The paternal inheritance of the centrosome, the cell's microtubule-organizing center, in humans, and the implications for infertility.

Successful fertilization in humans, achieved when parental chromosomes intermix at first mitosis, requires centrosome restoration and microtubule-mediated motility. Imaging of inseminated human oocytes reveals that the sperm introduces the centrosome. The centrosome then nucleates the new microtubule assembly to form the sperm aster--a step essential for successful fertilization. Oocytes from some infertile patients failed to complete fertilization because of defects in uniting the sperm and egg nuclei, indicating that failure to properly effect the cytoplasmic motions uniting the nuclei results in human infertility. These discoveries have important implications for infertility diagnosis and managing reproduction.

The role of thoracoscopic biopsies in the diagnosis of pleural tuberculosis.

Tuberculosis (TB) is a significant public health problem in many developing countries. In many cases, tuberculosis may present a significant diagnostic challenge. A 32-year-old male Chinese immigrant presented to our institution with a fever and non-productive cough. He was found to have a right pleural effusion, for which a chest drain was inserted. His tuberculin skin test was unreactive (0mm) although he was not immunocompromised (HIV negative). All cultures were negative, and 3 sputum samples and his pleural fluid sample tested negative for acid-fast bacilli. A computed tomography (CT) scan of his chest revealed features suggestive of an early empyema. There was no evidence suggestive of a malignant effusion. In an effort to attain a diagnosis, he underwent a video-assisted thoracoscopy (VATS) procedure with pleural drainage and biopsies. Anti-tuberculosis therapy (ATT) was commenced due to a high level of suspicion after failure of empirical therapy. Although the Ziehl-Neelsen stain for acid fast bacilli was negative, pleural biopsies demonstrated active chronic granulomatous pleuritis with many Langerhans type giant cells highly suggestive of tuberculosis. He was responsive to treatment and completed 6 months of ATT with complete clinical resolution. In young, immunocompetent patients with an exudative, culture-negative effusion, the diagnosis of pleural tuberculosis must be considered. Pleural biopsy is the gold standard for diagnosing pleural TB but demonstration of acid-fast bacilli or necrotizing granulomas in the specimen are not absolutely necessary to make the diagnosis.

Very low-dose (femtomolar) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disrupts steroidogenic enzyme mRNAs and steroid secretion by human luteinizing granulosa cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of the polyhalogenated aromatic hydrocarbons (PAH), which causes anatomical abnormalities and developmental defects, impairs ovulation and reduces fertility. TCDD's endocrine-disrupting effects are, in part, caused by a direct action at the ovary. Herein we investigated the in-vitro effects of environmentally relevant doses of TCDD on estradiol-17ß (E2) production by human luteinizing granulosa cells (hLGC) obtained from women stimulated for in-vitro fertilization (IVF). TCDD at all concentrations tested (3.1fM, 3.1pM and 3.1nM) significantly decreased E2 secretion when assayed for by radioimmunoassay (RIA). Herein we confirm that TCDD alters E2 secretion by hLGC in a time-, not dose-dependent fashion and are the first to show decreases in E2 secretion with fM concentrations of TCDD. Using real-time quantitative PCR (RT-qPCR), the decreased E2 secretion correlates with a decrease in the mRNA expression levels two enzymes in the estrogen biosynthesis pathway: CYP11A1 and CYP19A1.

Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the most toxic congener of a large class of environmental pollutants. Several studies have shown that TCDD exposure reduced fecundity and ovulatory rate in rats and increased the incidence of endometriosis in monkeys. Recent work suggests that TCDD's endocrine-disrupting effects are, at least in part, caused by a direct action at the ovary. Although the factors involved in TCDD-induced toxicity are still under investigation, several studies have shown that TCDD induces programmed cell death, or apoptosis, in various tissues and may act in a similar fashion in the ovary. In the present study, we set out to evaluate the in vitro effects of TCDD on steroid secretion, specifically estradiol-17beta (E2) and progesterone, by human luteinized granulosa cells (LGC), and to further determine whether TCDD is capable of inducing apoptosis in this cell type. Human LGC were obtained from women participating in an in vitro fertilization program. Medium, with or without three different concentrations of TCDD and substrates [androstenedione (A4) or pregnenolone], was added to each culture. The media were collected at 4, 8, 12, 24, 36, and 48 h and were assayed by RIA. At 24 and 48 h, the LGC were fixed for assessment of DNA fragmentation via an in situ immunofluorescence technique. Transmission electron microscopy was also performed on LGC after 24 and 48 h with TCDD. TCDD, at all concentrations tested (3.1 pM, 3.1 nM, and 3.1 microM), significantly reduced E2 accumulation in the media at 8, 12, and 24 h, compared with controls. At 36 and 48 h, TCDD treatment (at 3.1 microM) caused a significant increase in E2, compared with controls. The effect of TCDD on E2 was abolished with the addition of A4. TCDD treatment did not alter progesterone accumulation. Apoptosis increased at 24 h with 3.1 microM TCDD, with no apparent effect at 3.1 nM. By 48 h, however, TCDD increased apoptosis in a dose-dependent manner. Transmission electron microscopy showed ultrastructural differences in LGC with 3.1 microM TCDD at 24 and 48 h. Collectively, the results of the present study suggest that TCDD perturbs E2 secretion by depletion of A4 precursor and increases apoptotic cell death of human LGC in a dose- and time-dependent fashion.

Age- and pregnancy-related changes in serum total cholesterol and triglyceride levels in the Cayo Santiago rhesus macaques.

Ninety-six free-ranging rhesus monkeys were evaluated for age-, sex-, and pregnancy-related changes in total serum cholesterol and triglyceride levels and compared with previous studies. Our findings indicate that pregnancy depresses total cholesterol in females and that cholesterol levels tend to increase in males with age. Triglycerides decreased significantly with advancing age in males. The Cayo Santiago monkeys represent a unique opportunity to study the effects of age on population of nearly 1200 nonhuman primates on which there is accurate data on birth date, lineage, behavior, reproduction and post-mortem morphology (skeletons). Further gerontological studies are necessary to take full advantage of this resource and to increase the presently-scant body of information on aging in monkeys for comparative studies on humans and for the development of animal models of gerontological diseases of humans.

The relationship of human granulosa-lutein cell proliferative index to follicular diameter and serum estradiol.

OBJECTIVE: To examine the relationships of follicular diameter and serum estradiol (E2) to the percentage of granulosa cells undergoing mitosis as reflected by the proliferative index of granulosa cells. METHODS: In this prospective study, we enrolled 44 consecutive women undergoing controlled ovarian hyperstimulation for in vitro fertilization. Deoxyribonucleic acid histograms were generated by flow cytometry from granulosa cells isolated at the time of transvaginal aspiration. Proliferative index was defined as the sum of G2/M and S phases. We assessed the correlation between proliferative index and age, maximum serum E2, number of oocytes retrieved, percent mature oocytes, and follicular diameter. RESULTS: Follicles less than 16 mm had a significantly higher proliferative index (19.9 +/- 3.3%) than follicles 20 mm or greater (14.8 +/- 3.9%, P = .016). However, there was no significant difference between proliferative index of the latter group and proliferative index of follicles 16-19 mm (17.8 +/- 4.7%). An inverse correlation between patient age and proliferative index of granulosa cells was noted (r = -.39, P = .018). There was no significant relationship between serum E2 and proliferative index (P = .97). CONCLUSION: Mitotic activity tends to decrease as follicular diameter increases after a threshold diameter is achieved. Proliferative index of granulosa cells provides insight into the underlying cell biology of a follicle.

Objective assessment of meconium content of amniotic fluid.

