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There are 1,200 semen samples from banteng bulls, which are endangered. In all, there are 18,000 samples of blood and serum that can be used to study diseases and help medicate animals, including an itchy Andean bear at the St. Louis Zoo named Maria, who recently got the treatment she needs to help with her allergies. There are also samples and organs from animals who have died over the past 19 or so years, including Robert B., an endangered Sumatran orangutan who died in July from lung issues but whose organs have been frozen and preserved for study. The St. Louis Zoo's frozen zoo is like a bank, an important depository of tools that researchers can use to help animals now and in the future. It's housed in the Endangered Species Research Center and Veterinary Hospital, Home of the AZA Reproductive Center, behind River's Edge and within sight of the cheetah exhibit. It's only open to the public for special tours. "People have been blown away by what we've been able to achieve," said David Powell, who joined the zoo as its director of research in August. That's why they freeze so many things for a rainy day, knowing technology can only improve. "The science can proceed faster when you have the materials." The work they're doing there has implications worldwide, including helping some species from being wiped off the planet. For example, in the late 1980s, there were only seven known Mexican gray wolves left on the planet. The St. Louis Zoo worked with the Infertility Center of St. Louis to develop a way to freeze sperm and use it to inseminate wolves, explains the zoo's laboratory manager, Karen Bauman. The St. Louis Zoo has the largest sperm bank of Mexican gray wolves in the world, and - largely due to its work - there are now 250 of the animals in captivity and about 100 in the wild. Now that the zoo has done so much work on banking and perfecting their preservation recipe ("It makes beautiful sperm," Bauman said) they can now focus on genetic management. They haven't figured out how to produce embryos in wolves, but they have frozen eggs for when that day comes. When it does, the gene pool will broaden. "They're gorgeous animals in many respects," Bauman said. "They are just magnificent." About 15 years ago the zoo was asked whether it could transfer addax embryos and ship them overseas, which could be easier than shipping an entire 200- to 300-pound animal. They figured out how to produce addax embryos but got stuck trying to figure out controlling a female addax's ovulation cycle. So the embryos will sit in the tank until they figure that out. Meanwhile, conditions for addax in the wild continue to decline, Powell said. Every sample housed in the frozen zoo is listed in a database, and the zoo often shares samples with other zoos doing their own research and reproductive work. This fall, the St. Louis Zoo shipped frozen semen from an endangered Banteng bull, Bubba, to the zoo in Columbus, Ohio, to use to inseminate four cows. Bubba has been dead for several years. "He's been in our tank, waiting his turn," Bauman said. "And hopefully, four cows are pregnant." Some samples are collected during routine exams and others collected systematically. When Bauman isn't in the field with the wolves, she still acts as a keeper at the frozen zoo, answering middle-of-the-night calls from the alarm company if the electricity goes out. She still has to drive in to make sure everything's OK. The frozen zoo also helps keep live animals healthy. They'll run hormone panels and compare that to stress levels from five years ago to help animals adjust, or save blood to see if a drug works for a new species, said veterinarian Luis Padilla, head of animal health at the zoo. "When an animal gets sick, we could pull back samples from the frozen zoo and ask, is this something happening for a while or an emerging disease?" They will also pull serum samples to learn about the nutritional needs of endangered animals and are currently looking to see whether painted dogs need more fat in their diets, and whether there are differences between zoo gorillas with and without cardiac disease. Mary Duncan is the zoo's pathologist and performs biopsies on living animals and necropsies on ones that have died. Just recently, she removed a large mass from a gecko and froze a portion of it in case they need to test it later for a viral infection. Depending on the animal, they'll freeze the tumor. Jaguars are known for mammary tumors, so they'll freeze a portion of those to see whether they are associated with a gene mutation. During what was probably the St. Louis area's strangest freezer cleanout recently, she had to prioritize what to keep and what to throw out, and to make the decision that yes, they probably didn't need quite so many guinea pig tumors. (How many did she clean out? "A lot," she confessed.) She's interested in doing research on penguins, which tend to die from heart issues, so she has frozen samples of about 20 or so penguin hearts from the past two decades. "For me, having the frozen samples opens up the possibility of looking at so many things retrospectively," she said. "It gives you so much potential." She points to the example of a frozen human body in Alaska dug up from the 1918 flu epidemic and tested for research, possibly to help prepare for and maybe prevent another human pandemic. Powell, the zoo's director of research, said nobody could have predicted the advances that have taken place in research for the last 20 years. The frozen samples will help with the next breakthroughs. "We hit an inflection point," he said. "We don't know when that will come, but the frozen zoo has to be ready." Explore further: Klondike, puppy born from a frozen embryo, fetches good news for endangered animals

