Improved cryopreserved semen fecundability in an alternating fresh-frozen artificial insemination program.

This study with glycerol and increased number of motile sperm inseminated showed an increase in f to 10.4% for frozen semen and 27.4% for fresh semen. There is a significant (P less than 0.01) difference in pregnancy rate when greater than 100 million motile sperm are inseminated (31.3%) than when less than that number are inseminated (11.9%) in the first cycle when frozen semen is used.

Using videotaped specimens to test quality control in a computer-assisted semen analysis system.

OBJECTIVE: To determine the feasibility of using semen samples previously recorded on videotape for intralaboratory and interlaboratory quality control of computer-assisted semen analysis (CASA) systems. DESIGN: Blinded, controlled study. SETTING: Pooled semen specimens from two normal human volunteers in an academic research environment. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen parameters from a videotape analyzed internally and by four external laboratories. RESULT(S): Preliminary experiments designed to examine intralaboratory variation by repeated analysis of semen samples recorded on videotape revealed some significant differences for every variable examined. When these data were analyzed by using the larger biologic error caused by subsampling, no significant differences were found for any of the variables examined. When either a standard set or the specific laboratories' sets of parameters were used to analyze the same videotaped semen specimen, no statistically significant differences were detected for sperm concentration for motility among the five laboratories after the biological error caused by subsampling was applied to results. CONCLUSION(S): These data strongly suggest that videotaped semen specimens can serve as quality control for intralaboratory and interlaboratory testing of CASA equipment as long as the biologic error caused by subsampling is used to compare results.

Manual versus computer-automated semen analyses. Part III. Comparison of old versus new design MicroCell Chambers.

OBJECTIVE: To determine accuracy and precision of old and new design MicroCell counting Chambers analyzed manually and with a computer-automated semen analyzer (CASA; Hamilton-Thorn Research, Beverly, MA). DESIGN: Prospective study using comparative measurements of the concentration of latex beads with old and new design MicroCell Counting Chambers (Conception Technologies, Inc. La Jolla, CA). MAIN OUTCOME MEASURE: Beads were counted manually and with CASA to evaluate the accuracy of old and new design 20 MicroCell Chambers. RESULTS: The old design 20 MicroCell Chamber demonstrated a significant difference in bead concentration beginning at the 9.9 mm CASA stage position compared with stage positions of 0.0 to 9.5 mm. The new design 20 MicroCell Chamber demonstrated the same difference at the 8.0 mm CASA stage position compared with stage positions 0.0 to 7.6 mm. Similar findings were observed when the beads were analyzed manually. CONCLUSIONS: When analyzed within a specific range of stage positions, the old and new design 20 MicroCell Chambers are accurate and precise whether analyzed manually or with CASA.

Manual versus computer-automated semen analyses. Part I. Comparison of counting chambers.

OBJECTIVE: To determine the accuracy and precision of counting chambers analyzed manually and with a computer-automated semen analyzer (CASA; Hamilton-Thorne Research, Beverly, MA). DESIGN: Prospective study using comparative measurements of sperm concentration, motility and concentration of latex beads with three types of counting chambers: hemacytometers, 12 and 20 MicroCell (Conception Technologies, Inc., La Jolla, CA) Chambers, and Makler (Sefi-Medical Instruments, Haifa, Israel) Chambers. SETTING: A hospital-based Andrology laboratory. PATIENTS: Male partners of couples undergoing infertility evaluation. MAIN OUTCOME MEASURES: Experiment I: measurements of sperm concentration were evaluated within hemacytometers; Experiment II: measurements of sperm concentration in the 35 to 50 x 10(6)/mL range and sperm motility were determined using a known concentration of beads. RESULTS: Experiment I: differences were demonstrated within two of eight hemacytometers (side 1 versus side 2): however, no significant effect of hemacytometer on variation in sperm concentration measurements was observed. Experiment II: CASA-analyzed 20 MicroCell Chambers demonstrated the best precision for sperm concentration (intraclass correlation coefficient = 0.93) and motility (intraclass correlation coefficient = 0.88). Experiment III: 20 MicroCell Chambers most accurately determined known bead concentration (35 +/- 5 x 10(6)/mL) whether analyzed on CASA (34.9 x 10(6)/mL) or manually (35.2 x 10(6)/mL). CONCLUSIONS: 20 MicroCell Chambers proved to be accurate and precise for determining concentration and motility of semen specimens whether analyzed manually or with CASA.

Manual versus computer-automated semen analyses. Part II. Determination of the working range of a computer-automated semen analyzer.

