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Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P 0.05). This suggests NEAT is successful in determining blastocysts viability in cryopreserved mice blastocysts. At a commercial ovine facility, NEAT was performed on fourteen frozen and thawed ovine blastocysts. Blastocysts of similar descent times were paired and transferred into recipient ewes as twins. Pregnancy was later confirmed by blood test and multiple gestation outcomes were determined at lambing. Six of seven recipient ewes were pregnant and all pregnant ewes delivered lambs without complication. Four ewes delivered twin lambs and two ewes delivered singletons, which totals ten of the fourteen (71%) blastocysts surviving to term. This pregnancy rate is comparable to expected to pregnancy rates in a commercial setting. The blastocysts which did not establish pregnancy demonstrated less buoyancy versus those blastocysts which established pregnancies which survived to term (P 0.05). These results suggest NEAT can identify which blastocysts survive cryopreservation, thus significantly reduce the transfer of non-viable embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT.

Objective: The last twelve months have presented significant challenges for the ART laboratory. Mandatory shutdowns, lack of patient access, supply chain issues, and changing rules and recommendations brought on by COVID-19 have stretched laboratories to their limits in an attempt to maintain regular and required activities. One area of concern in the laboratory has been the lack of available fresh semen (FS) samples at the proper times for quality control (QC) and proficiency testing (PT). Cryopreserved semen (CS) would appear a reasonable alternative. However, the quality of CS is known to deteriorate much faster than FS, even in favorable culture conditions. The goal of the present study was to determine, given the limitations of CS samples, if a protocol could be developed for QC and PT testing using CS. Materials and Methods: Using materials of known quality from previous PT challenges, 7 commercial donor semen samples were thawed and prepared for quality control monitoring as follows. Samples were thawed using bank-specific protocols. Each thawed sample was split in half and prepared using an IUI wash protocol with the assigned PT challenge media, either tainted or un-tainted. Once prepared, samples were maintained at 37oC, room air, and 95% relative humidity. Starting at 0 hrs, the samples underwent a semen analysis hourly using an IVOS semen analyzer for a minimum of 6 hrs or until one sample in the pair reached 0% motility after the 6 hr time-point. The resulting data were compared using a paired student’s T-test. Further, results were compared with reports from laboratory PT to verify the efficacy of using frozen semen. Results: As expected semen parameters decreased over time regardless of treatment (P < 0.001). No pair of samples lasted more than seven hours of incubation. While sperm in the non-tainted media maintained at least 60% of its initial motility at 3 hrs (range 64-91%), none of the cells in the tainted media had more than 50% motility at that time point (range 12-43%;p < 0.001). Further by six hours, all but one of the seven samples in the tainted media had 0% motility (range 0-4%), while six of seven samples in the non-tainted media still maintained a minimum of 25% of their initial motility at thaw (22-37%: P < 0.001). Further, all samples correlated with previous PT results. Conclusions: The data suggest it is possible to perform a rapid sperm QC assay using CS. Having a secondary QC protocol would not only provide an alternative when fresh semen, mice embryos, or other methods are unavailable, it would also potentially allow for more standardized methods of QC and PT testing. Impact Statement: The past twelve months have taught that unexpected and uncontrollable events can disrupt routine procedures. Sperm QC assays, which are the mainstay for QC and PT in many andrology laboratories, are dependent on the availability of fresh semen. If a standardized CS method can be created, QC and PT could be done at the convenience of the lab without sacrificing quality or patient safety.

Several studies have evaluated the effects of excessive heat stress on chromatin damage; however, DNA damage due to ambient seasonal heat stress has yet to be evaluated. The objective of the present study was to evaluate the effects of natural heat stress on equine sperm chromatin structure by means of two tests: the sperm chromatin structure assay (SCSA) and Sperm-Halomax kit (Halo). Stallion semen samples were collected after a period of excessive heat load (31.05°C) and were compared with samples collected at lower ambient temperatures (14.26°C) to evaluate chromatin damage caused by ambient heat stress produced in the testes. The data indicate that there is no correlation between the SCSA and Halo assays ( r = 0.18 and F = 0.48). Additionally, no correlation for ambient heat stress was observed for any of the applied techniques—SCSA, Halo, and live:dead staining ( P = .2682, .4628, and .0377, respectively). These results suggest that ambient heat load has little effect on stallion chromatin damage. Other elements, such as relative humidity and heat index, were outside the realm of this study and should be assessed in future studies with a larger sample size.

