). A total of sixty women were randomly allocated according to the criteria for the treatment by either 100 IU(n = 30) or 200 IU (n = 30) of FSH. Although more cycle cancellations due to low response were observed in the 100 IU group (n = 9 vs n = 2), two cases of mild and moderate ovarian hyperstimulation syndrome were noted in the higher dose group. Subjects in the group treated with 200 IU appeared to yield more follicles > 17 mm (4.4 vs 3.3, p = 0.05) and more oocytes compared to the group treated with 100 IU (9.2 versus 6.0 oocytes, NS). The total dosage required to develop at least three follicles according to the protocol was significantly lower in the group treated with 100 IU (1203.33 versus 2106. 67, P < 0.0001). In conclusion, a fixed daily dose of 200 IU of rFSH Follitropin beta compared to a fixed daily dose of 100 IU is more effective in terms of follicles > 17 mm development and the number of oocytes retrieved along with a lower cancellation rate, but less efficient as indicated by a higher total rFSH dose needed

Endometrial progesterone and estrogen receptors and bleeding disturbances in depot medroxyprogesterone acetate users.

BACKGROUND: Depot medroxyprogesterone acetate (DMPA) is a very popular hormonal contraceptive. Unpredictable bleeding disturbances are the main reasons for discontinuation and may be mediated through endometrial hormone receptors. This study was aimed to compare the expression of progesterone and estrogen receptors in the endometrium of bleeding DMPA users with that of amenorrhoeic DMPA users. METHODS: Subjects were recruited between April 2000 and January 2001. On the day of the third DMPA injection, 42 amenorrhoeic DMPA users and 42 DMPA users who had frequent or prolonged endometrial bleeding and were bleeding on that day were matched by age and body mass index. Endometrial biopsies were collected for immunohistochemical study of progesterone receptor A plus B (PRAB) and B alone (PRB) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression. RESULTS: There were 23 adequate endometrial samples from each group. There were no differences in any of a series of comparisons of PRAB, PRB, ERalpha and ERbeta expression in glands or stroma between the groups. Serum estradiol and progesterone levels were also not different between the groups. CONCLUSIONS: Endometrial PRAB, PRB, ERalpha and ERbeta expression in glands and stroma was not different between DMPA users who had frequent or prolonged bleeding and amenorrhoeic DMPA users.

Correlation between human follicular diameter and oocyte outcomes in an ICSI program.

PURPOSE: To determine the correlation between the follicular sizes and oocyte recovery, metaphase II oocyte recovery, fertilization rate and good embryo quality from mature and immature oocytes in an intracytoplasmic sperm injection (ICSI) program. METHODS: 991 follicles obtained from 72 ICSI cycles were classified into three groups according to their diameters as measured by transvaginal ultrasound including group A (< 10 mm), group B (10-14 mm), and group C (> 14 mm). All obtained oocytes were classified according to their nuclear maturation: germinal vesicle (GV), metaphase I (MI) and metaphase II (MII). Mature oocytes underwent ICSI while immature oocytes were further cultured until maturity before ICSI was performed. The rates of fertilization and good quality embryos at day 3 were evaluated. RESULTS: A progressive and significant increase in the rates of oocyte recovery and MII oocyte recovery were observed from group A follicles compared to the other groups (p < 0.001). The fertilization rate of mature and in vitro matured oocytes, as well as the rate of good quality embryos showed a tendency to increase from group A to group C follicles, but not significantly. The corresponding fertilization rates were 78 and 55.3% (p < 0.001) for mature and in vitro matured oocytes, respectively. CONCLUSION: Collection of oocytes from small follicles, especially with a mean diameter less than 10 mm, and in vitro maturation of immature oocytes before fertilization may allow the total number of good quality and transferable embryos to be increased.

The effect of growth hormone on the development of in vitro matured unstimulated human oocytes.

OBJECTIVE: To investigate the effect of growth hormone on the development of in vitro matured unstimulated human oocytes. DESIGN: Randomized controlled study. SETTING: Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn university. MATERIAL AND METHOD: 108 germinal vesicle-stage oocytes were retrieved from 47 patients undergoing gynecologic surgery. They were aspirated either during gynecologic surgery or from excised ovaries. The oocytes were then cultured in vitro with or without growth hormone (1,000 ng/ml) in medium199 supplemented with sodium pyruvate, FSH, LH, antibiotic and synthetic serum. Incubation was done at 37 degree C with 5 per cent CO2 in air and nuclear stage was assessed after 18, 42, 66 and 90 h of incubation. MAIN OUTCOME MEASURE: Attainment of metaphase II and GVBD RESULTS: After in vitro culture, there were no significant differences in maturation and GVBD rate. 27 of 52 (51.9%) oocytes (GV) in growth hormone group matured to metaphase II compared with 25 of 53 (47.2%) GV in control group. GVBD rate for germinal vesicle-stage in growth hormone group was 76.9 per cent compared with 79.2 per cent in control group. CONCLUSION: Culture of immature oocytes in vitro with growth hormone results in similar maturation rate as that without GH.

Predictive value of human chorionic gonadotropin in the outcome of early pregnancy achieved by assisted reproductive technology.

This study was undertaken to evaluate the predictive value of the serum human chorionic gonadotropin (hCG) in pregnancies achieved by assisted reproductive techonology (ART). Two hundred and eighty-six pregnancies were studied retrospectively from September 1989 to June 1998. The serum hCG samples at 2-6 weeks after embryo transfer (ET) were analysed by fluoroimmunoassay. Pregnancy status was followed by ultrasonography. There were 100 nonviable pregnancies (NP), 140 viable single pregnancies (VSP) and 46 viable multiple pregnancies (VMP). The sensitivity, specificity, positive and negative predictive value of the D14 hCG (<160 mIU/ml) in distinguishing NP from VSP were 79 per cent, 75 per cent, 68 per cent and 84 per cent, respectively and of the D14 hCG (>350 mIU/ml) in distinguishing VMP from VSP were 82 per cent, 75 per cent, 56 per cent and 91 per cent, respectively. In conclusion, the serum hCG may be used to predict the outcome of early pregnancy achieved by ART.

Effects of initiation day of clomiphene citrate on the endometrium of women with regular menstrual cycles.

OBJECTIVE: To investigate the effects of initiation time of clomiphene citrate (CC) on the endometrium of women with regular menstrual cycles. DESIGN: Randomized, double-blind, cross-over study. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. PATIENT(S): Thirty-three healthy female volunteers with regular menstrual cycles. INTERVENTION(S): The volunteers were randomized to receive either 100 mg of CC on days 1-5 and placebo on days 5-9 (study group) or placebo on days 1-5 and CC on days 5-9 (control group). After a wash-out period of 1 month of CC treatment, the medication was switched in each group. Ultrasonography was performed daily after day 10 of the cycle to detect ovulation. Ultrasonography for endometrial appearance and thickness, endometrial sampling, and blood samples obtained for determination of E(2) and P levels were performed 7 days after ovulation in both groups. MAIN OUTCOME MEASURE(S): Morphometric analysis, histologic dating, and ultrasonographic appearance and thickness of the endometrium. RESULT(S): Morphometric parameters, histologic dating, and ultrasonographic appearance and thickness of the endometrium were similar in both groups. CONCLUSION(S): Starting CC on either day 1 or day 5 of the menstrual cycle did not have any differential effects on the endometrium of women with regular menstrual cycles, particularly regarding the morphometric analysis, histologic dating, or ultrasonographic appearance.