OBJECTIVE: To determine the accuracy and precision of a computer-automated semen analyzer (CASA; Hamilton-Thorne Research, Beverly, MA) over a wide range of sperm concentrations. DESIGN: Prospective study using comparative measurements of sperm concentration and motility were performed using manual and CASA methods of analyses. SETTING: A hospital-based Andrology laboratory. PATIENTS: Male partners of couples undergoing infertility evaluation. MAIN OUTCOME MEASURES: Measurements of sperm concentration in the range of 1 to 200 x 10(6)/mL were performed using hemacytometers and CASA-analyzed 20 MicroCell counting chambers (Conception Technologies, Inc., La Jolla, CA). Accuracy was assessed by comparison to the hemacytometer ("gold standard"). The MicroCell counting chambers also were used for manual and CASA sperm motility determinations. RESULTS: The CASA-analyzed 20 Microcell Chamber most precisely determined sperm concentration in the 1 to 200 x 10(6)/mL range (intraclass correlation coefficient = 0.94). The hemacytometer was least precise (intraclass correlation coefficient = 0.91). There was a high degree of correlation between the manually and CASA-analyzed MicroCell Chambers in the sperm concentration range of 20 to 149 x 10(6)/mL. Above and below this range, correlation was lower. Motility was determined most precisely using the CASA-analyzed 20 MicroCell Chamber. CONCLUSIONS: The Hamilton-Thorne CASA used with the 20 MicroCell Chamber is a precise and accurate method for determining sperm concentration and motility of semen specimens with concentrations in the 20 to 149 x 10(6)/mL range. This same combination of CASA and MicroCell Chamber provides precise sperm motility results.

Relative efficiency of therapeutic donor insemination using a luteinizing hormone monitor.

A prospective randomized study was performed to evaluate the use of a urinary luteinizing hormone (LH) detection kit with 1 insemination as compared with 2 alternate day inseminations with timing based on previous cycle length and basal body temperature changes. The study involved 60 patients who underwent a total of 264 therapeutic donor insemination cycles using cryopreserved semen specimens. Patients alternated LH-kit timed cycles with cycles timed by non-LH methods for a total of 6 cycles or until pregnancy was achieved. Fecundability rates were 12.3% for LH-kit cycles and 5.3% for non-LH method cycles. The difference in outcome was not statistically significant. However, when the LH kit plus 1 insemination was compared with 2 inseminations timed by conventional methods, there appeared to be a distinct monetary and time expenditure advantage. These findings suggest that sufficient advantage may be derived from use of an LH kit to recommend its use on a routine basis for the timing of therapeutic donor insemination.

Relationship of heparin binding with computer-analyzed physical traits of human sperm.

The objective of this study was to determine whether heparin affinity or the concentration of binding sites was related to sperm traits analyzed by a routine computerized semen analysis in human males requesting infertility evaluations. Saturation of heparin binding sites on sperm was achieved in 36 of 50 samples analyzed so that accurate estimates of dissociation constants (Kd) and binding site concentrations could be made. A broad range in Kds (18.2 to 284.5 nM/micrograms deoxyribonucleic acid [DNA]) and binding site concentrations (2.70 to 44.77 nM/micrograms DNA) was found. The binding affinity of sperm for heparin was significantly correlated with concentrations of cells in the ejaculate (r = 0.56), concentrations of motile sperm (r = 0.51), percentage of motile sperm (r = 0.33), and total numbers of ejaculated sperm (r = 0.37). The concentration of heparin binding sites also was correlated with concentration of cells in the ejaculate (r = 0.60), concentration of motile sperm (r = 0.50), percentage of motile sperm (r = 0.40), and total number of ejaculated sperm (r = 0.49).

Comparison of implantation and pregnancy rates in African American and white women in an assisted reproductive technology practice.

OBJECTIVE: To compare IVF outcomes between infertile African American and white women. DESIGN: Retrospective cohort study. SETTING: Hospital-based IVF practice. PATIENT(S): Women undergoing IVF procedures between November 1996 and June 2000. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates. RESULT(S): There were 24 African American and 273 white women < or =40 years of age who underwent 25 and 333 IVF cycles, respectively. African American women were more likely to have had tubal factor as a primary diagnosis, to have had a child, and to have undergone fewer previous assisted reproductive technology (ART) cycles as compared to white women. No differences between the two groups for clinical variables were noted with the exception of body mass index (BMI [kg/m(2)], 27.1 in African Americans vs. 24.8 in whites). Implantation rates were higher in African American than in white women (35% vs. 23%, respectively). Pregnancy rates were 71% in African Americans and 48% in whites. After adjustment for tubal factor, BMI, and parity, the odds ratio for pregnancy in African American women versus white women increased from 2.6 to 3.3. CONCLUSION(S): This is the first study to demonstrate a significantly higher clinical pregnancy rate in African American women as compared to white women undergoing ART. These data strongly contradict a recent study comparing the same two groups of women undergoing ART. We urge other ART centers to report their data pertaining to race.