Assisted reproductive technology (ART) laboratories represent the marriage of the most basic of biological activities with the most cutting edge technologies. While this association has worked well, the mixture of biology and technology can create risks to normal embryo development. Recently a significant amount of literature has explored the risks of manmade, electrically induced magnetic fields and carrier waves on reproduction, which some studies have suggested will lower functional gamete numbers in the males and potentially induce genetic issues in embryos. However, little is known about these phenomena within the ART laboratory, a laboratory filled with electronic equipment. The object of the present study was to explore the potential exposure of gametes and early stage embryos to two of the most prevalent fields and waves utilized in manmade technologies seen in the general environment, electromagnetic fields (EMF) and radio frequency waves (RF), and determine the effect varying levels of these energetic forces had on gamete function and embryo development. Results indicated that while extremely high concentrations of EMF (approximately 50-100X of laboratory background) caused negative outcomes in both gametes and embryos, levels consistent will the majority of lab equipment did not appear to impact growth, or function. Further, even extremely high RF appeared to have no impact cellular function. Results suggest few issues with EMF or RF on gamete and embryo function at normal laboratory levels for the relatively short exposure times seen in the ART laboratory.

Background The variability of published acupuncture protocols for patients undergoing In-Vitro Fertilization (IVF) complicates the interpretation of data and hinders our understanding of acupuncture’s impact. In 2012, an acupuncture treatment protocol developed by a Delphi consensus process was published to describe the parameters of best practice acupuncture for Assisted Reproductive Technology and future research. However, there has been no clinical trial utilizing this protocol to assess the effects of acupuncture. This study aims to a sse ss the implementation of Dephi consensus acupuncture protocol and to examine the impact of acupuncture on stress and uterine and ovarian blood flow among women between ages 21-42 years seeking IVF. Methods/Design This study is a one site prospective, two-arm randomized controlled non-blind clinical trial conducted in a medical school-affiliated fertility center . Participants will be randomized 1:1 into either the acupuncture group or the standard of care (no acupuncture) group using computer generated tables. Both groups will have 3 regular clinical visits as their standard IVF care during an approximately 2 to 3 weeks window. Women who are randomized into the acupuncture group would receive three sessions based on the Delphi consensus acupuncture protocol in addition to the standard care. The first treatment will be administered between days 6 to 8 of the stimulated IVF cycle. The second session will be performed on the day of embryo transfer at least 1 h prior to the transfer. The third session will be performed within 48 h post-embryo transfer. Participants will be followed for their pregnancy test and pregnancy outcome when applicable. The outcomes stress and blood flow will be measured by a validated perceived stress scale and vasoactive molecules, respectively. Discussion Although recruitment and scheduling could be challenging at times, the Delphi consensus acupuncture protocol was implemented as planned and well-accepted by the patients. Because of the time-specified sessions around patients’ IVF cycle, it is highly recommended to have on-site study acupuncturist(s) to accommodate the schedule. Trial Registration ClinicalTrials NCT02591186 registered on October 7, 2015. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1693-4) contains supplementary material, which is available to authorized users.

Obesity has become a well-recognized medical issue. However its exact role in male infertility remains unclear. The objective of the current study was to determine if an increase inBMIis associated with an increase in semen parameter abnormalities and if this relationship was influenced by other patient activities. Charts were reviewed for one hundred and thirty-three male patients who had also undergone a complete initial office face to face interview, as part of an infertility evaluation and a semen analysis. As part of standard patient care, all patients answered a detailed questionnaire regarding demographics, exposures, medical and reproductive history as part of their infertility evaluation. Patients were grouped according toBMIas normal (20 - 24 kg/m2), overweight (25 - 30 kg/m2), or obese (>30 kg/m2). Semen analysis parameters analyzed included: morphology, volume, concentration, percent motility, and agglutination. While some parameters suggested trends, results were similar between the normal, overweight, and obeseBMI, for concentration (P= 0.18), volume (P = 0.845), motility (P = 0.06); % Positive agglutination: 12%, 7%, 7% (P = 0.668) and % normal morphology (P = 0.083). Unlike a number of previous studies, results indicate that there is no statistically significant association betweenBMIand any of the individual semen parameters tested. Raw data suggested a trend for decreasing concentration with increasingBMI. Further, data also suggested equal numbers of oligospermics in each group. However, when the data looked at globally rather than on the effects on individually parameters (total number of normal motile sperm cells—NMS), functional sperm cells decreased with increasing BMI. None of these factors appeared to be affected by other patient factors. Collectively these data suggest that obesity has a multifactorial effect on male fertility; possibly due to relationships with the hormone cascade, body composition and potentially testis temperature regulation. Further study will be needed to confirm such relationships.

