Preconceptual female gender selection.
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OBJECTIVE: To evaluate the success for gender selection of insemination with a sample of semen separated by a serum albumin density separation gradient. DESIGN: We retrospectively compared the gender outcome of conceptions of couples who conceived spontaneously with those who conceived secondary to an insemination with separated semen. SETTING: Private practice of one author (M.A.K.). PATIENTS, PARTICIPANTS: The study group consisted of 48 pregnancies of couples who conceived by the separation technique. The control group consisted of 46 pregnancies of couples who initially presented for consultation for gender selection but conceived spontaneously. INTERVENTIONS: In the study group, one timed intrauterine insemination with separated semen was performed per cycle, with a mean of 2.3 cycles per couple. Patients desiring a female were also treated with clomiphene citrate and human chorionic gonadotropin. MAIN OUTCOME MEASURE: The gender outcome of the pregnancies of the two groups was evaluated based on the known desired gender. RESULTS: The success rate for conceiving a desired male was 56.5% in the study group and 60.9% in the control group (P = 1.000). Of couples seeking females, 78.6% of the procedure group versus 35.3% of the control group were successful (P = 0.019). CONCLUSIONS: This study debates the albumin gradient as definitively enriching the proportion of Y-bearing sperm after in vitro separation.
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OBJECTIVE: To perform preimplantation gender determination by a combination of polymerase chain reaction (PCR) sexing and fluorescent in situ hybridization technique using the directly labeled fluorescent alpha-satellite centromeric DNA probes for X and Y chromosomes. SETTING: The IVF program of Illinois Masonic Medical Center. PATIENTS: A couple requested preimplantation diagnosis because the mother is a carrier for hemophilia A. RESULTS: Two blastomeres were aspirated from each of the four- to eight-cell embryos, and only the embryos with both fluorescent in situ hybridization and PCR results indicating female sex chromosomal complement were transferred, resulting in a singleton pregnancy and delivery of a healthy female infant, after prenatal confirmation of the diagnosis as female. The male embryos or embryos diagnosed as females only by PCR were followed up by confirmatory fluorescent in situ hybridization analysis demonstrating a discrepancy of PCR and fluorescent in situ hybridization results in four embryos, presumably because of a possible sperm contamination of the PCR reaction or chromosomal mosaicism. CONCLUSION: The analysis of two blastomeres from the same embryo by a combination of PCR sexing and fluorescent in situ hybridization increases the reliability of preimplantation gender identification at the cleavage stage.
Fluorescent (F) body distribution was determined in a group of men who did not have a fertility problem, but rather had fathered exclusively female offspring. The study was designed to analyze spermatozoa for the frequency of zero F-body (X-bearing) and one F-body (Y-bearing) spermatozoa. Semen samples were separated (processed) for Y-bearing spermatozoa enrichment and reanalyzed for fluorescent body distribution. The study consisted of 50 control (10 males) samples (unprocessed), 35 preseparation (35 patients) samples (unprocessed), and 18 postseparation (18 patients) samples (processed). A significantly higher frequency (P less than 0.05) of zero F-body spermatozoa were observed in the preseparation samples when compared with the control samples. The presence of more spermatozoa without fluorescent body correlates with the occurrence of more female births.
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Over 20,000 calves have resulted from artificial insemination (AI) of cattle with sexed, frozen/thawed sperm in the course of experimentation in several countries, and from commercial sales in the United Kingdom. This technology likely will become commercially available in many countries within a few years. Accuracy of the process is about 90% for either sex, and resulting calves appear to be no different from non-sexed controls in birthweight, mortality, rate of gain, and incidence of abnormalities. The main determinants of the extent of use of sexed sperm will be pregnancy rate and cost. Fertility of low doses (1.5 x 10(6)-2 x 10(6)) of sexed, frozen sperm for AI of heifers usually has been in the range of 70-80% of unsexed sperm at normal doses (10 x 10(6)-20 x 10(6) sperm) in well managed herds; it has been lower in poorly managed herds, and likely will be lower with lactating dairy cows. It is expected that fertility of sexed sperm will increase significantly due to very recent improvements in the hydrodynamics of the sexing process and potential improvements in cryopreservation procedures. It is unclear how sexed sperm will be priced; the cost of sexed sperm for cattle will likely be more than double the cost of unsexed sperm in most markets. The economic benefit of using sexed sperm also will depend on the baseline fertility of the herd since at lower fertility, it takes more doses of semen per calf produced. It is noted that for a small percentage of elite cattle, the economics of using sexed sperm do not depend primarily on increased production or efficiency of producing meat or milk, but rather on factors such as scarcity, tradition, cattle show winnings, and biosecurity during herd expansion. Until sorting efficiencies improve and costs decline, sales likely will be limited primarily to these niche markets. With near normal fertility and a premium for sexing in the range of US$ 10 per insemination dose, sexed sperm likely would become economically and environmentally beneficial for many, if not most populations of cattle being bred by AI.