Freezing of mammalian embryos without the aid of a programmable freezer.

A simple, inexpensive and repeatable method of freezing/thawing (F/T) mammalian concepti was developed with the use of 2-cell mouse embryos. Cryoprotectants, length of exposure to protectants, and subzero holding times before liquid nitrogen (LN2) exposure were examined in an effort to obtain an effective freezing protocol. Sequential examination of these variables provided data suggesting that 3.5 M dimethyl sulfoxide (DMSO) plus 0.25 M sucrose exposure for 5 minutes at room temperature, followed by a -30 degrees C environment for 90 minutes, best prepared embryos for LN2 storage. After thawing and culture, 48 of the 93 (52%) embryos developed to the blastocyst stage.

Control of air quality in an assisted reproductive technology laboratory.

OBJECTIVE: To investigate the effect of improved air quality on IVF and subsequent embryo development. DESIGN: Retrospective cohort study. SETTING: Hospital-based IVF facility composed of an anteroom, a cleanroom, and an adjacent operating room. PATIENT(S): Two-hundred seventy-five couples requesting IVF between 1993 and 1997. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Particle counts (sizes 0.3, 0.5, 1.0, and 5.0 microm); IVF rates; and embryo quality (stage and grade). RESULT(S): Clinical pregnancy rates decreased from 35% in 1993 to 16% in 1994 (numerous construction odors were detected during 1994) and increased steadily after the cleanroom was built (rates for 1995-1997 were 20%, 32%, and 59%, respectively). Fertilization rates decreased between 1993 (74%) and 1994 (60%) and then steadily increased after cleanroom installation (62% in 1995, 71% in 1996, and 69% in 1997). The proportion of embryos past the four-cell stage decreased from 66% in 1993 to 61% in 1994 but then increased steadily in the years after the cleanroom was built (78%, 77%, and 83% in 1995, 1996, and 1997, respectively). During the same 5-year period, there were no differences in embryo quality or number of embryos transferred. CONCLUSION(S): Construction of a Class 100 cleanroom improved air quality and IVF rate and increased the number of embryos past the four-cell stage available for transfer.

Validity and cost-effectiveness of antisperm antibody testing before in vitro fertilization.

OBJECTIVE: To determine the usefulness of and cost-effectiveness of antisperm antibody testing in the prediction of poor fertilization rates in couples undergoing IVF. DESIGN: Retrospective cohort study. SETTING: A hospital-based reproductive endocrinology and infertility practice. PATIENT(S): Male partners of 251 couples undergoing IVF between 1992 and 1997. MAIN OUTCOME MEASURE(S): Fertilization rates in couples undergoing conventional IVF. RESULT(S): One hundred nineteen couples were evaluated for antisperm antibodies; fertilization rates were similar in those couples whose husbands were and were not tested (64% versus 68%). Antisperm antibodies were detected in 16 men. Four (25%) of the 16 couples whose husbands had antisperm antibodies fertilized < or = 50% of oocytes, compared with 31 (30%) of the 103 couples whose husbands did not have these antibodies. Overall, 21 couples (8.4%) experienced complete fertilization failure. In a program that included antisperm antibody testing for selected couples and intracytoplasmic sperm injection (ICSI) for those who tested positive, it would cost $11,735 to prevent a fertilization failure (assuming ICSI were 100% effective), whereas it would cost $9,250 to perform ICSI in a second IVF cycle for those who initially failed. CONCLUSION(S): In this practice setting, antisperm antibody testing has low sensitivity in predicting low or no fertilization and does not appear to be cost-effective when selectively ordered as part of an IVF workup.

Serum progesterone, oestradiol, luteinizing hormone and prolactin profiles in the female black bear (Ursus americanus).