The management of diabetes in pregnancy varies depending on whether the condition was first diagnosed during pregnancy (gestational diabetes) or was diagnosed before pregnancy (pregestational diabetes). Little has been published comparing the relative efficacy of various oral agents for the treatment of gestational diabetes and the reported experience with the insulin pump in pregnancy for pregestational diabetes remains meager. We conducted a retrospective chart review of women managed in a specialized diabetic clinic to compare the results of treatment of gestational diabetes with oral agents, glyburide and acarbose, to those treated with split-mixed insulin and treatment of pregestational diabetes with either the insulin pump or conventional splitmixed insulin. Gestational diabetics treated with split-mixed insulin were hospitalized significantly more often (p < 0.001) than those treated with oral agents only. The incidence of several important pregnancy complications (growth restriction, preterm labor, preeclampsia, oligohydramnios) did not differ between groups. Pregestational diabetics managed with an insulin pump had comparable glycemic control, as measured by hemoglobin A1c, to those managed with split-mixed insulin. Infant birth weights and Apgar scores were similar in each group. There were no perinatal deaths in either group. Acarbose and glyburide showed comparable efficacy in treating gestational diabetics. In addition, our experience adds to the small number of pregnant women with pregestational diabetes who were managed with an insulin pump that have been reported in the literature.

Cryopreservation is the main functional means for storage of excess embryos produced from artificial reproductive technologies; the process assumes embryos chemical nature is highly conserved across embryos of the same species. However, in practice there appears to be a high degree of variability in embryo tolerance to cryopreservation, suggesting potential differences in embryo chemistry. The objective of the current study was to develop reproducible means of estimating relative embryos weight and associating those weights to lipid content. Relative embryo weights of frozen/thawed in-vivo Jersey and Crossbred beef breed embryos were determined using a modified specific gravity chamber. Embryo weights were then correlated with lipid content. Results suggest that beef cattle embryos are 20% - 27% heavier than Jersey embryos (P

Cyathostome resistance to the benzimidazole fenbendazole (FBZ) and other anthelmintic medication has been documented worldwide. Parasite resistance to anthelmintic medication is of great concern to the anthelmintic industry and to horse owners. The present study examines the efficacy of FBZ anthelmintic treatments in horse herds from ranches in four different geographical locations within Texas. In addition, the load reduction method was compared with the traditional fecal egg count reduction test to determine the incidence of parasite resistance to FBZ. Four ranches in different areas of Texas were surveyed to determine cyathostome resistance to FBZ. Two of the four ranches had young (aged ≤2 years) and older animals (aged >2 years). The number of animals with parasites varied widely between the study's locations. Differences were observed in both the fecal egg count reduction test and load reduction method across the four study sites ( P P = .081). These results show the importance of testing anthelmintic medication effectiveness.

There can be little argument that embryo quality is one of the most critical factors in the success of assisted reproductive techniques. Yet the current methods of grading embryos are subjective at best. While a number of different groups have described more qualitative means of assessing embryo quality, the current standard remains morphology. Morphology has proven a good standard, but it does not allow for the detection of chromosomal abnormalities nor can it assess the biochemical status of the embryo prior to transfer. This laboratory recently described a method to estimate embryo weight and suggested weight might be a good indicator of biochemical status. The objective of the present study was to determine the relationship between maternal body composition and embryo weight and determine the influence maternal chemistry had on embryo development. The data continue to suggest that maternal body composition, especially body fat, influences the chemical nature of the embryo and may play a critical role in long-term survival.

