Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol.

RESEARCH QUESTION: Can immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGN: This study examined modifications of oocyte vitrification and warming protocols that reduce the length of exposure to vitrification and warming solutions. In total, 561 germinal vesicles and 218 metaphase I oocytes that were immature at oocyte retrieval were vitrified at room temperature for 2 min. Warming was performed at 37°C for 2 min. Resumption of meiotic activity was evaluated after 24 and 48 h of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTS: Ninety-five percent of germinal vesicles survived, with no difference observed between the kits. The survival of metaphase I oocytes was, on average, 95.4% and did not differ significantly between the kits. Of the 533 germinal vesicles that survived, 491 converted to metaphase I oocytes (92.1%). After culture for 48 h, 54.4% converted to metaphase II oocytes. In addition, of the 208 metaphase I oocytes that survived warming, 84.1% converted to metaphase II oocytes after 24 h of culture. These maturation rates were similar to those of non-vitrified oocytes. CONCLUSIONS: Vitrification and warming of oocytes at different nuclear maturation stages can be performed with 2 min of exposure to hypertonic solution and 2 min of exposure to hypotonic solution, respectively. This approach reduces exposure of the oocytes to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential of oocytes to resume nuclear meiotic activity, and conversion from germinal vesicles to metaphase I and metaphase II oocytes.

The effect of nonsteroidal anti-inflammatory drugs on ovulation: a prospective, randomized clinical trial.

OBJECTIVE: To assess the effect of ibuprofen, a nonspecific inhibitor of prostaglandin synthesis, on ovulation. DESIGN: Prospective, randomized, double-blind, placebo-controlled cross-over study. SETTING: University Medical Center. PATIENT(S): Twelve normally cycling women between ages 20 and 40. INTERVENTION(S): Subjects were randomized to either oral ibuprofen (800 mg) or placebo three times per day, beginning when the maximum diameter of the leading follicle reached 16 mm by ultrasound, and continuing for 10 days total. The second cycle was a washout period, and in the third cycle, the subjects were crossed over to the alternate regimen from the first cycle. The probability of delayed follicular collapse was determined using the binomial distribution, and changes in P levels were compared using the paired t test. MAIN OUTCOME MEASURE(S): Urinary LH surge, follicular collapse by serial transvaginal ultrasonography, and serum midluteal P levels. RESULT(S): Eleven of 12 subjects detected an LH surge with both ibuprofen and placebo. Five of 11 women demonstrated a >or=2-day increase in time interval from detection of the LH surge to follicular collapse, and 3 of those 5 had been randomized to ibuprofen. This represents a 27% (3 of 11; 95% confidence limits: 1%, 53%) rate of delay for follicular collapse for ibuprofen. There was no difference in average midluteal P levels for ibuprofen or placebo. CONCLUSION(S): If ibuprofen inhibits follicular collapse, this effect is seen in a small group of study subjects, and this information should be clinically reassuring to patients who take nonsteroidal anti-inflammatory drugs. Serum midluteal P levels were unaffected by administration of ibuprofen.

Perforated duodenal ulcer associated with ovarian hyperstimulation syndrome: Case Report.

Ovarian hyperstimulation syndrome (OHSS) remains the most serious medical complication of controlled ovarian stimulation. An unusual case of perforated duodenal ulcer following critical OHSS is presented. A 29 year old nulligravid woman with polycystic ovarian syndrome underwent her first attempt at in-vitro fertilization. She was admitted to the hospital with critical OHSS and subsequently found to have a perforated posterior duodenal ulcer. She underwent exploratory laparotomy, antrectomy and gastrojejunostomy. Pathological analysis of her gastric antrum confirmed chronic gastritis and Helicobacter pylori. She required prolonged assisted ventilation, vasopressor support, multiple i.v. antibiotics, blood product replacement and nutritional support. The patient was hospitalized for a total of 47 days and then transferred to a rehabilitation facility for an additional 30 days before being discharged to home. In this critically ill patient with OHSS, severe stress associated with invasive monitoring and multiple medical therapies in the intensive care unit as well as H. pylori infection appear to be the most probable causative factors of her perforated viscus. Prompt recognition of potential complications and proper medical intervention are essential in the management of patients with OHSS. Avoidance strategies are still needed.

Estrogen replacement therapy and gallbladder disease in postmenopausal women.

OBJECTIVE: To examine the role of estrogen replacement therapy on the development of gallbladder disease in postmenopausal women. DESIGN: Systematic review of the English literature was conducted. All studies that addressed the association between hormone replacement therapy and gallbladder disease published from 1970 to the present were reviewed. RESULTS: Seven observational studies, two clinical trials, two case series, and one nonrandomized and three randomized investigations were reviewed. The results of each study were reported and analyzed. CONCLUSIONS: Estrogen replacement therapy in postmenopausal women increased the chances for gallstone formation.