Hodes-Wertz B.,New York University | Grifo J.,New York University | Ghadir S.,Reproductive Center | Kaplan B.,Fertility Centers of Illinois | And 3 more authors.
Fertility and Sterility | Year: 2012

Objective: To determine any beneficial effects of preimplantation genetic screening (PGS) of all chromosomes by array comparative genomic hybridization (aCGH), with either day 3 or blastocyst biopsy, for idiopathic recurrent pregnancy loss (RPL) patients compared with their expected loss rate. Design: Case series report. Setting: Multiple fertility centers. Patient(s): A total of 287 cycles of couples with idiopathic RPL (defined as two or more losses). Intervention(s): PGS was done with day 3 biopsy (n = 193) or blastocyst biopsy (n = 94), followed by analysis with aCGH. Main Outcome Measure(s): Spontaneous abortion rate, euploidy rate. Result(s): A total of 2,282 embryos were analyzed, of which 35% were euploid and 60% were aneuploid. There were 181 embryo transfer cycles, of which 100 (55%) became pregnant with an implantation rate of 45% (136 sacs/299 replaced embryos) and 94 pregnancies (92%) were ongoing (past second trimester) or delivered. The miscarriage rate was found to be only 6.9% (7/102), compared with the expected rate of 33.5% in an RPL control population and 23.7% in an infertile control population. Conclusion(s): Current PGS results with aCGH indicate a significant decrease in the miscarriage rate of idiopathic RPL patients and high pregnancy rates. Furthermore, this suggests that idiopathic recurrent miscarriage is mostly caused by chromosomal abnormalities in embryos. © 2012 by American Society for Reproductive Medicine.

Colls P.,Reprogenetics | Escudero T.,Reprogenetics | Fischer J.,Reprogenetics | Cekleniak N.A.,IRMS | And 6 more authors.
Reproductive BioMedicine Online | Year: 2012

Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were ∼6 Mbp and ∼5 Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy. © 2012, Reproductive Healthcare Ltd.

Ata B.,McGill University | Ata B.,Uludag University | Kaplan B.,Fertility Centers of Illinois | Danzer H.,Reproductive Center | And 5 more authors.
Reproductive BioMedicine Online | Year: 2012

This study retrospectively analysed array comparative genomic hybridization (CGH) results of 7753 embryos from 990 patients to determine the frequency of embryonic euploidy and its relationship with the cohort size (i.e. the number of embryos available for biopsy and array CGH analysis). Linear regression analysis was performed to assess the effect of cohort size on euploidy rate adjusted for the effect of female age. While increasing female age was associated with a significant decrease in euploidy rate of day-3 and day-5 embryos (P < 0.001 for both groups), cohort size was not significantly associated with euploidy rate. Logistic regression analysis was performed to assess the effect of cohort size, adjusted for maternal age, on the likelihood of having at least one euploid embryo available for transfer. The odds of having at least one euploid embryo in an assisted cycle was significantly decreased by increasing female age (P < 0.01 for both day-3 and day-5 embryos) and was significantly increased by every additional embryo available for analysis (P < 0.001 for both day-3 and day-5 embryos). © 2012, Reproductive Healthcare Ltd.

Simerman A.A.,University of California at Los Angeles | Hill D.L.,Reproductive Center | Grogan T.R.,University of California at Los Angeles | Elashoff D.,University of California at Los Angeles | And 5 more authors.
Fertility and Sterility | Year: 2015

Objective: To determine whether follicular fluid (FF) cortisol levels affect cumulus cell (CC) lipid content during oocyte meiotic resumption, and whether CCs express genes for glucocorticoid action. Design: Prospective cohort study. Setting: Academic medical center. Patient(s): Thirty-seven nonobese women underwent ovarian stimulation for in vitro fertilization (IVF). Intervention(s): At oocyte retrieval, FF was aspirated from the first follicle (>16 mm in size) of each ovary and pooled CCs were collected. Main Outcome Measure(s): Follicular fluid cortisol and cortisone analysis was performed with the use of liquid chromatography-tandem mass spectrometry. CCs were stained with lipid fluorescent dye Bodipy FL C16 to determine lipid content with the use of confocal microscopy. Quantitative real-time polymerase chain reaction was used to detect CC gene expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, glucocorticoid receptor (NR3C1), lipoprotein lipase (LPL), and hormone-sensitive lipase (HSL). Result(s): Adjusting for maternal age, FF cortisol levels negatively correlated with CC lipid content and positively correlated with numbers of total and mature oocytes. CCs expressed genes for 11β-HSD type 1 as the predominant 11β-HSD isoform, NR3C1, LPL, and HSL. Conclusion(s): FF cortisol levels may regulate CC lipolysis during oocyte meiotic resumption and affect oocyte quality during IVF. ©2015 by American Society for Reproductive Medicine.

