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Female Sex Selection Using Clomiphene Citrate and Albumin
Separation of Sperm
A. Y. Silverman1, S. R. Stephens2, M. T. Drouin3,
R. G. Zack4, J. Osborne5, and S.A. Ericsson6,7. 112 Greenridge Avenue, Suite 400 White Plains, NY 10605 USA. Walnut Hill Lane, Suite 324, Dallas, TX 75231 USA. 3Women's Health Center, 287 Main Street, Suite 201 Lewiston, ME 04240 USA. 4Midwest Fertility and Sex Selection Center, 18211 West Twelve Mile Road Lathrup Village, MI 48076 USA. 5Birmingham Gender Clinic, Eldon House, Central Square High Street, Erdington, 6, 7Department of Animal Science, Box C-11, Sul Ross State University, Alpine, Texas, USA. Running title: Sex selection using clomiphene and albumin separated sperm.
Key words: albumin separation/ clomiphene citrate/ sex ratio/ sex selection/ sperm.
7To whom correspondence should be addressed at: Background - This study was undertaken to assess whether the use of clomiphene
citrate in conjunction with albumin-separated sperm wil alter the sex ratio, proportion of males, towards females and if this skewing is due solely to induction of ovulation. Methods - The sex ratios of 184 single and 42 twin births born at 5 assisted
reproductive biology clinics were determined. The normal approximation to the
binomial distribution was used to determine significant differences between these sex ratios and the established sex ratios for single, twin and combined (single and twin) non- and ovulation induced births. Results - The non-induced ovulation sex ratio for singletons (51.4%) and twins
(50.2%) were greater than the treatment singleton (27.7%; P < 0.001) and twin sex
ratios (33.3%; P < 0.01), respectively. Correspondingly, the non-induced sex ratio for combined (51.4%) births was greater than the treatment sex ratio (28.8%; P < 0.001). The induced singleton and twin sex ratios (48.1%) were lower than the non-induced sex ratio (51.4%), but higher than the treatment singleton (27.7%; P < 0.001) or twin (33.3%; P < 0.03) sex ratios. The induced combined (48.1%) was less than the non-induced combined (51.4%) sex ratio, although greater than the treatment combined sex ratio (28.8%; P < 0.001). Conclusions
Clomiphene citrate in conjunction with albumin-separated sperm will decrease the sex ratio and this reduction is not exclusively due to induction of ovulation. Introduction
The secondary sex ratio, the proportion of males to females at the time of birth, can be expressed as the percentage of males and is conventionally thought to be similar (51.4%) for both singleton births (Maconochie and Roman, 1997) and for the general population, which includes multiple births (Ericsson and Ericsson, 1999). The twin birth sex ratio (50.2%) is slightly less than the singleton and general population sex ratios (NCHS, 1995). Many factors have been shown to slightly alter sex ratios. These include ethnicity, season of the year, birth order, maternal and paternal age, specific diseases and nutrition (Ericsson and Ericsson, 1999). An additional variable which has been identified as skewing the sex ratio is gonadotropin and/or clomiphene induced ovulations resulting in either single or twin births (48.1%) (James, 1985). Treatment with clomiphene citrate (CC) prior to artificial insemination with albumin-separated sperm is currently used clinically for couples desiring a female child. This procedure involves isolation of progressively motile spermatozoa that are morphologically normal using an albumin column (Ericsson and Ericsson, 1999) with the resulting pregnancy and spontaneous abortion rates similar to those observed in the general population (Beernink et al., 1993). These selected spermatozoa are artificially inseminated into a woman who has been treated with the ovulatory drug CC. The purpose of this study was to determine if CC in combination with albumin- separated sperm will reduce the sex ratio and if this decline is due entirely to induction Materials and methods
Experimental design
The sex ratios of offspring of couples treated with CC prior to artificial insemination with albumin-separated sperm were compared to established sex ratios. Differences from the treatment sex ratios for the general population, single and twin births were established through comparison with the corresponding control sex ratios for combined (single and twin births) (51.4%) (Ericsson and Ericsson, 1999), single births (51.4%) (Maconochie and Roman, 1997), and twins (50.2%) (NCHS, 1995), respectively. In addition, the treatment singleton and twin sex ratios were compared to the sex ratio (48.1%) identified for gonadotropin and/or CC induced ovulations resulting in either single or twin births (48.1%) (James, 1985).
Subjects