Comparison of the impact of transdermal versus oral estrogens on biliary markers of gallstone formation in postmenopausal women.

This prospective, randomized, double blind, parallel study was undertaken to elucidate further the potential mechanisms through which estrogens could promote the formation of cholesterol gallstones and to compare the impact of nonoral (transdermal) and oral estrogens on serum, hepatic, and biliary markers of estrogen action. Ninety-seven postmenopausal women were randomized to receive either transdermal estradiol (E2; 0.1 mg every 3.5 days; n = 48) or oral conjugated equine estrogens (1.25 mg every day; n = 49) for 8 weeks. Blood samples were drawn, and bile samples were obtained by cholecystokinin-stimulated duodenal drainage before and after 8 weeks of estrogen administration. The main outcome measures included serum FSH, LH, E2, estrone, estrone sulfate, sex hormone-binding globulin, lipid profiles, biliary cholesterol saturation index, cholesterol nucleation time, presence of cholesterol crystals in bile, as well as biliary arachidonate, PGE2, and mucous glycoproteins. Estrogens administered by both routes increased circulating estrogens and resulted in similar suppression of both gonadotropins. Sex hormone-binding globulin was clearly increased, and the changes in serum lipids were more pronounced with oral conjugated equine estrogens than with transdermal E2. The biliary cholesterol saturation index was significantly increased compared to the baseline values with both transdermal E2 (1.08 +/- 0.04 vs. 1.00 +/- 0.03; mean change, 8%) and oral conjugated equine estrogens (1.04 +/- 0.03 vs. 0.99 +/- 0.03; mean change, 6%); however, there was no difference between the treatments. The number of patients with cholesterol crystals detected in bile was similar after both estrogen regimens. Transdermal and oral estrogens decreased nucleation time in vitro, increased arachidonate and PGE2 levels, and minimally raised total glycoprotein concentrations. In conclusion, transdermal and oral estrogens exerted comparable nonhepatic effects, as evidenced by similar reductions of gonadotropin levels, but oral therapy exhibited substantially greater actions on hepatic markers of estrogen action. Both transdermal E2 and oral conjugated equine estrogens significantly elevated the biliary cholesterol saturation index and reduced the nucleation time. These results suggest that estrogens at the doses studied could promote gallstone formation by alteration of biliary lipids and cholesterol nucleation time that have been incriminated in this process.

Nucleation of biliary cholesterol, arachidonate, prostaglandin E2, and glycoproteins in postmenopausal women.

BACKGROUND & AIMS: Among dieting obese patients, cholesterol gallstone formation is preceded by increases in levels of biliary cholesterol saturation, arachidonate, prostaglandin E2, total glycoproteins, and rapid nucleation of cholesterol. The aim of this study was to determine if similar increases occur among postmenopausal women with cholesterol crystals in their bile. METHODS: In 101 postmenopausal women without gallstones, gallbladder bile was sampled via nasoduodenal tube and analyzed. RESULTS: Nineteen of the women had saturated bile and crystals. Levels of cholesterol saturation, arachidonate, prostaglandin E2, and total glycoprotein were highest among women with cholesterol-saturated bile and cholesterol crystals and lowest among women with unsaturated bile. Levels were intermediate among women with saturated bile but no crystals. CONCLUSIONS: Among postmenopausal women, increases in levels of biliary cholesterol saturation, arachidonate, prostaglandin E2, and total glycoproteins may be important pathophysiologically in the rapid nucleation of cholesterol crystals.

The relationship of the postcoital test and semen characteristics to pregnancy rates in 200 presumed fertile couples.

OBJECTIVE: To evaluate the postcoital test (PCT) and semen analysis (SA) in the prediction of pregnancy in 200 potentially fertile couples. METHODS: 200 couples without risk for infertility were prospectively followed for 1 year. Couples were attempting pregnancy for 12 menstrual cycles. In the first three cycles, the women underwent monthly PCTs and collected daily urines while the men provided bimonthly semen analyses. For the next nine cycles, the couples were monitored for pregnancy. The PCT included hours post-coitus, amount of mucus, spinnbarkeit, number of motile sperm, and percent of motile sperm. Since multiple PCTs and SAs were available for each couple, values were averaged to provide one mean value per couple. The Wilcoxon ranked sum test was used to detect differences in PCT and SA. RESULTS: Pregnancy occurred in 163/200 couples (82%) in 12 cycles. Mean sperm count per high-power field (p = 0.01) and mean number of highly motile sperm (p = 0.03) were higher among women in whom pregnancy occurred. Amount of mucus and spinnbarkeit were similar between women who became pregnant and those who did not. Semen concentration, motile sperm count, and percent motile sperm were significantly higher among men whose partner conceived (P < .02). Only 93 couples (47%) had PCTs that were correctly timed. CONCLUSIONS: Measures predictive of pregnancy included vigorously moving sperm per high-power field, sperm concentration, motile sperm count, and percent motility. Mucus characteristics were not predictive of pregnancy. Additionally, a high number of sperm seen in the PCT correlated with a high number of motile sperm in the SA. These results support the use of the PCT for initial evaluation of the infertile couple.