Commercialization of animal biotechnology is a wide-ranging topic for discussion. In this paper, we will attempt to review embryo transfer (ET) and related technologies that relate to food-producing mammals. A brief review of the history of advances in biotechnology will provide a glimpse to present and future applications. Commercialization of animal biotechnology is presently taking two pathways. The first application involves the use of animals for biomedical purposes. Very few companies have developed all of the core competencies and intellectual properties to complete the bridge from lab bench to product. The second pathway of application is for the production of animals used for food. Artificial insemination (AI), embryo transfer, in vitro fertilization (IVF), cloning, transgenics, and genomics all are components of the toolbox for present and future applications. Individually, these are powerful tools capable of providing significant improvements in productivity. Combinations of these technologies coupled with information systems and data analysis, will provide even more significant change in the next decade. Any strategies for the commercial application of animal biotechnology must include a careful review of regulatory and social concerns. Careful review of industry infrastructure is also important. Our colleagues in plant biotechnology have helped highlight some of these pitfalls and provide us with a retrospective review. In summary, today we have core competencies that provide a wealth of opportunities for the members of this society, commercial companies, producers, and the general population. Successful commercialization will benefit all of the above stakeholders.
The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen-thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 microg LH, 1.0 microg E2/ml) for 22-24h at 39 degrees C in 5% CO(2) in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6-7h after insemination and cultured for 65-66h. Embryos that had cleaved by 72h post-insemination were cultured an additional 96h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7-8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.
Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.
A decision tree for genetics or sperm-sexing entities considering sales of sexed sperm is discussed in terms of: (a) how best to avoid harm; (b) how best to do good; (c) needed synergy with other assisted reproductive technologies; (d) constraints on biotechnology; and (e) costs with current and likely technologies versus potential benefits to producers. The sexed-sperm industry might wish to take a pro-active stance on societal issues potentially affecting use of sexed sperm. For most sales in animal agriculture, cost of added value must be < 50% of benefit. Cost is less important for emotionally-driven uses with horses and human beings. Current procedures for flow-sorting allow most sperm to retain their fertilizing potential. Added cost to produce and package 2 x 10(6) sperm is estimated at US $30 to US $46 with flow sorted sperm. Estimating cost of any alternative technology is premature. For IVF/embryo transfer (ET), cost and numbers of flow-sorted sexed sperm are appropriate for commercial use. For use in low-dose AI, however, added cost to supply one insemination dose must be near US $12. Flow-sorting instruments with higher throughput and lower purchase and operating costs are obligatory for economic application in most AI situations. Developers of antibody-based separations also will face issues of retention of fertilizing potential while minimizing cell loss, separation of living from dead sperm concurrent with sperm sexing, output, and cost. To benefit producers and consumers in a changing world, genetics and sperm-sexing companies will have to collaborate and interface to provide funding for needed research and development and to recover these costs, using mechanisms not yet obvious.
Preimplantation genetic diagnosis is a valuable addition to the repertoire of reproductive medicine because it gives individuals with a documented history of a genetic disorder the opportunity to begin a wanted pregnancy with little or no fear that they are transmitting this disorder to their offspring. An outstanding example of this is the recent report of PGD being successfully used to avoid the conception of a child that could have inherited a predisposition to early-onset Alzheimer disease. However, if this service becomes embroiled in the detection of conditions having little or nothing to do with health and disease in order to satisfy patient demand or to be a profitable business, its activities will be much more ethically suspect.
Preimplantation genetic diagnosis (PGD) was introduced at the beginning of the 1990s as an alternative to prenatal diagnosis, to prevent termination of pregnancy in couples with a high risk for offspring affected by a sex-linked genetic disease. At that time, embryos obtained in vitro were tested to ascertain their sex, and only female embryos were transferred. Since then, techniques for genetic analysis at the single-cell level, involving assessment of first and second polar bodies from oocytes or blastomeres from cleavage-stage embryos, have evolved. Fluorescence in-situ hybridisation (FISH) has been introduced for the analysis of chromosomes and PCR for the analysis of genes in cases of monogenic diseases. In-vitro culture of embryos has also improved through the use of sequential media. Here, we provide an overview of indications for, and techniques used in, PGD, and discuss results obtained with the technique and outcomes of pregnancies. A brief review of new technologies is also included.
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