Couples who participated in this study were self selected from 5 assisted reproductive biology clinics and were provided information about risks involved prior to using CC and albumin column separated sperm. Clinics did not terminate pregnancies due to the presence of a male fetus. Female patients received 50 mg of CC on cycle days 5 through 9. A total of 184 single and 42 twin births resulted from the use of this procedure. The singleton (n = 10; sex ratio 30.0%) and twin (n = 4; 0.0% sex ratio) subject groups included births from couples seeking female sex selection due to their Female sex selection techniques
Each semen sample was diluted with an equal amount of Tyrode’s salt solution (Sigma Chemical Co., St. Louis, MO) and centrifuged at 2800-3200 rpm for 10 min. The washed spermatozoa were resuspended in Tyrode’s salt solution at a concentration of 30 x 106 sperm per ml. Aliquots of 0.5 ml portions, containing 15 x 106 washed spermatozoa with at least 40% motility, were then layered over a 1 ml solution of 7.5% human serum albumin (HSA) over 0.5 ml of 17.5% HSA (Alpha Therapeutic Corp., Los Angeles, CA) for 1 h in an 8 x 75 mm glass column at room temperature. The 0.5 ml layer of sperm was removed and the columns allowed to stand for another 30 min. The remaining 7.5% layer then was removed and discarded; the 17.5% layer was pooled, diluted with an equal volume of Tyrode’s salt solution and centrifuged at 2800-3200 rpm for 10 min. The sperm pellet was resuspended in 0.25 ml of Tyrode’s salt solution in preparation for artificial insemination (17% motile sperm recovery). The mean number of motile sperm (95%) used for insemination was 38 x 106 (Beernink et al., 1993). Intracervical or intrauterine insemination was utilized just prior to a rise in basal body temperature on the day following a luteinizing hormone (LH) surge in CC treated patients.
Statistical analysis
Significant differences between treatment and established sex ratios were assessed using the normal approximation to the binomial distribution (Zar, 1999). The uniformity among centers in the single birth and twin sex ratios of children born to couples who used clomiphene citrate and albumin column separated sperm for female sex selection is illustrated in Table 1. The significant differences between established control sex ratios (with and without CC) and the treatment sex ratios are shown in Table 2. The control (non-CC) sex ratios for singletons (51.4%) (Maconochie and Roman, 1997) and twins (50.2%) (NCHS, 1995) were greater than the treatment singleton (27.7%; P < 0.001) and twin sex ratios (33.3%; P < 0.01), respectively. Similarly, the control (non-CC) sex ratios for combined (single and twin) (51.4%) (Ericsson and Ericsson, 1999) births were greater than the treatment sex ratio (28.8%; P < 0.001). The CC control sex ratios for singleton and twin (48.1%) births (James, 1985) were higher than the corresponding treatment singleton (27.7%; P < 0.001) and twin (33.3%; P < 0.03) sex ratios. This same trend was again observed when the CC control for combined (single and twin) (48.1%) births (James, 1985) was compared to the treatment combined sex ratio (28.8%; P < 0.001). Discussion
Some clinicians are doubtful as to the efficacy of using CC prior to artificial insemination with albumin-separated sperm (Ericsson and Ericsson, 1999) as a method of skewing the sex ratio towards females. In addition, other practitioners question whether the alteration of the sex ratio is due solely to the use of CC and not albumin- separated sperm (Martin, 1994). The reduction in the established sex ratio for the general population and single births from 51.4% (Ericsson and Ericsson, 1999) to 28.8% and 27.7%, respectively, illustrates that this methodology is effective in increasing the number of females born. A similar reduction in the twin sex ratio from 50.2% (NCHS, 1995) to 33.3% shows that this sex selection procedure is effective for improving the chance of twin female births. The skewing of the sex ratio in favor of females by all centers illustrates that this procedure can be used successfully by different clinicians for female sex preselection. The ovulatory agent, CC, does reduce the sex ratio as shown by the singleton, twin and combined (single and twin) CC induced births (48.1%) (James, 1985) being lower than those identified for similar non-induced births (51.4%) (Ericsson and Ericsson, 1999; Maconochie and Roman, 1997). The further decrease in the sex ratio for single (27.7%), twin (33.3%) and combined (28.8%) (single and twin) births attributable to the use of CC and albumin separated sperm reveals that sperm treatment is an essential component to this procedure for reducing the sex ratio. We conclude that the use of CC in conjunction with albumin-separated sperm
will alter the sex ratio towards females and that this skewing is not

exclusively due to induction of ovulation.
References
Beernink, F.J., Dmowski, W.P., and Ericsson, R.J. (1993) Sex preselection through separation of sperm. Fertil. Steril., 59, 382-386.
Ericsson, R.J. and Ericsson, S.A. (1999) Sex ratios. In: Encyclopedia of Reproduction volume 4. Eds: Knobil,E. and Neill, J.D. Academic Press. Pp. 431-437. James, W.H. (1985) The sex ratio of infants born after hormonal induction of ovulation. 92, 299-301.
Maconochie, N. and Roman, E. (1997) Sex ratios: are there natural variations within the human population? Br. J. Obstet. Gynaecol., 104(9):1050-1053.
Martin, R.H. (1994) Human sex pre-selection by sperm manipulation. Hum. Reprod. 9,
1790-1791. NCHS. 1995. Matched Twins. National Center for Health Statistics. Hyattsville, MD. Zar, J. H. (1999) Biostatistical Analysis. 4th edition. Prentice Hall. Upper Saddle River. Table 1. Single birth and twin sex ratios reported by individual centers for children born to couples who used clomiphene citrate and albumin column separated sperm Table 2. The sex ratio of children born to couples who used clomiphene citrate and albumin column separated sperm for female sex selection.

Source: http://www.gender-select.com/pdf/Dr.Silverman's_article.pdf

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