Evaluation of computer-assisted semen analysis (CASA) with IDENT stain to determine sperm concentration.

This study was undertaken to compare a new fluorescent stain-based computer-assisted semen analysis (CASA) system (IDENT) for determining human sperm concentration to the manual hemacytometric method and to conventional CASA (CASA-CONV). Normal healthy semen donors as well as patients provided samples that were evaluated for sperm concentration with the CASA-IDENT method, the hemacytometer method, and CASA-CONV. Each field was examined visually to determine the sources of overcounting and undercounting for the two CASA methods. Four ranges of sperm concentration were examined: 0-10, > 10-30, > 30-100, and > 100 x 10(6)/ml. The main outcome measures were sperm concentration, debris counted as sperm, and missed sperm. Our results showed that significantly more debris was counted as sperm and more sperm were missed with CASA-CONV than CASA-IDENT. As the sperm density increased, so did the number of counting errors for the CASA-CONV system. The error rate was much greater using CASA-CONV (12.1 +/- 42.2%) than with CASA-IDENT (0.4 +/- 0.7%) when compared to hemacytometer counts (P = 0.068). We conclude that the CASA-IDENT method of sperm counting is highly accurate and less time-consuming when compared to the hemacytometer method. There are significant differences in the amount of debris counted as sperm and number of missed sperm between CASA-CONV and CASA-IDENT with varying sperm density. With both parameters, the counts are more accurate using the CASA-IDENT method.

The effect of varying inseminating sperm concentration in male factor and non-male factor infertility during in vitro fertilization.

OBJECTIVE: To assess the effect of varying inseminating sperm concentrations on fertilization rates and polyspermy in human in vitro fertilization (IVF). SUBJECTS AND METHODS: Eighty-six couples who completed 107 consecutive IVF cycles were assigned to one of three groups according to the results of their semen analysis (SA), sperm penetration assay (SPA), and titers of antisperm antibodies (ASA). Group 1 (non-male factor) had normal results for SA, SPA and ASA; group 2 had one abnormal result; and group 3 had two or more abnormal results. Inseminating concentrations of 50,000, 250,000, or 500,000 progressively motile sperm/oocyte were prospectively assigned to groups 1, 2 and 3, respectively. MAIN OUTCOME MEASURES: Incidence of polyspermy and fertilization rates. RESULTS: A total of 992 oocytes were available for analysis. The fertilization rate of 61% for non-male factor patient (group 1) was significantly higher than for male-factor patients [group 2 (48%) and group 3 (43%; P < .01)]. The incidence of polyspermy was 3.3%, 5.5%, and 0% for groups 1, 2 and 3, respectively, and did not differ significantly between the non-male factor and male factor groups (P = .16). Polyspermic fertilization was increased in both mature (4.1%) and postmature (5.7%) as compared to immature oocytes (1.4%; P < .05). CONCLUSION: In male factor infertile couples, increasing the inseminating concentration to 250,000 or 500,000 motile sperm/oocyte does not result in an increase in the incidence of polyspermy but does not improve fertilization rates.

Assessment of human sperm acrosome reaction by flow cytometry: validation and evaluation of the method by fluorescence-activated cell sorting.

OBJECTIVE: To determine the applicability of flow cytometry to assess human sperm acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated overnight for the development of spontaneous acrosome reaction or exposed to the calcium ionophore A23187 (5 microM) for 3 hours for induction of the acrosome reaction. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: The spermatozoa were stained with fluorescein isothiocyanate (FITC)-labeled pea agglutinin. The labeled samples were assessed visually and also subjected to analytic flow cytometry and fluorescence-activated cell sorting. MAIN OUTCOME MEASURES: Acrosome reaction assessed visually and by flow cytometry. RESULTS: Flow cytometric analysis showed a single peak of FITC fluorescence in the washed semen samples. A second peak of lower FITC fluorescence intensity was noted after overnight incubation or exposure to A23187, suggesting loss of fluorescence, which indicated the occurrence of the acrosome reaction. There was a statistically significant correlation between the assessment by the two methods (n = 41). However, although the mean difference between the methods was small (2.49%), the difference between the two methods for each individual sample can vary between -24% to +29%. When the sperm cells were subjected to cell sorting based on green fluorescence intensity, reanalysis and visual scoring verified that the low intensity peak contained a majority of acrosome-reacted spermatozoa (77.52% +/- 2.39%). CONCLUSION: These results validated the flow cytometric method for assessment of acrosome-reacted spermatozoa. Although flow cytometry is more objective and less time consuming when many samples are assessed at the same time, the visual method remains a useful and practical procedure in the clinical andrology laboratory.

