Tracking of insemination outcomes: a novel approach.
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Female↗
Biomedical subjects
G M Centola
Publications and source records attributed to G M Centola.
Metal ions and human sperm mannose receptors.
Zinc and lead concentrations were measured in seminal plasma from fertile donors, infertile men with varicocoele and men undergoing work-ups for in vitro fertilization. Ejaculated spermatozoa from these subjects were incubated in vitro with various metal ions and/or dibromoethane and dibromochloropropane. Mannose receptor expression was correlated with metal and toxicant levels. Sperm distributions of potassium channels were compared with lead ions and calcium channels with zinc ions. Mannose receptor expression by capacitated spermatozoa increased linearly with seminal plasma zinc levels, and correlated inversely with lead levels. Cobalt had no effect on mannose receptor expression, but nickel had a concentration-dependent biphasic effect. Mannose receptor expression was not affected by dibromoethane and dibromochloropropane if the cholesterol content of the sperm membrane was high, but mannose receptor expression was decreased in low cholesterol spermatozoa by exposures below estimated permissive exposure limits. Potassium channels and lead ions co-localized over the entire head of human spermatozoa, while both calcium channels and zinc ions were confined to the equatorial segment of the head. Mannose receptor expression on the external surface of the human sperm plasma membrane is a biomarker for the effects of transition and heavy metals and organic toxicants on sperm fertility potential.
Cobalt↗
Seasonal variations and age-related changes in human sperm count, motility, motion parameters, morphology, and white blood cell concentration.
OBJECTIVE: To determine the presence of any seasonal variations and age-related changes in sperm parameters in andrology patients and fertile donors. DESIGN: Retrospective analysis. SETTING: University medical center andrology laboratory. PATIENT(S): The database of 2,065 semen analyses was retrospectively reviewed for the period of March 1, 1996, to October 31, 1998. INTERVENTION(S): None. MAIN OUTCOME MEASURES(S): The sperm count, motility, motile count, progressive straightline velocity, and percentage of rapid sperm were determined with the Hamilton-Thorne IVOS analyzer with standard setup parameters. RESULT(S): There were no significant seasonal differences in the patient's volume, sperm count, motility, motile count, whereas the percentage of rapid sperm and progressive straightline velocity were significantly lower in the spring. Correlation analysis of patient semen parameters versus age implied that as age increases there is a tendency for these semen parameters to decrease, whereas percent tail defects showed a significant positive correlation with age. CONCLUSION(S): Age-adjusted analyses of seasonal variations in andrology patient semen parameters showed significant seasonal variation in the percentage rapid motile sperm and straightline velocity, as well as the percent tail defects, percent immature sperm, and the percent tapered sperm. Such seasonal variations might prove to be clinically relevant and important when designing experimental protocols.
Adult↗
Dose-response effects of gramicidin-D, EDTA, and nonoxynol-9 on sperm motion parameters and acrosome status.
Previous reports showed that gramicidin-D (G-D), a polypeptide with antiviral and antimicrobial properties, nonoxynol-9 (N9), a common spermicidal detergent, and EDTA, a Ca-Mg chelating agent, inhibited sperm motility and cervical mucus penetration. The purpose of this study was to determine the dose-response effects of G-D, N9, EDTA and G-D + EDTA on sperm motion parameters and acrosome status. Semen specimens from known fertile donors were subjected to computer-assisted semen analysis of motility, path velocity, progressive velocity, and hyperactivation prior to and after incubation with varying concentrations of gramicidin-D, EDTA and nonoxynol-9. Each specimen was also prepared for acrosome status using rhodamine isothiocyanate conjugated pisum sativum agglutinin (RITC-PSA). There was a significant decrease in motility by G-D, EDTA, G-D + EDTA, and N9 at all doses as compared to the fresh specimen. N9 completely immobilized all sperm at each dose. Progressive velocity and path velocity also decreased in a dose-response manner. Sperm hyperactive motility also significantly decreased in all groups. The majority of sperm remained acrosome intact following exposure to all doses tested, whereas N9 resulted in complete breakdown/release of the acrosomal contents. This study confirms previous reports that G-D, EDTA, and N9 significantly impair sperm motility and motion parameters. The effective 100% inhibitory concentration was seen only with N9, whereas G-D, EDTA, and G-D + EDTA resulted in incomplete impairment of sperm motion parameters. At the concentrations used, N9 demonstrated potent spermostatic activity. Gramicidin-D and EDTA should be further studied for their potential contraceptive spermostatic activity.
Acrosome↗
Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation.