The amount of meconium in amniotic fluid is subjectively estimated by visual inspection and classified as thin (light), moderate, or thick (heavy). This estimate may be important for assessing the neonatal risk of perinatal asphyxia and meconium aspiration syndrome. This study reports on the "meconium-crit," a simple, rapid, inexpensive, and reproducible method of quantifying meconium concentration in amniotic fluid. The purpose of the study was to determine the relationship between the meconium-crit and meconium concentration. Specimens were prepared by placing 3.0 g of fresh neonatal meconium into clear amniotic fluid and vortexing for 15 minutes to obtain a stock solution of 15.0 g meconium/100 mL amniotic fluid. Stock solutions were then diluted with clear amniotic fluid to obtain concentrations of 10.0, 7.5, 5.0, 3.0, and 1.5 g/100 mL. One-tenth milliliter of the amniotic fluid/meconium mixture was drawn into a standard hematocrit tube and centrifuged. The meconium-crit was then measured directly as with a hematocrit. Regression analysis indicated that meconium-crit values were linearly related to meconium concentration (r = 0.901-0.995). This method provides a reproducible means of quantifying meconium in amniotic fluid.

The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo.

OBJECTIVE: To evaluate the impact of the oviduct, uterine, and in vitro environments on zona pellucida thinning in the mouse embryo. DESIGN: Female mice were stimulated with pregnant mare serum gonadotropin and mated and hCG injection. Unilateral oviduct ligation was performed on day 2 of gestation using the dorsal approach. The mice were divided into equal groups and killed on days 2, 3, 4, 5, and 10 of gestation. In vitro incubated embryos served as controls. Average daily zona thickness measurements were subjected to analysis of variance and paired Student's t-test. SETTING: The laboratory of the assisted reproductive program of Rush University Medical Center. MAIN OUTCOME MEASURE(S): Progressive daily decrease in average zona thickness. RESULT(S): Zona measurements of embryos flushed out of uterine horns, ligated oviducts, and in vitro incubation demonstrated statistically significant decreases in zona thickness, from 9.6 +/- 1.6 microns (day 3) to 6.0 +/- 0.8 microns (day 5), from 11.6 +/- 2.2 microns (day 2) to 6.0 +/- 1.6 microns (day 5), and from 11.1 +/- 2.0 microns (day 2) to 6.0 +/- 1.6 microns (day 5), respectively. There were no differences in average zona thickness for embryos in the same cell stage and same protocol day in all three locations. CONCLUSION(S): Zona thinning seems to be induced primarily by the dividing embryo before implantation. A substantial tubal and uterine contribution to zona thinning was not detected in this mouse embryo model.

Steroidogenesis of cultured granulosa cells in women at risk for ovarian hyperstimulation syndrome.

OBJECTIVE: To determine if cultured human granulosa cells (GCs) obtained from women at risk for ovarian hyperstimulation syndrome (OHSS) possess altered steroidogenic capacity. DESIGN: Prospective analysis of 28 consecutive in vitro fertilization-gamete intrafallopian transfer (IVF-GIFT) cycles. SETTING: In Vitro Fertilization Program at Rush Presbyterian St. Luke's Medical Center in Chicago, Illinois. PATIENTS: Eighteen patients (group I) with serum estradiol (E2) levels > 7,342 pmol/L on the day of exogenous human chorionic gonadotropin (hCG) administration (day 0) with > 10 ovarian follicles present (high risk for OHSS); 10 patients (group II) with E2 < or = 7,342 pmol/L on day 0 and < or = 10 follicles. INTERVENTIONS: Human GCs obtained during gonadotropin-releasing hormone agonist-pretreated IVF-GIFT cycles were cultured in the absence (control) or presence (hCG) of hCG, 1 IU/mL, and/or androstenedione (A) 10(-7) M. Granulosa cells obtained from follicles < or = 15 mm diameter were cultured separately from those obtained from follicles > 15 mm diameter. MAIN OUTCOME MEASURES: Estradiol (E2) and progesterone were measured in tissue-culture medium by a solid-phase direct radioimmunoassay. RESULTS: In vitro E2 production by cultured GCs was significantly increased in follicles < or = 15 mm diameter from women considered at risk of developing OHSS (group I). Estradiol response to hCG and/or A appeared enhanced in all follicles in group I. Progesterone production in the basal and hCG challenged state was greater in cells obtained from large follicles in group I than in group II. CONCLUSION: Ovarian hyperstimulation syndrome appears to be a function of an increased number of follicles that express an enhanced steroidogenic capacity.