Hepatic rupture in pregnancy associated with cocaine use.

BACKGROUND: Hepatic rupture during pregnancy has a high rate of maternal and fetal mortality. Most cases occur as a complication of severe pregnancy-induced hypertension, and maternal survival has been highest in patients managed with conservative surgical therapy. CASE: A woman in late pregnancy experienced hepatic rupture associated with cocaine use. She underwent emergency cesarean delivery and was treated with topical hemostatic agents, perihepatic packing, and hepatic artery embolization. Reexploration with perihepatic packing was performed three times during the first 48 hours after delivery to control hepatic hemorrhage. Vasopressor support, blood product replacement, and prolonged assisted ventilation were used, and the patient was discharged on hospital day 42. CONCLUSION: Liver damage may result from the potent vasoconstrictor property of cocaine, leading to vasospasm and ischemia. Conservative surgical management of hepatic rupture and supportive measures resulted in maternal survival.

Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction.

OBJECTIVE: To study the direct effects of progesterone (P) and its antagonist RU486 (mifepristone) on sperm hyperactivation (HA) and acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated in capacitation media with P and/or RU486. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: Semen samples were incubated in media with P or RU486 alone or in combination, and aliquots were taken at 10 minutes, 1, 5, and 24 hours for HA analyses by computer-aided sperm analysis system, and at 0, 5, and 24 hours for assessment of acrosome reaction by fluorescein-labeled Pisum sativum (pea) agglutinin. MAIN OUTCOME MEASURES: HA and acrosome reaction. RESULTS: Sperm HA was significantly increased at 10 minutes by P both at 10(-7) M (9.27 +/- 1.59%; mean +/- SEM) and 10(-6) M (9.39 +/- 1.94%) when compared with untreated spermatozoa (5.62 +/- 1.59%). The stimulatory effect of P on sperm HA was transient because this was not observed after 1, 5, and 24 hours of incubation. The antiprogesterone RU486 (10(-6) M) alone had no effect and did not abolish the stimulatory effect of P on HA. The %HA was further enhanced by the addition of RU486 at 10(-6) M to P at 10(-7) M (12.43 +/- 3.31%) or P at 10(-6) M (13.52 +/- 4.10%); however, this effect was not significantly different from P alone. Coincubation of P or RU486 with spermatozoa during capacitation did not stimulate the acrosome reaction in the concentrations tested. CONCLUSION: Progesterone directly stimulates human sperm HA transiently. Progesterone has no significant effect on acrosome reaction in capacitating spermatozoa. The effects of P are rapid and not counteracted by RU486, suggesting that the mechanism of action of P may not be mediated by specific P nuclear receptors.

PIP: Reproductive endocrinologists at the University of California, Los Angeles Medical Center, obtained semen samples from healthy volunteers to examine the direct effects of progesterone and the antiprogesterone RU-486 on sperm hyperactivation and acrosome reaction, which they hoped would elucidate progesterone's mechanism of action. They used progesterone concentrations which match those of the female reproductive tract. Progesterone induced a considerable increase in sperm hyperactivation at 10 millionths M and 1 millionth M after 10 minutes (9.27% and 9.39% vs. 5.62% for untreated spermatozoa; p .05). The ability of progesterone to increase sperm hyperactivation was only temporary, however. In fact, progesterone had no effect at 1, 5, and 24 hours. RU-486 alone did not affect sperm hyperactivation. Addition of RU-486 at 1 millionth M to progesterone further did strengthen the stimulatory effect, however, but it was not significant (13.52% vs. 9.39%). When progesterone or RU-486 were coincubated with spermatozoa during the process by which they become able to fertilize the ovum after they are at the ampullar portion of the uterine tube, neither chemical stimulated the acrosome reaction, regardless of the concentration. These results indicated that progesterone can only directly stimulate sperm hyperactivation temporarily and rapidly (within about 10 minutes) and does not influence acrosome reaction during capacitation. They also demonstrated that RU-486 cannot counteract progesterone. Thus, specific progesterone receptors do not mediate progesterone's mechanism of action.