The effectiveness of flow cytometric sorting of human sperm (MicroSort®) for influencing a child’s sex

Reproductive Biology and Endocrinology

Joseph D Schulman²  Lawrence A Johnson¹  Marisa L Cole²  Chrispo I Opanga²  Daniel A Potter³  Harvey J Stern²  Donald P Marazzo²  David S Karabinus² 

[1] 16920 Hardy Rd, Mount Airy, MD 21771, USA;Genetics & IVF Institute, 3015 Williams Dr, Fairfax, VA 22031, USA;Huntington Reproductive Center, 23961 Calle de la Magdalena, Suite 503, Laguna Hills, CA 92653, USA

关键词: ART procedures;    FET;    IVF/ICSI;    IUI;    Sex selection;    Flow cytometry;    Human sperm, sperm sorting;   

Others  :  1139750
DOI  :  10.1186/1477-7827-12-106

 received in 2014-05-03, accepted in 2014-09-11,  发布年份 2014

【 摘 要 】

Background

Flow cytometric sorting can be used to separate sperm based on sex chromosome content. Differential fluorescence emitted by stained X- vs. Y-chromosome-bearing sperm enables sorting and collection of samples enriched in either X- or Y-bearing sperm for use to influence the likelihood that the offspring will be a particular sex. Herein we report the effectiveness of flow cytometric sorting of human sperm and its use in human ART procedures.

Methods

This prospective, observational cohort study of the series of subjects treated with flow cytometrically sorted human sperm was conducted at investigational sites at two private reproductive centers. After meeting inclusion criteria, married couples (n = 4993) enrolled to reduce the likelihood of sex-linked or sex-limited disease in future children (n = 383) or to balance the sex ratio of their children (n = 4610). Fresh or frozen-thawed semen was processed and recovered sperm were stained with Hoechst 33342 and sorted by flow cytometry (n = 7718) to increase the percentage of X-bearing sperm (n = 5635) or Y-bearing sperm (n = 2083) in the sorted specimen. Sorted sperm were used for IUI (n = 4448) and IVF/ICSI (n = 2957). Measures of effectiveness were the percentage of X- and Y-bearing sperm in sorted samples, determined by fluorescence in situ hybridization, sex of babies born, IVF/ICSI fertilization- and cleavage rates, and IUI, IVF/ICSI, FET pregnancy rates and miscarriage rates.

Results

Sorted specimens averaged 87.7 ± 5.0% X-bearing sperm after sorting for X and 74.3 ± 7.0% Y-bearing sperm after sorting for Y. Seventy-three percent of sorts were for girls. For babies born, 93.5% were females and 85.3% were males after sorting for X- and Y-bearing sperm, respectively. IUI, IVF/ICSI, and FET clinical pregnancy rates were 14.7%, 30.8%, and 32.1%, respectively; clinical miscarriage rates were 15.5%, 10.2%, and 12.7%.

Conclusions

Flow cytometric sorting of human sperm shifted the X:Y sperm ratio. IUI, IVF/ICSI and FET outcomes were consistent with unimpaired sperm function. Results provide evidence supporting the effectiveness of flow cytometric sorting of human sperm for use as a preconception method of influencing a baby’s sex.

Trial registration

NCT00865735 (ClinicalTrials.gov)

