Sperm Spinning for Gender Selection:
Density Gradient Centrifugation (Percoll)
Density gradient centrifugation, or the Percoll method, is a standard procedure for preparing sperm for IUI or IVF; in the early 1980s, researchers reported that it might also cause separation of X and Y-sperm. Later investigation proved that this was not the case, but the method has been promoted in some clinics for gender selection.
Gradient Density Centrifugation Overview
Just as with the Ericsson method, the father provides a semen sample which is processed for boy or girl gender selection, and then used to inseminate the mother. This is done on the same day that the mother is expected to ovulate, by a simple IUI (intra-uterine insemination).
COST: $600 to $900 per attempt. The cost is negligible compared to MicroSort. The fee seems to include sperm separation for gender selection as well as insemination, but this will vary by clinic. Most couples will require multiple attempts to get pregnant.
SUCCESS RATE: 60% - 70% is claimed.
SAFETY: Gradient density centrifugation is deemed to be safe, depending on the density medium used. This method is a standard procedure for sperm preparation before IUI or IVF (without gender selection).
Sperm Preparation by Gradient Density Centrifugation
In this procedure, sperm is centrifuged through increasingly dense layers of a solution, on the theory that heavier X-sperm will sediment to the bottom, and lighter Y-sperm will migrate to top.
- A density medium, a liquid solution available in various thicknesses, is layered in a conical centrifuge tube, with the most dense layer at the bottom and the least dense layer at the top. In the standard procedure of preparing sperm for assisted reproduction, 2 layers are used. For gender selection, 3 to 12 layers are used.
- Diluted semen is layered on top, and centrifuged for 30 minutes.
- To conceive a girl, only the bottom layer, or "pellet", is retained, in which the heavier X-sperm have sedimented. To conceive a boy, only the top layer of the liquid "supernatant" is retained, containing the lighter Y-sperm.
- The remainder is washed and centrifuged again to remove the density medium.
Percoll, and Other Mediums for Density Gradient Centrifugation
This procedure is often called just the "Percoll method" because Percoll was so often used as the density medium, but other density media are now used. Two other media, Ficoll and Sephadex microbead solution, have been attempted with density gradient centrifugation for gender selection.
The Percoll product was withdrawn from the market in 1996 because of its risk of contamination with endotoxins. PureSperm® or other products are now commonly used. However, pregnancy rates may be lower than with Percoll or swim-up. See:
- Comparison of Four Methods for Sperm Preparation for IUI
Jun-2004, Archives of Andrology
Published Medical Studies on Density Gradient Centrifugation for Gender Selection
Separating X-bearing human spermatozoa through a discontinuous
Percoll density gradient proved to be inefficient
Oct-1998, Journal of Assisted Reproductive Medicine (Taiwan)
- The Percoll method didn't work for X-sperm separation.
- X-sperm was separated in a 12-step discontinuous Percoll density gradient.
- X:Y ratios were verified by double-label fluorescence in situ hybridization (FISH)
- The resulting X:Y ratio was 52.2:46.4 (53% X-sperm).
- "The enrichment of X-bearing spermatozoa is insufficient for clinical use in
preconceptional sex selection"
- Separation of sperm through a 12-layer percoll column
Feb-2000, Archives of Andrology (New Jersey)
- After X-sperm separation by the Percoll gradient, the quinacrine-staining technique apparently showed that Y-sperm in the sample had been reduced to 10% to 19%. However, the quinacrine-staining method is considered unreliable.
- X-sperm separation by a 12-layer Percoll gradient centrifugation was performed on 31 semen samples.
- Y-sperm percentage was determined using quinacrine staining, which stains Y-bearing sperm.
- Only 10% to 19% of the separated sperm was stained.
- However, because the accuracy of the quinacrine method has been questioned when used with sperm separation, the researchers couldn't conclude whether there was actually less Y-bearing sperm, or if the procedure had only caused some of the Y-bearing sperm to resist quinacrine staining. (Why the heck didn't they also FISH test the samples to find out???)
- "Thus, 12-layer Percoll separation may actually enrich for X-bearing sperm or
possibly this procedure somehow nonspecifically inhibits the ability of
quinacrine to stain the Y chromosome."
- Assessment of sex chromosome ratio and aneuploidy rate in motile spermatozoa selected by three different methods
Nov-1997, Human Reproduction (Japan)
- Three standard procedures for isolating motile sperm for use in IUI or IVF were used: swim-up, glass wool, and 2-layer Percoll. The X:Y ratio was verified using fluorescence in situ hybridization (FISH), and was found to remain essentially 1:1 in all three cases.
- Enhanced separation of X and Y bearing sperm cells by a combined density gradient centrifugation
Feb-1997, Acta Obstet Gynecol Scand. (Denmark)
- This study found that adding a final layer of media, even denser than 100% Percoll, could significantly decrease the Y-sperm ratio. The results look promising, but there seem to be no additional studies investigating NycoPrep.
- X-sperm separation was performed on 7 semen samples using a 7-step Percoll gradient alone, or with NycoPrep, a denser media.
- The remaining Y-sperm ratio was determined by fluorescence in situ hybridization (FISH).