OBJECTIVE: To compare the Enhance (Percoll; Conception Technologies, San Diego, CA) and PureSperm (Gen X International, Madison, CT) sperm preparation methods with respect to recovery (percentage of motile sperm), motility (%), path and progressive velocities (microm/s), and hyperactivation (%). DESIGN: Comparison of sperm processing methods. SETTING: University medical center-based clinical andrology laboratory and infertility program. PATIENT(S): Twenty-five men who presented for semen analysis. INTERVENTION(S): Each of 25 semen specimens were divided and each aliquot was prepared using two different density gradient centrifugation methods. MAIN OUTCOME MEASURE(S): The motile sperm recovery, percent motility, motion parameters, and percent hyperactivation were measured for each semen specimen (n=25) before and after separation with the use of the two methods. RESULT(S): There was no difference in the percent motility and motile count between specimens prepared with Enhance (Percoll) and PureSperm and fresh specimens. Statistically significant differences were found (fresh versus test) in the velocities and in hyperactivation (PureSperm only), and no differences were found between the processing methods. CONCLUSION(S): PureSperm appears to be as effective as Percoll (Enhance) for the recovery of good, progressively motile sperm for use in IUI or other assisted reproductive techniques.
Cell Separation↗
Comparison of the immunobead binding test (IBT) and immunospheres (IS) assay for detecting serum antisperm antibodies.
PROBLEM: The IBT is considered the gold standard of sperm antibody assays. This test uses polyacrylamide beads labeled with antiglobulins (anti-IgG, anti-IgA, and anti-IgM), which bind to the corresponding antibody on the sperm surface. The IS uses color-coded latex beads of uniform 3.0 microns size coated with the antiglobulins which can be viewed with brightfield light microscopy. The purpose of the present study was to compare the IBT and IS in an indirect test using human serum. METHOD: Serum specimens (n = 42) were tested for the presence of antibody isotypes IgG, IgA, and IgM to sperm using the standard protocol for IBT and IS. Donor sperm was washed in BWW with 5% BSA and diluted to a final concentration of 50 x 10(6) motile sperm/ ml. The sperm were incubated with a 1:10 dilution of test serum for 30 min to 1 h at 37 degrees C and then washed by three cycles of centrifugation. The sperm and beads (IBT, IS) were mixed on a glass slide, covered with a coverslip, and observed within 5 min. At least 100 motile sperm were counted and scored for bead binding. A specimen was considered positive if 20% or more of the sperm were coated with one or more beads. The data were analyzed using calculation of the non-parametric kappa statistic with correction for chance expected agreement, and by calculating the proportion of specific agreement between the two methods. RESULTS: The results are summarized in the following table: [table see text]; The IS was able to detect 94% of IgG antibodies, 91% of IgA antibodies, and 100% of IgM antibodies. One serum specimen was IgG negative by IS (14% binding), but positive by IBT (20%). A second serum specimen was IgA negative by IS (16%) yet positive by IBT (29%). There were no false positives with the IS assay. Of the IgM positives (five of six) occurred alone and not with IgG or IgA, suggesting the necessity for testing all specimens also for IgM. CONCLUSION: Antisperm antibody test results obtained by the IS assay are in agreement with the results obtained with the IBT test. The Immunospheres are monodispersed, color coded, and can be visualized with brightfield microscopy.
Acrylic Resins↗
Selecting against "unfit sperm".
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Humans↗
Methods of semen preparation for intrauterine insemination and subsequent pregnancy rates.
Semen for insemination, either intrauterine or in vitro, must be prepared to remove seminal plasma products and/or select the healthier population of sperm prior to use. Traditionally, a double wash technique is performed, with or without subsequent swim-up to isolate the motile fraction if necessary. More recently, the use of the SperPrep filtration method has gained acceptance, with the benefits of removal of leukocytes and seminal debris from the specimen as well as enhancement of overall sperm quality. In the current study we compared the traditional double wash method without the swim-up to Sperm-Prep filtration. Intrauterine inseminations (IUI's) were performed in 307 cycles on 148 infertile couples at two different infertility centers in the USA. After complete diagnostic evaluation the couples were offered IUI before proceeding to any other form of assisted reproductive technologies. Semen samples were prepared in human tubal fluid media supplemented with 5% human serum albumin (HSA; location 1) or in Ham's F-10 media supplemented with 3% HSA (location 2), either with the SpermPrep filtration method (ZBL, Inc., Lexington, KY 40523, USA) or the double sperm wash (SW) procedure. Similar sperm numbers were used for the IUI procedure in both treatment groups and locations. The Sperm-Prep method resulted in significantly higher pregnancy rates (PR) than the SW procedure, independent of location. The clinical pregnancy rates per cycle were statistically lower (p < 0.05) in the SW group (20-22% vs. 9-10%). Of significant clinical importance, almost twice as many cycles were required in the SW group to achieve these pregnancies when compared to the SpermPrep group of patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.