Background The Accreditation Council for Graduate Medical Education requires scholarly activity for both faculty and residents in obstetrics and gynecology (Ob-Gyn). There is little evidence on the most effective method to train, recruit, and retain research faculty who can mentor resident researchers at small programs. Innovation To address this problem, we created the “Baby Steps” program for a small university-based Ob-Gyn program. Methods After a thorough assessment of existing resources, a postdoctoral researcher was recruited and coupled with an established researcher to raise the standards of resident research, facilitate and coordinate resident projects, and support clinical faculty participation in research activities. Grant submissions, grants awarded, publications submitted, presentations, and awards were tracked before and after the implementation of the Baby Steps program for faculty and residents. Results After 2 years the program has already begun to show an increase in scholarly activity. In a program of 12 residents, 8 made one or more presentations at regional or national meetings within the previous 24 months. Additionally, 8 of 12 clinical faculty members were engaged as mentors in resident research, compared with only 3 in past years. Further, abstract, paper, and grant submissions by faculty increased approximately 25%. Conclusion The addition of a mentored postdoctoral researcher was associated with improvements to both resident and faculty research activities. Based on this success, a sister residency program has incorporated the Baby Steps approach into its training.

So while it is generally agreed that AI is the most efficient means currently available for rapid and widespread dissemination of superior male genetics and embryo transfer represents the equivalent for the female of the species; why has their use not been more widely accepted across the entire field of agriculture? Quite simply it can be reduced to a few factors; some technical, some logistical and some based on upfront cost.

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.

With recent large-breed organization acceptance, the use of frozen semen is gaining more attention in the equine industry. However, cryopreserved stallion semen is commonly associated with poor quality and decreased pregnancy rates as compared with those produced during normal mating or with cooled semen techniques. Therefore our objective was to investigate a new unique freezing technique (UFT) with the intent of improving fertility outcomes. A series of experiments tested the UFT compared with traditional liquid nitrogen methodology in combination with influence of the extenders and stallions used. In Experiment 1, post-thaw motility results of UFT variations were compared with those from liquid nitrogen methods. The averaged post-thaw motility percentages of the 4 UFT treatments were similar when compared with the traditional liquid nitrogen control group (P = .845). In Experiment 2, 2 egg-yolk–based freezing extenders, Biladyl AB intended for bovine samples and Freezing Medium Test Yolk Buffer used for human samples, were compared. A significant difference in the average post-thaw motilities was found between Biladyl AB (17%) and Freezing Medium Test Yolk Buffer (25%) (P < .002). In the third experiment, we compared variability among stallions using the UFT with the intention of creating a more consistent outcome. Post-thaw motilities and percent of original motility returns among the 4 stallions were significantly different (P < .001). In a field trial using shipped semen from a regional stallion station, the UFT demonstrated very promising results. In conclusion, the UFT may potentially be used as an alterative freezing method to replace current liquid nitrogen methodology. However, further investigation is needed to refine techniques.

Within the equine industry, overly aggressive stallion behavior related to various factors including genetics, dominance, or mating can create a dangerous environment and present several training challenges. In attempt to reduce this behavior, some performance trainers are administering altrenogest, an oral synthetic progestogen (Regu-Mate®, Intervet Inc, Millsboro, Del) in an off-label use. Regu-Mate® is commonly used in the equine industry to suppress estrus in performance mares and to synchronize estrous cycles for breeding. Although several articles have been published concerning the possible negative effects of this off-label use of altrenogest in stallions on spermatogenesis and steroid production with varying behavioral effects, the use within the industry continues. However, there are currently no known estimates of the extent to which this exogenous hormone is being utilized in stallions. Our objective was to survey the rate of Regu-Mate use in Quarter horse stallions during 2002. Two hundred questionnaires were sent to Quarter horse stallion owners, trainers, and exhibitors in the United States. The answers presented give a type reference of the equine facilities that responded, as well as details concerning those that administered Regu-Mate®. Sixty-three of the surveys were competed and returned (32%), with ReguMate® use occurring in stallions identified by 18 of 63 facilities (29%). Most of the stallions receiving ReguMate® were not used for breeding at the time of dosing; however most were intended for breeding at a later point. The primary use of altrenogest given to stallions 2