- The Y-sperm ratio did not decrease when separated with Percoll only.
- The Y-sperm ratio decreased significantly, to 39%, in the samples processed with Percoll + NycoPrep.
- "The described centrifugation technique can be applied in connection with
preimplantation diagnosis in order to create more pre-embryos of the desired
- [Why did this experiment test only for a reduction in Y-sperm, not verify an increase in X-sperm?]
- Sperm kinetics and morphology before and after fractionation on discontinuous Percoll gradient for sex preselection: computerized analyses
Aug-1996, Archives of Andrology
- Sperm was processed with the 8-layer Percoll method, then computer analyzed to see if sperm movement and shape in the upper and lower layers matched X and Y-sperm, respectively.
- Sperm in the lower layer was found to have higher motility, progression, longevity, and shorter tails, than sperm in the top layer.
- However, sperm head dimensions were not found to be significantly different in the top and bottom layers, discounting the idea that X-sperm are larger and settle to thebottom.
- Male:female sex ratio in births resulting from IVF according to swim-up versus Percoll preparation of inseminated sperm,
Archives of Andrology, Jul-1994 (Full Text)
- IVF births after the use of the Percoll method or the standard swim-up technique were both found to be close to 50% boy/girl.
- Sperm samples were prepared for use in IVF in one of two ways: a) by the standard swim-up technique (the standard technique used to prepare sperm for IUI or IVF, not the modified swim-up attempted for gender selection); and b) by a 3-layer Percoll density gradient centrifugation.
- Swim-up: Of the 103 resulting pregnancies, 125 babies were born (82 singletons, 20 sets of twins and 1 set of triplets). Boy births accounted for 53% of the babies (52% of the singletons, and 51% of multiples).
- Percoll: Of the 34 resulting pregnancies, 41 babies were born (26 singletons, 6 sets of twins, 2 sets of triplets). Boy births accounted for 50% of the babies (54% of the singletons and 40% of multiples).
- [Don't read anything in to the fact that only 40% of the Percoll multiples were boys; with only 18 babies, just one baby more or less of either gender can significantly skew the percentage.]
- Discontinuous Percoll gradients enrich X-bearing human
Jul-1994, Human Reproduction (Australia)
- Percoll separation only increased the ration of X-sperm to 57%, insufficient for gender selection.
- A 12-step discontinuous Percoll gradients
- double-label fluorescence in-situ hybridization and chromosome-specific DNA probes
- Before separation, the X:Y ratio was 49:48.2.
- After separation, the X:Y ratio was 55:41, or 57% X-sperm. The increase in X-sperm was concluded to be insufficient for use in preconceptual gender selection.
- The sex ratio of normal and manipulated human sperm
Feb-1993, Fertility & Sterility
- Semen samples were analyzed after swim-up or Sephadex filtration.
- The ratio of Y-sperm was determined using quantitative polymerase chain reaction (PCR), considered to be a much better method than quinacrine but less accurate than double-label FISH.
- Before processing, the X:Y ratio was close to 1:1. Neither swim-up or Sephadex filtering altered the ratio.
- Assessment of the sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies
Nov-1989, Fertility & Sterility
- 182 semen samples were processed with Sephadex gel filtration, and then tested with both DNA probing and Y-body tests (quinacrine staining). No higher ratio of X-sperm was observed.
- Sexing of human sperm by discontinuous Percoll density gradient and its clinical application
Oct-1987, Human Reproduction (Tokyo)
- In this strange report, X-sperm separation by the Percoll method seems to have resulted in 6 of 6 female births.
- Percoll density gradient was used for X-sperm separation, resulting in 6 pregnancies.
- All 6 pregnancies resulted in the birth of a healthy girl.
- Separation of human X- and Y-bearing sperm using percoll
density gradient centrifugation
Nov-1983, Fertility & Sterility
This seems to be the first report of using Percoll density gradient centrifugation to separate X and Y-sperm.
- Sperm was separated using Percoll density gradient centrifugation.
- The percentage of remaining Y-sperm was determined using quinacrine mustard fluorescent staining.
- After centrifugation, the bottom portion was found to have only 28% Y-sperm, while Y-sperm in the top portion had increased to 71%.
- [The quinacrine staining method used to detect Y-sperm has since been criticized as ineffective; however, the FISH method was not available at the time of this study.]
- Failure to separate human x- and y-chromosome bearing spermatozoa by Sephadex gel-filtration
- Reported a failure of the Sephadex gel filtration method to separate X and Y-sperm.
- A simple and reproducible method for separating Y-bearing spermatozoa from human semen
May-1977, Indian Journal of Medical Research
- Seems to be the first mention of using gradient density centrifugation (with Ficoll) to separate Y sperm, using the ineffective quinacrine staining method.
- The separation of X-and Y-spermatozoa with regard to the
possible clinical application by means of artificial insemination
A report of successful separation of X and Y-sperm, but used the flawed quinecrine staining method for testing
- Separation of X- and Y-bearing human spermatozoa with the sephadex gel-filtration method
- A report of successful separation of X and Y-sperm, but used the flawed quinecrine staining method for testing.