Effects of induced hyperprolactinemia on in vitro fertilization cycles.

OBJECTIVE: To investigate the effect of induced endogenous hyperprolactinemia on the luteinization process, as expressed by the shift in the P:E2 ratio after hCG injection in IVF cycles. PATIENTS AND INTERVENTIONS: Serum PRL, E2, and P levels were measured in 49 IVF patients (leuprolide acetate and hMG protocol) on the day of hCG injection. Estradiol and P also were measured on the day after hCG. Serum P:E2 ratios were calculated for two groups of patients; group I (control): PRL < or = 20 ng/mL (conversion factor to SI unit, 1.00); group II (hyperprolactinemia): PRL > 20 ng/mL. Estradiol and P also were measured in follicular fluid (FF) and the gamete performance was compared between groups. RESULTS: Data analysis showed no significant differences in the mean +/- SD serum peak E2 (pg/mL; conversion factor to SI unit, 3.671) between groups: group I, 1,769 +/- 843; group II, 2,333 +/- 1,194; the mean FF E2 (pg/mL) group I, 351 +/- 221; group II, 370 +/- 186; or the mean FF P (ng/mL; conversion factor to SI unit, 3.180) group I, 8,357 +/- 3,127; group II, 11,354 +/- 12,888. No significant differences were found between groups in the P:E2 ratios on days 1 or 2: group I, 78 +/- 48 and 209 +/- 137; group II, 70 +/- 47 and 224 +/- 197, respectively. The magnitude of the P shift also showed no significant difference between the two groups; the mean +/- SD shift in the P level was 2.9 +/- 2.2 for group I, and 4.3 +/- 5.1 for group II. The serum PRL level had no effect on the fertilization rate (60% for group I and 70% for group II) or on the pregnancy rate (17% for group I and 23% for group II). CONCLUSION: These findings suggest that mild endogenous hyperprolactinemia induced by ovarian stimulation does not affect granulosa cell luteinization and gamete performance in humans.

Effects of danazol, gonadotropin-releasing hormone agonist, and estrogen/progestogen combination on experimental endometriosis in the ovariectomized rat.

Direct effects of a gonadotropin-releasing hormone agonist (GnRHa), danazol, or estrogen/progestogen (E/P) on experimental endometriosis were evaluated in castrated female rats. Endometrial explants decreased in size following castration, but there was no further change in the treatment groups. Histologic examination indicated atrophy and regression of experimental endometriosis in all groups of castrated animals. As expected, following castration, serum estradiol (E2) became undetectable, serum progesterone (P4) decreased, and cytosolic E2 and P4 binding capacity in the endometrial explants was lower. However, in danazol-treated animals, serum P4 and E2 receptor concentrations were significantly higher than in all other castrated groups, and in both danazol and E/P treated animals, concentrations of P4 receptor were significantly higher than in castrated controls. In contrast, GnRHa treatment had no effect on serum E2 and P4 levels nor on E2 or P4 receptors. The authors conclude that danazol and E/P preparations may have a direct effect on the ectopic endometrium through interaction with steroid receptors.

Human chorionic gonadotropin levels in conception cycles after uterine and tubal embryo transfer.

OBJECTIVE: To describe the rate of rise of beta-hCG levels in normal single gestations after uterine and tubal embryo transfer. DESIGN: A prospective study from 1991 through 1992 in which infertility patients who conceived after ovulation induction and ET were monitored with serum beta-hCG levels. SETTING: Infertility unit at Rush-Presbyterian-St. Luke's Medical Center in Chicago, Illinois. PATIENTS: Ninety-eight women conceived after ovarian stimulation and ET. This study included only patients who conceived after uterine ET (n = 12) and tubal ET (n = 25) and had an exponential rise of beta-hCG levels and sonographic evidence of an intrauterine gestation. RESULTS: The rate of rise of mean beta-hCG levels after ET preceded that of tubal ET by 24 hours until day 14 when the two approximate each other. CONCLUSION: There appears to be a unique interaction between the trophoblast and endometrial surface that may account for the early detection of beta-hCG after uterine versus tubal ET.