The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.
Acrosome↗
Pregnancy rates after double versus single insemination with frozen donor semen.
Ninety-nine females (mean age 34 years +/- 1) were seen for therapeutic donor insemination using frozen semen. All patients used over-the-counter urinary ovulation predictor kits, with the insemination scheduled either the day after the positive test (group I; n = 46) or consecutively the day of and the day after the positive test (group II; n = 53). Group I patients underwent a total of 113 cycles of insemination (mean 2.5 cycles/patient). Seven pregnancies were achieved in group I for an overall success of 15.2% and a monthly fecundability of 0.06. Group II patients underwent a total of 100 cycles of therapeutic donor insemination (mean of 1.9 cycles/patient). Twenty-one pregnancies were achieved in this group for an overall success rate of 39.6% and fecundability of 0.21. The estimated cumulative probability of conception (F) for 6 months was 0.32 in group I and 0.78 in group II. These data indicate that the F after therapeutic donor insemination with frozen semen is greater if two consecutive inseminations are performed at midcycle.
Cryopreservation↗
Relationship between testicular volume and presence of varicocele. A comparative study.
The varicocele, present in many of the male partners of infertile couples continues to generate controversy, particularly as related to its diagnosis and pathophysiology. The purpose of our study was to determine the relationship between testicular volume and the presence or absence of a varicocele. Testicular volume was determined by the use of an orchiometer; the presence of a varicocele was determined by palpation during a Valsalva maneuver. The patients utilized in the study were those seen in our Andrology and General Urology Clinics; 291 patients with varicoceles and 83 control patients (no evidence of varicocele) were used. The left (mean = 21.4 mL) and right (mean = 23.4 mL) testicular volumes of patients with a varicocele were significantly reduced compared with that of the control group (left, mean = 23.4 mL; right, mean = 26.2 mL; p = 0.0041 and p = 0.002, respectively). The testicular volume corrected on the basis of the body mass (V/m2) also was significantly reduced in the varicocele group compared with controls (left p = 0.007; right p = 0.001). Reduced testicular volume relative to body size may be detected prior to actual demonstration of the stress pattern and the presence of a varicocele, and may be useful in early diagnosis. It is suggested that measurement of testicular volume may be a useful adjunct to routine examination of the infertile male.
Adult↗
Mixed mesodermal tumor of the uterus (RJ-984): case report and in vitro study.
This report describes the establishment of a cell line of a human uterine mixed mesodermal tumor. The tumor of origin derived from a hysterectomy specimen, has been maintained for 14 months in vitro and continues to grow as an established cell line. The original tumor as well as the cell line exhibited no estrogen receptors. alpha-Fetoprotein was not detected in the cultured cells or in the spent culture medium. Karyotyping revealed 46 XX chromosome complement with a balanced 11-16 translocation. This is the first documentation of such a chromosome abnormality in a genital tract carcinoma. Steroids inhibited cell growth at high (10.0 micrograms/ml) concentrations. This cell line continues to be studied and further characterized. The cell line is readily available for study of this aggressive human neoplasm.
Cell Division↗
Long-term in vitro growth of a heterologous mixed Müllerian tumor of the ovary.
This paper describes the long-term culture and characterization of a human ovarian heterologous mixed Müllerian tumor, initially acquired from a nude mouse heterotransplant. Microscope examination of the heterotransplants revealed that epithelial and heterologous elements of the original tumor were serially transplanted. After an initial 2- to 4-day lag period, the cells entered a linear growth phase until confluence was reached and the cells passaged. The cells have been in culture for 15-18 months, and passaged approximately 8-10 times. DNA flow cytometry of the cultures revealed the presence of two hyperdiploid cell populations, presumably the epithelial and heterologous portions of the tumor. The cells remain tumorigenic in the nude mouse and form colonies on semisolid medium, attesting to the maintenance of their neoplastic identity following long-term culture. Immunofluorescent labeling for collagen revealed the presence of types I, III, V. The presence of types I, III and V collagen was also confirmed using gel electrophoresis of radiolabeled collagen from these cells. This is consistent with bony tissue, since fibroblasts make types I and III, chondrocytes, type II. In view of the poor prognosis of patients bearing these aggressive tumors, this in vitro tumor cell line could serve as a useful model for the study of this type of carcinoma.