The objective of this study was to ascertain whether vaginal preparation with povidone-iodine before Cesarean delivery would reduce the incidence of post-Cesarean related infection. Participants were randomized to vaginal preparation with either povidone-iodine (n = 80) or saline (n = 80). Post-cesarean related infections included 1) endometritis, defined as fever of >100.4°F on two separate occasions, 6 hours apart, >24 hours postoperatively or >101°F at any time with abdominal/uterine tenderness or 2) cellulitis, defined as advancing erythema around the incision. We calculated overall rates of post-cesarean related infection, relative risk, and 95% confidence intervals for the effect of vaginal preparation. As designed and reported, the trial had at least an 85% power to detect a 30% or greater absolute difference in rates of overall infection (two tailed, α = 0.05). There was no significant difference among group demographics (maternal age, parity, anesthesia, labor before current cesarean delivery, number of vaginal examinations during labor, prophylactic antibiotic use, or gestational age at delivery). The post-cesarean endometritis rate was (9.4%). The post-cesarean cellulitis rate was (6.8%). Vaginal preparation with povidone-iodine before Cesarean delivery reduced the rate of post-cesarean endometritis (P

Previous research has suggested a correlation between the subtle rise in progesterone induced by the administration of human chorionic gonadotropin (hCG) and cycle outcome in patients undergoing in vitro fertilization with embryo transfer (IVF-ET). The data suggest that patients who have2-fold increase in progesterone levels will not conceive as a result of that IVF-ET attempt (poor responders). Further, if the luteinizing-granulosa cells of poor responders are placed in culture, their steroid hormone production will be significantly less than that of the luteinizing-granulosa cells of patients with a normal response to hCG (3-fold; normal responders). Recent studies have demonstrated that steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis. The objective of the present study was to determine whether StAR expression differed in normal responders and poor responders undergoing IVF-ET.The luteinizing-granulosa cells of 6 patients were isolated after follicular aspiration and assayed for StAR expression. Three sets of cells were from patients exhibiting a normal response to hCG administration and 3 sets were from patients exhibiting a poor response.Data suggest a 17% drop in StAR protein in patients with a poor response to hCG administration when compared with those with a normal response.Although StAR protein was expressed in all 6 cellular extracts, expression appeared greatest in cells recovered from normal responders. Further, although two thirds of the patients with a normal response reported term pregnancies, no pregnancies were reported in the poor responders. Low levels of progesterone and absence of pregnancy may be due to a defect in the mechanism that converts granulosa and theca-lutein cells to luteal cells after the luteinizing hormone surge (or hCG administration). Data from the current study would suggest that the StAR protein may be involved.

To date, the common thread in the use of semen extenders/collection techniques, whether they are being used for fresh extended semen, chilled semen, or cryopreserved semen, is that the extenders have all traditionally been added post-collection. The objective of this study was to determine if modifying the method of collection/ extension of semen to include a warmed media environment would improve semen parameters by lessening cold and pH shock. Ten canine semen samples were collected with a modified artificial vagina to allow for a true split collection into to collection containers at one time. The treatment half of the sample was collected into a measured amount of warmed extension media. The control half was collected into a dry container and no attempts to maintain temperature was used. Standard semen parameters, available sperm pool, and number of inseminations were evaluated at specific time intervals and evaluations continued until samples reached zero percent motility. Data analysis was performed with SPSS using the general linear model and appropriate t-tests. There was a difference between the treatment and control groups for Motility (P .05). Modification of the semen collection/extension procedure resulted in improved semen parameters for extended time-periods post-collection. The data suggest the described collection technique can yield significantly more motile sperm by placing the sample into a physiologically favorable environment (eliminating pH and cold shock and allowing osmoregulation to begin), thus providing more available sperm for breeding.