Identifying steroid and pituitary hormone profiles in the female black bear (Ursus americanus) throughout pregnancy may provide a greater understanding of the reproductive cycle and indicate which hormones are required for implantation. Our objective was to assess endocrine activity in black bears oestrus onset, at oestrus, during pregnancy and after parturition. Serum samples were obtained from 12 captive, 16 uncollared and five radiocollared free-ranging female black bears from March through the end of December and assayed for serum progesterone, oestradiol, luteinizing hormone (LH) and prolactin (PRL). In captive bears, progesterone concentrations were low at days 0-10 after oestrus and increased significantly days 25-35 and 45-52 after oestrus. Oestradiol concentrations were high at oestrus (day 0) and days 4-10 after oestrus and then decreased days 25-35 and 45-52 after oestrus. LH concentrations were not significantly different throughout the sampling period. Changes in PRL concentrations pattern were similar to those of oestradiol, with elevated levels at oestrus and days 4-10 after oestrus, followed by a significant decrease 45-52 days after oestrus. In non-collared free-ranging bears, progesterone concentrations increased gradually after mating with a further significant increase in November-December. Oestradiol concentrations were highest in March (before mating) and in June (during mating) followed by a significant decrease in July (early delay period) and November-December (peri-implantation period). LH concentrations were low until November-December and then increased significantly. PRL concentrations were low in March (before mating), increased significantly during the mating season in June, decreased slightly in July, and were low in November-December (peri-implantation period). In radiocollared free-ranging bears, serum progesterone concentrations were elevated in pregnant bears in December and extremely low in lactating and non-lactating bears in March. Oestradiol levels were slightly higher in pregnant bears in December than in non-lactating or lactating bears in March. PRL concentrations were considerably higher in lactating bears in March than in pregnant bears in December. Our results suggest that: (1) serum progesterone concentrations are low, but detectable during the early delay implantation period and greatly elevated during the peri-implantation period; (2) serum oestradiol concentrations are elevated at oestrus and decline during the delay period; (3) LH may be involved in luteal activation; and (4) the decline of serum PRL concentrations during short days may be necessary for implantation to occur.

Development of screening systems for evaluation of materials used in mammalian embryo transfer.

Embryo transfer units use a wide variety of materials that come in contact with embryos. Studies were conducted to evaluate procedures that could be utilized to determine the toxicity of some commonly used materials in embryo collection, culture and transfer. Forty-five female mice were sacrificed on Day 3 or 4 of gestation (Day 1 = vaginal plug), and the uterus and oviducts were removed and minced. A total of 522 embryos was collected (4-cell to blastocyst stages). Four to 16 cell embryos were cultured in Phosphate Buffered Saline (PBS) plus 20% fetal bovine serum. Morula to blastocyst stage embryos were cultured in Nutrient Mixture F10 (HAM) plus 20% fetal bovine serum gassed with 5% CO(2), 5% O(2) and 90% N(2). In Experiment I, embryos and culture media were placed in a covered embryological watch glass (EWG, control) or sealed in the lumen of a siliconized Foley catheter or a section of 1) latex tubing, 2) tygon tubing or 3) silastic tubing. In Experiment II, embryos were placed in EWG and cultured alone (control) or cocultured with sections of 1) tygon tubing, 2) silastic tubing or 3) latex tubing. In Experiment III, embryos were cultured in covered plastic petri dishes containing 15 ml of media, alone (control) or co-cultured with two new plunger tips from sterile Monoject syringes. All embryos were cultured at 32 to 34 degrees C for 24 h. The Criterion used for development was two or more cellular divisions within the 24-h period. Embryo development in Experiment I was lower (P<0.05) in latex (0%) and tygon (24%) tubing and in the siliconized Foley catheter (2%) than in silastic tubing (51%) and the EWG (46%), which did not differ. Experiment II embryos that were co-cultured with latex tubing (5%) showed very little development as compared with those co-cultured with tygon tubing (76%), silastic tubing (76%) and EWG (93%), the last of which were not significantly different. Embryos co-cultured with Monoject syringe plunger tips had a reduced embryo development rate compared to embryos in the control group (0% vs 52%). Although the embryos did not remain in contact with these seemingly toxic materials for prolonged periods, our results indicate that a significant reduction in embryo viability may occur due to this exposure.

Live birth of a bear cub following nonsurgical embryo collection.

In the near future, 6 of 8 bear species will face extinction mainly because of loss of their natural habitat. This loss of habitat will ultimately require some of these bears to be maintained in zoos and wildlife preserves in the hope of conserving genetic diversity. If the giant panda is representative of other bear species, reproductive performance will be inhibited in such an environment. In this study, we used the nonendangered American black bear (Ursus americanus) as the model for developing appropriate embryo transfer procedures. The donor bear mated numerous times between late May and early June. In late July we anesthetized her and used a series of telescoping sheaths to gain access to the uterus Then we passed a catheter through the largest sheath, inflated the balloon, and, using a 20-mL syringe, repeatedly infused into and then aspirated from the uterus PBS + BSA. We emptied the syringe into Petri dishes and observed 2 embryos. We rinsed the embryos, placed them in human tubal fluid + HSA + HEPES and then held them at 35 degrees C for 5 h. The recipient mated during mid-June; in late July we anesthetized her and, with the aid of laparoscopy, transferred an embryo into the cranial portion of the uterine horn ipsilateral to the ovary containing a CL. The recipient delivered 2 cubs in January. Necropsy results indicated that the neonates lived for 6 to 8 wk before succumbing to flooding in the den. The DNA from hair samples belonging to the neonates indicated that the male cub belonged to the donor, the female cub to the recipient. The delayed implantation mechanism in bears probably allowed for the successful development of the embryo in the presence of a substantial asynchrony between the donor and the recipient (13 d). We conclude that embryo transfer is possible in the American black bear and can lead to the birth of live cubs.