【 授权许可】

   
2014 Karabinus et al.; licensee BioMed Central Ltd.

【 参考文献 】

• [1]Pinkel D, Gledhill BL, Van Dilla MA, Stephenson D, Watchmaker G: High resolution DNA measurements of mammalian sperm. Cytometry 1982, 3:1-9.
• [2]Johnson LA, Welch GR, Keyvanfar K, Dorfmann A, Fugger EF, Schulman JD: Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases. Hum Reprod 1993, 8:1733-1739.
• [3]Sumner AT, Robinson JA: A difference in dry mass between the heads of X- and Y-bearing human spermatozoa. J Reprod Fertil 1976, 48:9-15.
• [4]Johnson LA, Flook JP, Look MV, Pinkel D: Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res 1987, 16:1-9.
• [5]Johnson LA, Flook JP, Hawk HW: Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989, 41:199-203.
• [6]Morrell JM, Dresser DW: Offspring from inseminations with mammalian sperm stained with Hoechst 33342, either with or without flow cytometry. Mutat Res 1989, 224:177-183.
• [7]Cran DG, Johnson LA, Miller NGA, Cochrane D, Polge CE: Production of bovine calves following separation of X- and Y-chromosome bearing sperm and in vitro fertilization. Vet Rec 1993, 132:40-41.
• [8]Cran DG, Cochrane DJ, Johnson LA, Wei H, Lu KH, Polge C: Separation of X- and Y-chromosome bearing bovine sperm by flow cytometry for use in IVF. Theriogenology 1994, 41:183.
• [9]Johnson LA: Sex preselection in swine: altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm. Reprod Domest Anim 1991, 26:309-314.
• [10]Levinson G, Keyvanfar K, Wu JC, Fugger EF, Fields RA, Harton GL, Palmer FT, Sisson ME, Starr KM, Dennison-Lagos L, Calvo L, Sherins RJ, Bick D, Schulman JD, Black S: DNA based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease. Hum Reprod 1995, 10:979-982.
• [11]Fugger EF, Black SH, Keyvanfar K, Schulman JD: Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection. Hum Reprod 1998, 30:2367-2370.
• [12]Vidal F, Fugger EF, Blanco J, Keyvanfar K, Catala V, Norton M, Hazelrigg WB, Black SH, Levinson G, Egozcue J, Schulman JD: Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X- or Y-chromosome haplotypes: a blind trial assessed by double and triple colour fluorescent in-situ hybridization. Hum Reprod 1998, 13:308-312.
• [13]Muller W, Gautier F: Interactions of heteroaroatic compounds with nucleic acids; A-T specific nonintercalating DNA ligands. Eur J Biochem 1975, 54:385-394.
• [14]Arndt-Jovan DJ, Jovin TM: Analysis and sorting of living cells according to deoxyribonucleic acid content. J Histochem Cytochem 1977, 25:585-589.
• [15]Lalande ME, Ling V, Miller RG: Hoechst 33343 dye uptake as a probe of membrane permeability changes in mammalian cells. Proc Natl Acad Sci U S A 1981, 78:363-367.
• [16]Latt SA, Stetten G: Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. J Histochem Cytochem 1976, 24:24-33.
• [17]Cosa G, Focsaneanu KS, McLean JRN, McNamee JP, Scaiano JC: Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution. Photochem Photobiol 2001, 73:585-599.
• [18]Johnson LA, Pinkel D: Modification of a laser-based flow cytometer for high resolution DNA analysis of mammalian spermatozoa. Cytometry 1986, 7:268-273.
• [19]Pieters MHEC, Geraedts JPM, Meyer H, Dumoulin JCM, Evers JLH, Jongbloed RJE, Nederlof PM, van der Flier S: Human gametes and zygotes studied by nonradioactive in situ hybridization. Cytogenet Cell Genet 1990, 53:15-19.
• [20]Landis RJ, Heyman ER, Koch GG: Average partial association in three-way contingency tables: A review and discussion of alternative tests. Int Stat Rev 1978, 45:237-254.
• [21]Fugger EF: Clinical experience with flow cytometric separation of human X- and Y-chromosome bearing sperm. Theriogenology 1999, 52:1435-1444.
• [22]Schulman JD, Karabinus DS: Scientific aspects of preconception gender selection. Reprod Biomed Online 2005, 10:111-115.
• [23]Karabinus DS: Flow cytometric sorting of human sperm: MicroSort® clinical trial update. Theriogenology 2009, 71:74-79.
• [24]Online Mendelian Inheritance in Man. OMIM Statistics for the week of January 7, 2013. [http://www.ncbi.nlm.nih.gov/Omim/mimstats.html webcite]
• [25]O’Brien P, Vandekerckhove P: Intra-uterine versus cervical insemination of donor sperm for subfertility. Cochrane Database Syst Rev 2000, 2:CD000317.
• [26]Le Lannou D, Gastard E, Guivarch A, Laurent MC, Poulain P: Strategies in frozen donor semen procreation. Hum Reprod 1995, 10:1765-1774.
• [27]Ferrara I, Balet R, Grudzinskas JG: Intrauterine insemination with frozen donor sperm. Pregnancy outcome in relation to age and ovarian stimulation regime. Hum Reprod 2002, 17:2320-2324.
• [28]Ombelet W, Dhont N, Thijssen A, Bosmans E, Kruger T: Semen quality and prediction of IUI success in male subfertility: a systematic review. Reprod Biomed Online 2014, 28:300-309.
• [29]Burris AS, Clark RV, Vantman D, Sherins RJ: A low sperm concentration does not preclude fertility in men with isolated hypogonadotropic hypogonadism after gonadotropin therapy. Fertil Steril 1988, 50:343-347.
• [30]Trout S, Bohrer M, Corsan G, Sachdev R, Kemmann E: Is it worthwhile to perform intrauterine insemination of less than one million motile sperm? Fertil Steril 1995, 64(Suppl 1):s132.