STUDY QUESTION Can a modified specific gravity technique be used to distinguish viable from nonviable embryos? SUMMARY ANSWER Preliminary data suggests a modified specific gravity technique can be used to determine embryo viability and potential for future development. WHAT IS KNOWN ALREADY Single embryo transfer (SET) is fast becoming the standard of practice. However, there is currently no reliable method to ensure development of the embryo transferred. STUDY DESIGN, SIZE, DURATION A preliminary, animal-based in vitro study of specific gravity as a predictor of embryo development using a mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS After a brief study to demonstrate embryo recovery, experiments were conducted to assess the ability of the specific gravity system (SGS) to distinguish between viable and nonviable embryos. In the first study, 1-cell mouse embryos were exposed to the SGS with or without previous exposure to an extreme heat source (60°C); measurements were repeated daily for 5 days. In the second experiment, larger pools of 1-cell embryos were either placed directly in culture or passed through the SGS and then placed in culture and monitored for 4 days. MAIN RESULTS AND THE ROLE OF CHANCE In the first experiment, viable embryos demonstrated a predictable pattern of descent time over the first 48 h of development (similar to previous experience with the SGS), while embryos that were heat killed demonstrated significantly altered drop patterns (P < 0.001); first descending faster. In the second experiment, average descent times were different for embryos that stalled early versus those that developed to blastocyst (P < 0.001). Interestingly, more embryos dropped through the SGS developed to blastocyst than the culture control (P < 0.01). LIMITATIONS, REASONS FOR CAUTION As this is a preliminary report of the SGS technology determining viability, a larger embryo population will be needed. Further, the current in vitro study will need to be followed by fecundity studies prior to application to a human population. WIDER IMPLICATIONS OF THE FINDINGS If proven, the SGS would provide a noninvasive means of assessing embryos prior to transfer after assisted reproductive technologies procedures, thereby improving fecundity and allowing more reliable SET. STUDY FUNDING/COMPETING INTERESTS The authors gratefully acknowledge the funding support of the U.S. Jersey Association, the Laura W. Bush Institute for Women's Health and a Howard Hughes Medical Institute grant through the Undergraduate Science Education Program to Texas Tech University. None of the authors have any conflict of interest regarding this work. TRIAL REGISTRATION NUMBER none.

To suppress sexual and aggressive behavior in performance stallions, many trainers administer oral altrenogest in an off-label regimen of short-term, repetitive dosages despite the absence of any research to examine possible side effects. In this study, nine 3-yr-old Quarter-Horse stallions were initially blocked by sire and randomly allotted to two groups, treatment (n = 5) and control (n = 4). The treatment group received a total of three 10-d (d 1 to 10, 21 to 30, and 41 to 50) regimens of 0.088 mg/kg BW Regu-Mate® (Intervet, Millsboro, DE) followed by 10 d of nontreatment (d 11 to 20, 31 to 40, and 51 to 60). Following this 60-d period, there was a 36-d recovery period in which no treatments were administered. No significant changes were found between the groups in BW or body condition score (BCS). Of the testicular parameters analyzed, scrotal width (SW) and gross testicular parameters were unaffected by the altrenogest; however, mean spermatid counts were decreased in the testicular biopsies of the treated animals (P

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Discharge of man-made organic compounds into the environment poses unknown risks to human reproduction. With less than 10% of the estimated 80,000 man-made chemical compounds having been specifically tested for toxic reproductive effect, an accurate and routine testing procedure is critical to further evaluate the risks associated with these compounds. Previous research from the Department of Obstetrics/Gynecology, Texas Tech University and other laboratories has suggested that cultured human luteinized-granulosa cells (HLGC) may be useful as a model for toxicology studies. In the current study, HLGC were exposed to varying levels of four organic solvents with known or suspected carcinogenic potential. Using the established culture system, HLGC collected from patients in the in vitro fertilization program were collected and cultured at known densities. Once the cultures were established, the wells were rinsed and the media replaced with media containing increasing concentrations of the organic solvents acetone, benzene, carbon tetrachloride, and toluene. After 48 hours the media was removed for hormonal analysis and the cells were prepared for electron microscopy. Using a random complete block design with factorial arrangement of treatments, the resulting data were statistically analyzed using analysis of variance and Fisher's polynomial least significant difference for mean separation. There was an overall trend toward decreased progesterone production (P = .14) with increasing compound concentration. As might be expected, these effects were compound specific (P < .004), with increasing levels of toluene appearing to have the most deleterious effect (P < .001). Ultrastructural examination revealed leaching of cell lipids by all compounds at concentrations of 10 ppm and higher. This decreased production of progesterone would affect embryo implantation and uterine viability, which could explain the increased incidence of spontaneous abortions in women routinely exposed to these compounds.