Instructing the animal physiology graduate student in human assisted reproductive technology.

Animal physiology graduate students provide an excellent personnel resource for laboratories performing human assisted reproductive technology (ART) procedures. However, the basic training of these students falls short of what is required for this highly specialized field. We designed a course to enhance their education in this area via classroom and hands-on laboratory instruction in a hospital and university setting. Topics covered in the course included in vitro maturation, in vitro fertilization, embryo culture, embryo transfer, quality control, quality assurance, micromanipulation, and cryopreservation. These techniques were applied to a group project to evaluate the influence of spermatozoal quality and quantity on early embryonic development in cattle and humans. Student grades were based on 1) oral and written examinations; 2) demonstrated competency in laboratory techniques; 3) presentation of class project data at a state academy of science meeting; and 4) initiative, determination, and interest in the coursework. Three aspects of the course stood out as very positive. First, the team approach to accomplishing a class project was new to some of the graduate students. Second, a bond was formed between hospital- and university-based faculty that did and will continue to foster unique teaching and research opportunities between the two groups. Third, the opportunity for students to present research data in a formal setting was very rewarding. This course made the students keenly aware of the many aspects of ART and provided them with specialized skills that should make them more marketable in the field of reproductive technology.

Thermal patterns and health perceptions.

INTRODUCTION: Thermal pattern analysis is thought to be an indicator of health. However, the validity of this concept has not been established. To further investigate the relationship between thermal pattern analysis and health perceptions, thermal scans were assessed in conjunction with results from the SF-12 health survey. METHODS: Sixty-eight chiropractic students were recruited to receive two paraspinal thermal scans, 5 minutes apart, on three visits that were 1 week apart. Each scan produces three graphs or channels; one for each of left and right sides of the spine and a delta or difference between left and right. The scans were imported into a thermal pattern calculator (TPC) providing a percent similarity between the two. The TPC percent were compared with to their corresponding SF-12 scores. RESULTS: There were no significant findings in the left or delta channel. For the right channel, there was a decrease in mental health perception in participants having a TPC percent of 70.8 or higher (32.3% of all visits). CONCLUSION: Participants in this study who had right channel TPC percent of 70.8 or higher were associated with lower mental health perception scores. The study is considered a preliminary inquiry only due to the small sample size.

Reliability of three methods of computer-aided thermal pattern analysis.

BACKGROUND: The objective of this study was to assess three methods of computer-aided thermal pattern analysis for a) examiner reliability, b) inter-method differences, and c) determine which method yields the highest percent-similarity between paired test-retest scans. METHODS: Three examiners compared two sets of thermal scans from the same 30 subjects using three different methods of scan alignment. The results were evaluated by the Intraclass Correlation Coefficient and the Wilcoxon signed-rank test, at the 5% level of significance. RESULTS: Intra and inter-examiner ICC scores for all methods were acceptable (> 0.75). There were no statistically significant differences (at the Bonferroni-corrected level of significance of 0.0004%) in percent similarity of the scans between the three methods CONCLUSIONS: The results contribute evidence to the reliability of TPC program software. Manually aligning the readings plays an important role in obtaining precise TPC percent-similarities.

Effects of a chiropractic adjustment on changes in pupillary diameter: a model for evaluating somatovisceral response.

The relationship between a cervical chiropractic adjustment, in subluxated vs. unsubluxated subjects, and autonomic response monitored as change in pupillary diameter was evaluated in 15 subjects. The results indicate that: a) a successful adjustment elicits either a parasympathetic or sympathetic response; b) the vertebral level at which the adjustment is administered has undetectable specificity for the parasympathetic or sympathetic input to the pupil; c) unsubluxated subjects generally exhibit no change in pupillary diameter following a sham adjustment and d) subluxated subjects exhibit variable preadjustment pupillary diameters, with significant pupillary diameter changes in response to an adjustment. These data suggest that autonomic input to the pupil may be influenced by subluxation, as well as the magnitude and direction of force exerted during the chiropractic adjustment. An anatomical pathway through which the observed responses may occur is proposed.