The relationship between total motile sperm count and the success of intrauterine insemination.

OBJECTIVES: To determine if the total motile sperm count is predictive of IUI success and to apply life-table analysis to a large database of IUI procedures. DESIGN: Contingency table and life-table analyses were used to analyze a large group of couples (n = 546) undergoing 1,205 cycles of therapeutic IUI with husband's sperm. SETTING: Clinical infertility practice at a medical school referral center. PATIENTS: Five hundred forty-six couples with varying etiologies of infertility. RESULTS: There was a trend toward an increasing percentage of conception with increasing total motile sperm count. Significance was reached when the total motile sperm count used for IUI exceeded 20 x 10(6). The cumulative pregnancy rate calculated from this data reached 52% after the sixth attempted cycle of IUI.

Xenogenous and in vitro fertilization of frozen/thawed primate oocytes and blastomere separation of embryos.

Little research has been done on the in vitro and xenogenous fertilization of cryopreserved primate oocytes. This study reports the development of freezing and thawing methods for squirrel monkey oocytes with subsequent successful fertilization by these two methods. Preliminary results on techniques for blastomere separation using the hamster and squirrel monkey as models are also given. These studies have important implications relative to the long-term frozen storage of human oocytes, their subsequent thawing, in vitro fertilization and embryo transfer, and the use of the blastomere separation procedure, in conjunction with in vitro fertilization, in the diagnosis of embryonic normality and possible congenital defects prior to implantation.

Induction of the human sperm acrosome reaction by human oocytes.

The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ (P greater than 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar (P greater than 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization.

Microsurgical enucleation of tripronuclear human zygotes.

Polyspermic fertilization of human oocytes in vitro produces genetically abnormal embryos whose replacement in utero represents a potential obstetrical risk. Microsurgical removal of extra male pronuclei offers the possibility that normal ploidy can be restored in these zygotes. Pronuclear removal was attempted in three tripronuclear human oocytes fertilized in vitro. Male pronuclei were distinguished by their larger size and an associated sperm tail piece. Zygotes were pretreated with cytochalasin B and colcemid in phosphate-buffered saline before microsurgery. Enucleation was completed in all embryos; syngamy occurred in one embryo, but cleavage was not observed.

Elevated follicular fluid angiotensin II and pregnancy outcome.

OBJECTIVE: To assess whether a relationship exists between follicular fluid (FF) angiotensin II (AII) concentration and pregnancy outcome or earlier fecundity parameters and whether correlations exist among FF AII concentrations and P, E2, T, androstenedione (A), or various ratios of these. DESIGN: Retrospective study in which hormone concentrations in FF samples were measured. SETTING: In vitro fertilization clinic-Assisted Reproductive Technology Program, Rush Medical Center. PATIENTS: Twenty-six female patients underwent ovarian stimulation for IVF. INTERVENTION: Leuprolide acetate was combined with hMG and FSH for ovarian stimulation. MAIN OUTCOME MEASURE: Follicular fluid aspirates were collected and oocytes were recovered 34 to 36 hours after hCG injection. The patients proceeded to undergo IVF and ET. Follicular fluid hormones were measured using standard RIA. Angiotensin II and steroid hormone concentrations in FF were compared for pregnant versus nonpregnant women using the Student's t-test and rank-sum test. Pearson multiple-correlation analysis was performed to calculate correlation coefficients among AII concentrations and steroid concentrations in FF aspirates. RESULTS: Mean FF concentration of AII was significantly lower in samples from women showing clinical pregnancies (112.2 +/- 13.9 pg/mL [107.3 +/- 13.3 pmol/L]) compared with samples from women who did not achieve pregnancy (217.1 +/- 23.8 pg/mL [207.5 +/- 22.7 pmol/L]) (mean +/- SE). A negative correlation was observed between FF concentrations of AII and P. Correlations of AII with E2, T, A, or with ratios of these did not show significance. CONCLUSION: These data suggest that high AII concentration at time of oocyte recovery may indicate poor pregnancy outcome in women undergoing ovarian stimulation for IVF. These data corroborate previous results in animal models showing that AII predisposes follicles to undergo atresia-like conditions.