The purpose of this study was to evaluate the effect of hemoglobin (Hb) on an enzyme-linked immunosorbent assay (ELISA) for the detection of tumor necrosis factor-alpha (TNF-alpha). Detection of TNF-alpha was performed by using the commercially available ELISA systems Factor-Test ttm hTNF-alpha and Predicta tm Tumor Necrosis Factor-alpha Kit (Genzyme, Cambridge, MA) in buffered samples containing 0.00 pg ml −1 or 200 pg ml −1 of recombinant human TNF-alpha, spiked with human Hb. The results suggest that Hb interferes with the detection of TNF-alpha by ELISA. A more pronounced effect was observed with the Factor-Test tm hTNF-alpha system. The observed effects were directly proportional to the concentration of Hb ranging from 1 to 20 mg ml −1 . It appeared that in freshly spiked samples, Hb cross-reacted with monoclonal anti-TNF-alpha antibodies. When such an interaction occurred, Hb underwent a chemical reaction with hydrogen peroxide to yield a potent oxidant, capable of oxidizing the assay's substrates o -phenylenediamine dihydrochloride or 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate. The fact that Hb might interact with other proteins and possesses catalytic, peroxidase-like activity, suggests the possibility that this molecule may mimic the action of horseradish peroxidase in peroxidase-based ELISA systems and produce false positive results. It was also found that incubation of the TNF samples with Hb outside the ELISA system may have caused its instability. Non-denaturing size-exclusion chromatography revealed that incubation of recombinant human TNF-alpha with Hb caused its partial dissociation and consequent formation of immunologically less reactive TNF monomers possibly producing false negative results.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hb's inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.

Objective To evaluate coagulation parameters during IVF cycles with elevated E2. Design Prospective clinical study. Setting Human volunteers in an IVF clinic. Patients Infertile women undergoing IVF procedures. Interventions Coagulation factors were measured in blood along with E2 and P after singular hMG or leuprolide acetate (LA) plus hMG up to 14 days after hCG. Main outcome measures Plasma coagulation factors. Results Some coagulation factors were statistically but not clinically elevated after LA-hMG-induced hyperestrogenism. For the most part, this was not correlated with E2. Conclusion This study suggests that endogenous E2 increases due to fertility drugs cause a molecular activation of some coagulation factors, which do not result in an increased thrombosis.

To determine safety and efficacy of induction with prostaglandin E2 gel, we compared the outcome of 25 patients (study group) with an unfavorable cervix, a medical indication for delivery, and one prior low cervical transverse cesarean section to 56 patients (comparison group) with one prior low cervical transverse cesarean section and spontaneous labor. We placed 1 mg of prostaglandin E2 in gel intracervically in the 25 study patients. Common indications for delivery were: diabetes, post dates, and preeclampsia. Although most labor and delivery variables were similar, the study group had a longer mean latent phase (14.2 +/- 13.8 versus 7.3 +/- 3.7 hours: p less than 0.002), but had a shorter mean length of active phase (4.0 +/- 3.5 versus 5.7 +/- 3.0 hours; p less than 0.02). None of the patients in either group had a dehiscence of the uterine scar, nor rupture of the uterus. Both groups had a similar cesarean section rate. Since from the few reported, nonrandomized studies it appears that prostaglandin E2 gel use in patients with a prior low cervical transverse cesarean section may be useful and relatively safe, it may be time to attempt randomized trials of prostaglandin E2 gel versus oxytocin for induction of patients with a prior low cervical transverse cesarean section, unfavorable cervix, and a medical indication for delivery.

In this study, both IVF-ET cycle and pregnancy outcome were compared with increases seen in P concentration during the 24-hour period from 12 hours before to 12 hours after hCG administration. A significantly higher PR (P < 0.001) was seen in women who had at least a threefold increase in P during this time period. Further, no pregnancies were reported from women with a less than twofold increase in P.