Animals↗
Serial transplantation of a heterologous, mixed Müllerian tumor in the nude (athymic) mouse: growth characteristics and morphology.
In view of the poor prognosis for patients with mixed Müllerian tumors, transplantation of these tumors into nude mice offers an important model for the study of the physiologic processes occurring in this type of malignancy. We report the growth and serial transplantation of all elements of this tumor in the nude mouse. The lag period for establishment of the original tumor was 5 weeks. The tumor required 11 weeks to achieve a 2 X 2 X 1 cm volume at the initial pass, but on subsequent passes reached this volume in 6-12 weeks (mean = 8.3). Microscopic examination revealed that the epithelial, stromal and heterologous elements were serially transplanted. An osteoid matrix, confirmed by special staining for bone, was identifiable at both the light and electron microscopic levels. Throughout successive passages, the tumor retained the morphologic features of the original tumor. This tumor line is stable and reproducible, providing a readily available source of this ovarian carcinoma for clinical research studies.
Adenocarcinoma↗
Inhibition of endometrial carcinoma cell cultures by a synthetic androgen.
This paper describes the dose-response inhibitory effects of a synthetic androgen, methyltrienolone, on the growth of a grade II endometrial adenocarcinoma in vitro. Derived from a nude mouse heterotransplant, this cell line has maintained the morphological characteristics of the original human tumor, remains tumorigenic in the nude mouse, forms colonies on soft agar, and exhibits low levels of estrogen receptors. Data reported in this paper indicate that the cells contain androgen binding sites. At low doses of 0.25 micrograms/ml methyltrienolone had no effect on these cells, whereas at 1.0 and 10.0 micrograms/ml there was significant dose dependent inhibition of cell growth and an increase in the cell doubling time. Both testosterone and dihydrotestosterone were not inhibitory to the cells except at high doses where inhibition was slight. Methyltrienolone was not inhibitory to normal human foreskin fibroblast cultures tested as a control for cytotoxicity of the synthetic androgen. These data suggest that androgens may play a role in the treatment of tumors not responsive to conventional forms of hormonal therapy.
Adenocarcinoma↗
Establishment and morphologic characterization of normal human endometrium in vitro.
Tissue culture offers a model system with which to study the endocrine-mediated growth, differentiation, and metabolic activities of the endometrium. We have established and continue to maintain monolayer cultures of normal human endometrial epithelial cells from each phase of the menstrual cycle. At present, eight proliferative, two secretory, and two menstrual phase cultures have been established. These have been passed at least three times. One proliferative phase culture has been growing for 18 mo, and passed 10 times. Colonies of epithelioid cells as well as single cells appear in the cultures within 2 to 8 h of initial culture and maintain this appearance throughout long-term growth. The cells are periodic acid Schiff positive for carbohydrates and positive for keratin, an immunochemical marker for epithelial tissues. Studies comparing the ultrastructure of the cultures with fresh endometrial tissue revealed morphologic features common to both, including prominent nucleoli, Golgi, mitochondria-rough endoplasmic reticulum complexes, and abundant glycogen. The cells are not tumorigenic in the nude mouse and do not form colonies on soft agarose, confirming the nonneoplastic identity of the cells.
Culture Techniques↗
Esophageal contraction pressures are not affected by normal menstrual cycles.
Previous studies of lower esophageal sphincter pressures during the menstrual cycle and pregnancy have suggested that the smooth muscle relaxing effect of progesterone depresses sphincter tone. Results, however, have been contradictory and limited to a small number of patients. We studied lower esophageal sphincter and distal peristaltic pressures during the follicular (days 2-8) and luteal (days 20-28) phases of the menstrual cycle. Twenty normal menstruating women (mean age 31 yr) not using oral contraceptives were evaluated. A low compliance pneumohydraulic infusion system was used for all studies. Lower esophageal sphincter pressure was determined by both rapid and station pull-through techniques. Distal peristaltic pressures were recorded 2 and 7 cm above the sphincter in response to 10 wet swallows (5 cc of H2O). Mean amplitude and duration in the distal esophagus were evaluated. Plasma progesterone and estrogen concentrations were obtained and correlated with changes in esophageal pressures. The results were as follows: (a) unlike prior investigations, we found the menstrual cycle had no effect on lower esophageal sphincter pressure and (b) likewise, no change in esophageal contractions in the distal esophagus was found during the cycle. We concluded that changes in female sex hormone concentrations that characterize the menstrual cycle are not associated with changes in parameters of esophageal motor function.
Adult↗
Hormone requirements for long-term maintenance of rat granulosa cell cultures.
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Animals↗