Biancotti J.-C.,Cedars Sinai Medical Center | Narwani K.,Cedars Sinai Medical Center | Buehler N.,Reproductive Center | Mandefro B.,Cedars Sinai Medical Center | And 8 more authors.
Stem Cells | Year: 2010

Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans. © AlphaMed Press.

Son W.-Y.,Reproductive Center | Chung J.-T.,Reproductive Center | Henderson S.,Reproductive Center | Reinblatt S.,Reproductive Center | And 3 more authors.
Fertility and Sterility | Year: 2013

Objective To evaluate the fertilization rate and embryo development resulting from intracytoplasmic sperm injection (ICSI) of spermatozoa retrieved by testicular sperm extraction (TESE) in hCG-primed in vitro maturation (IVM) cycles. Design Case-control study. Setting University teaching hospital. Patient(s) Twenty-four IVM cycles were performed in 21 patients (mean age, 32.3 ± 2.4 years) with polycystic ovaries (PCO) whose partners were nonobstructive azoospermic. Twelve cycles where IVM oocytes were also retrieved were compared with a control group consisting of age-matched IVM cycles with ICSI using ejaculated spermatozoa (n = 12). Intervention(s) In vitro maturation treatment with TESE sperm. Main Outcome Measure(s) Fertilization and embryo development between sibling oocytes matured in vivo and in vitro. Result(s) Eight singleton pregnancies and one twin pregnancy were obtained after ET (9/24, 37.5%). In the 12 IVM cycles where in vivo-matured oocytes were also obtained, the fertilization rate after TESE-ICSI was significantly higher in in vivo-matured oocytes than in sibling in vitro-matured oocytes (84.2% vs. 53.2%). The proportion of good quality embryos was also higher (63.5% vs. 40.2%). In the control group of cycles with ejaculated spermatozoa, there was no difference in fertilization rates between sibling oocytes matured in vivo and in vitro (84.6% vs. 79.6%). Conclusion(s) Our results suggest that IVM of immature oocytes combined with TESE-ICSI is an option for couples with PCO and azoospermia. However, there are lower fertilization and good quality embryo rates achieved when TESE-ICSI was done with in vitro-matured oocytes. Additional studies are necessary to determine the role of this treatment combination. Copyright © 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.

Iwata K.,Reproductive Center | Yumoto K.,Reproductive Center | Sugishima M.,Reproductive Center | Mizoguchi C.,Reproductive Center | And 3 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2014

Purpose: To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. Methods: One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Results: Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. Conclusions: The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis). © 2014 Springer Science+Business Media New York.

Iwata K.,Reproductive Center | Mio Y.,Reproductive Center
Reproductive Medicine and Biology | Year: 2016

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes. © 2015 Japan Society for Reproductive Medicine

Santibanez A.,Reproductive Center | Garcia J.,Reproductive Center | Pashkova O.,Reproductive Center | Colin O.,Reproductive Center | And 3 more authors.
Reproductive Biology and Endocrinology | Year: 2014

Background: The implantation process after embryo transfer depends on the embryo quality and endometrial receptivity. It is estimated that fifty to seventy-five per cent of pregnancies are lost due to a failure of implantation. There is evidence that there is an early secretion of human chorionic gonadotrophin before embryo implantation, and this secretion has been linked to an important function in angiogenesis and the inflammatory response that promotes the implantation process. Our objective was to determine the effects of intrauterine injection of human chorionic gonadotropin (hCG) before the embryo transfer in an in vitro fertilisation cycle.Methods: A prospective randomised study was conducted in Reproductive Medicine Centre PROCREA in Mexico City. Infertile patients who had a medical indication for in vitro fertilisation were studied. Two groups were included (n 210); the intervention group received an intrauterine injection of 500 IU of hCG before the embryo transfer (n 101). The control group (n 109) did not receive hCG. Comparisons were performed using a chi-square test.Results: The clinical pregnancy rate (CPR) was our principal outcome. The implantation rate was a secondary outcome. The implantation rate was significantly higher in the hCG group compared to the control group (52.4% vs 35.7%, p 0.014). The clinical pregnancy rate was also significantly higher (50.4 vs 33.0%, p 0.010). No adverse effects were observed.Conclusions: The intrauterine injection of hCG before embryo transfer showed a significant increase in the clinical pregnancy rate. More clinical trials are needed to reproduce these results on this promising intervention. The live birth rate must be included in subsequent studies. © 2014 Santibañez et al.; licensee BioMed Central Ltd.

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