Persistent ovarian cysts following administration of human menopausal and chorionic gonadotropins: an attenuated form of ovarian hyperstimulation syndrome.

Ovarian cysts persisting after the onset of menses were demonstrated by ultrasound (US) in 40 of 71 (56%) nonconception cycles following ovulation induction with human menopausal gonadotropins (hMG) and human chorionic gonadotropin (hCG). Persistent cysts were self-limited and all resolved spontaneously within two cycles. They developed more frequently during stimulation cycles with (1) higher mean pre-hCG serum estradiol (E2), (2) a greater number of medium and large follicles at peak pre-hCG E2, and (3) a larger leading follicle diameter at peak pre-hCG E2. Persistent ovarian cysts frequently occurred despite a peak pre-hCG E2 lower than 1000 pg/ml. Although ovarian enlargement in the presence of cysts exceeded 5 X 5 cm in 25% of cases, no patient developed clinical symptoms of ovarian hyperstimulation syndrome (OHSS). Repeated induction of ovulation with hMG/hCG in the presence of nonfunctional, persistent cysts resulted in pregnancies in 6 of 15 cases (40%). Asymptomatic persistent ovarian cysts frequently follow an hMG/hCG regimen and, when nonfunctional, are not a contraindication to repeated ovarian stimulation. Persistent ovarian cysts appear to be an attenuated form of OHSS.

Successful use of gonadotropin-releasing hormone agonist leuprolide for in vitro fertilization in a patient with polycystic ovarian disease and infertility unresponsive to standard treatment.

A patient with PCO and primary infertility had undergone numerous failed attempts of ovulation induction. She then was treated with GnRHa leuprolide 500 micrograms subcutaneously daily for 4 weeks, later combined with hMG 225 IU IM daily for 8 days and hCG 5000 IU IM. Six oocytes were retrieved for IVF, four fertilized and two were replaced. Twin pregnancy was established and delivered at term. Hyperstimulation syndrome was managed conservatively.

Effects of dioxin, an environmental pollutant, on mouse blastocyst development and apoptosis.

OBJECTIVE: To evaluate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) on mouse embryo development and apoptosis. DESIGN: Controlled animal study. SETTING: Academic research environment. ANIMAL(S): Female mice (CB6F1) at 3 to 6 weeks of age and proven breeders (C578B46). INTERVENTION(S): Mouse embryos were obtained at the morula stage and cultured to the blastocyst stage in a pharmacologic dose of TCDD (3.1 microM) or a control medium. The morphology was assessed, and staining for apoptosis was performed. Immunohistochemistry for the presence of aromatic hydrocarbon receptor (AhR) was performed in another set of morula-stage embryos. MAIN OUTCOME MEASURE(S): The number of embryos developing from the morula to the blastocyst stage and number of apoptotic blastomeres in control vs. TCDD culture conditions. RESULT(S): No statistically significant differences were observed in the percentage of embryos reaching the blastocyst stage: 80.9% (115 of 142) in the TCDD-treated group, vs. 82.9% (121 of 146) in the control group. There was also no difference in the degree of apoptosis: 22.6 +/- 7.3% apoptotic cells (TCDD) vs. 25.3 +/- 9.7% (controls). Staining indicated the slight presence of aromatic hydrocarbon receptor in the morula-stage mouse embryos. CONCLUSION(S): TCDD at 3.1 microM did not alter the development of early mouse morula to blastocysts and did not significantly induce apoptosis in vitro.