• [31]Cressman BE, Pace-Owens S, Pliego JF, Wincek TJ, Kuehl TJ: Effect of sperm dose on pregnancy rate from intrauterine insemination: a retrospective analysis. Tex Med 1996, 92:74-79.
• [32]Centola GM: Successful treatment of severe oligozoospermia with sperm washing and intrauterine insemination. J Androl 1997, 18:448-453.
• [33]Marshburn PB, Alanis M, Matthews ML, Usadi R, Papadakis MH, Kullstam S, Hurst BS: A short period of abstinence before intrauterine insemination is associated with higher pregnancy rates. Fertil Steril 2010, 93:286-288.
• [34]Botchan A, Hauser R, Gamzu R, Yogev L, Paz G, Yavetz H: Results from 6139 artificial insemination cycles with donor spermatozoa. Hum Reprod 2001, 16:2298-2304.
• [35]Jurema MW, Viera AD, Bankowski B, Petrella C, Zhao Y, Wallach E, Zacur H: Effect of ejaculatory abstinence period on the pregnancy rate after interuterine insemination. Fertil Steril 2005, 84:678-681.
• [36]Randall GW, Gantt PA: Double vs. single intrauterine insemination per cycle: use in gonadotropin cycles and in diagnostic categories of ovulatory dysfunction and male factor infertility. J Reprod Med 2008, 53:196-202.
• [37]ESHRE Capri Workshop Group: Intrauterine insemination. Hum Reprod Update 2009, 15:265-277.
• [38]Merviel P, Heraud MH, Grenier N, Lourdel E, Sanguinet P, Copin H: Predictive factors for pregnancy after intrauterine insemination (IUI): An analysis of 1038 cycles and review of the literature. Fertil Steril 2010, 93:79-88.
• [39]Chavkin DE, Molinaro TA, Roe AH, Sammel MD, Dokras A: Donor sperm insemination cycles: Are two inseminations better than one? J Androl 2012, 33:375-380.
• [40]Ventura SJ, Curtin SC, Abma JC, Henshaw SK: Estimated pregnancy rates and rates of pregnancy outcomes in the United States, 1990–2008. Nat Vital Statistics Rep 2012, 60:1-22.
• [41]Tournaye H, Verheyen G, Albano C, Camus M, Van Landuyt L, Devroey P, Van Steirteghem A: Intracytoplasmic sperm injection versus in vitro fertilization: a randomized controlled trial and a meta-analysis of the literature. Fertil Steril 2002, 78:1030-1036.
• [42]Shen S, Khabani A, Klein N, Battaglia D: Statistical analysis of factors affecting fertilization rates and clinical outcome associated with intracytoplasmic sperm injection. Fertil Steril 2003, 79:355-360.
• [43]Rosen MP, Shen S, Rinaudo PF, Huddleston G, McCulloch CE, Cedars MI: Fertilization rate is an independent predictor of implantation rate. Fertil Steril 2010, 94:1328-1333.
• [44]Johnson LNC, Sasson IE, Sammel MD, Dokras A: Does intracytoplasmic sperm injection improve fertilization rate and decrease the total fertilization failure rate in couples with well defined unexplained infertility? A systematic review and meta analysis. Fertil Steril 2013, 100:704-711.
• [45]Cook S, Quinn P, Kime L, Ayres C, Tyler JPP, Driscoll GL: Improvement in early human embryo development using new formulation sequential stage-specific culture media. Fertil Steril 2002, 78:1254-1260.
• [46]Johnson JE, Higdon HL, Blackhurst DW, Boone WR: Expectations for oocyte fertilization and embryo cleavage after whole sperm versus sperm head intracytoplasmic sperm injection. Fertil Steril 2004, 82:1412-1417.
• [47]Ciray HN, Karagenc L, Ulg U, Bener F, Bahceci M: Early cleavage morphology affects the quality and implant potential of day 3 embryos. Fertil Steril 2006, 85:358-365.
• [48]Centers for Disease Control and Prevention, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology: 2011 Assisted Reproductive Technology Fertility Clinic Success Rates Report. Atlanta (GA): US Dept of Health and Human Services; 2013.
• [49]Belloc S, Cohen-Bacrie P, Benkhalifa M, Cohen-Bacrie M, De Mouzon J, Hazout A, Menezo Y: Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination. Reprod Biomed Online 2008, 17:392-397.
• [50]Bellver J, Garrida N, Remoni J, Pellicer A, Meseguer M: Influence of paternal age on assisted reproductive outcome. Reprod Biomed Online 2008, 17:595-604.
• [51]Brandes M, Verzijden JCM, Hamilton CJCM, de Weys NPC, de Bruin JP, Bots RSGM, Nelen WLDM, Kremer JAM: Is the fertility treatment itself a risk factor for early pregnancy loss? Reprod Biomed Online 2011, 22:192-199.
• [52]Riggs R, Mayer J, Dowling-Lacey D, Chi T-C, Jones E, Oehninger S: Does storage time influence postthaw survival and pregnancy outcome? An analysis of 11,768 cryopreserved human embryos. Fertil Steril 2010, 93:109-115.
• [53]Hill MJ, Miller KA, Frattarelli JL: A GnRH agonist and exogenous hormone stimulation protocol has a higher live-birth rte than an natural endogenous hormone protocol for frozen-thawed blastocyst-stage embryo transfer cycles: an analysis of 1391 cycles. Fertil Steril 2010, 93:416-422.
• [54]Dasig J, Zhao J, Reddy V, Gebhardt J, Suarez M, Behr B: Spontaneous abortion rates following frozen embryo transfer: preliminary experience comparing slow and vitrified cryopreservation methods. Fertil Steril 2011, 86:s417.
• [55]Tomas C, Alsbjerg B, Martikainen H, Humaidan P: Pregnancy loss after frozen-embryo transfer – a comparison of three protocols. Fertil Steril 2012, 98:1165-1169.