View source for Sperm sorting

'''Sperm sorting''' is a means of choosing what type of [[sperm cell]] is to [[Fertilisation|fertilize]] an [[egg cell]] using several conventional techniques of [[density gradient centrifugation|centrifugation]] or [[swim-up]]. Newly applied methods such as flow cytometry expand the possibilities of sperm sorting and new techniques of sperm sorting are being developed.

It can be used to sort out sperm that are most healthy, as well as for determination of more specific traits, such as [[sex selection]] in which spermatozoa are separated into [[X chromosome|X-]] (female) and [[Y chromosome|Y-]] (male) chromosome bearing populations based on their difference in DNA content. The resultant 'sex-sorted' spermatozoa are then able to be used in conjunction with other assisted reproductive technologies such as [[artificial insemination]] or [[in-vitro fertilization]] (IVF) to produce offspring of the desired sex - in farming animals but also in human medical practice.

== Methods ==

=== Conventional techniques ===
Several methods have been used to sort sperm before the advent of flow cytometry. [[Density gradient centrifugation]] (in a continuous or discontinuous gradient) can concentrate semen samples with low concentration of sperm, using the density of sperm as a measure of their quality.<ref name=":0" /><ref name=":3">{{Cite journal|last1=Ribeiro|first1=Sofia C.|last2=Sartorius|first2=Gideon|last3=Pletscher|first3=Flurina|last4=Geyter|first4=Maria de|last5=Zhang|first5=Hong|last6=Geyter|first6=Christian de|title=Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting|journal=Fertility and Sterility|volume=100|issue=3|pages=686–694.e4|doi=10.1016/j.fertnstert.2013.05.030|pmid=23809499|date=September 2013|doi-access=free}}</ref> Similarly, so-called [[swim-up]] techniques apply a centrifugation step and then sperm is allowed to swim up into a medium, thus enriching a subpopulation of motile sperm. However, use of sperm centrifugation is detrimental to the sperm viability and elicits production of reactive oxygen species.<ref name=":0">{{Cite journal|last1=Rappa|first1=Kari L.|last2=Rodriguez|first2=Harold F.|last3=Hakkarainen|first3=Gloria C.|last4=Anchan|first4=Raymond M.|last5=Mutter|first5=George L.|last6=Asghar|first6=Waseem|date=2016-09-01|title=Sperm processing for advanced reproductive technologies: Where are we today?|journal=Biotechnology Advances|volume=34|issue=5|pages=578–587|doi=10.1016/j.biotechadv.2016.01.007|pmid=26845061|s2cid=41862891 }}</ref> Conventional techniques are routinely used in assisted reproductive technology.<ref name=":2">{{Cite journal|last1=Paasch|first1=Uwe|last2=Grunewald|first2=Sonja|last3=Glander|first3=Hans-Juergen|date=2007-01-01|title=Sperm selection in assisted reproductive techniques|journal=Society of Reproduction and Fertility Supplement|volume=65|pages=515–525|issn=1747-3403|pmid=17644989}}</ref>

=== Flow cytometry ===
[[Flow cytometry]] is another method used to sort sperm and adaptations of this technique opens new opportunities in sperm sorting. However, because flow cytometry-based sperm sorting often uses fluorescent dyes that often stain DNA, the safety of this technique in human reproductive medicine is a matter of scientific discussion.<ref>{{Cite journal|last=Caroppo|first=Ettore|title=Sperm sorting for selection of healthy sperm: is it safe and useful?|journal=Fertility and Sterility|volume=100|issue=3|pages=695–696|doi=10.1016/j.fertnstert.2013.06.006|pmid=23809504|date=September 2013|doi-access=free}}</ref><ref>{{Cite journal|last1=Ribeiro|first1=Sofia|last2=Sartorius|first2=Gideon|last3=Geyter|first3=Christian de|title=Sorting of spermatozoa with flow cytometry|journal=Fertility and Sterility|volume=100|issue=3|doi=10.1016/j.fertnstert.2013.06.039|pmid=23876533|page=e15|date=September 2013|doi-access=free}}</ref>

However, flow cytometry is the only currently used technique able to determine the sex of future progeny by measuring DNA content of individual sperm cells. It evaluates if they contain the larger [[X chromosome]] (giving rise to a female offspring) or smaller [[Y chromosome]] (leading to male progeny). It then allows separation of X and Y sperm.<ref>{{Cite journal|last1=Johnson|first1=L. A.|last2=Welch|first2=G. R.|last3=Rens|first3=W.|date=1999-01-01|title=The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI|journal=Journal of Animal Science|volume=77 Suppl 2|pages=213–220|issn=0021-8812|pmid=15526798|doi=10.2527/1999.77suppl_2213x}}</ref> The so-called ''[[Beltsfield Sperm Sexing Technology]]'' was developed by USDA in conjunction with Lawrence Livermore National Laboratories, relying on the DNA difference between the X- and Y- chromosomes.<ref>Garner DL, Seidel GE. History of commercializing sexed semen for cattle. Theriogenology 2008;69: 886-895.</ref> Prior to flow cytometric sorting, semen is labeled with a fluorescent dye called [[Hoechst 33342]] which binds to the DNA of each spermatozoon. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the "female" (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than its male (Y-chromosome bearing) counterpart.  As a consequence, when exposed to UV light during flow cytometry, X spermatozoa fluoresce brighter than Y- spermatozoa.  As the spermatozoa pass through the flow cytometer in single file, each spermatozoon is encased by a single droplet of fluid and assigned an electric charge corresponding to its chromosome status (e.g. X-positive charge, Y-negative charge).  The stream of X- and Y- droplets is then separated by means of electrostatic deflection and collected into separate collection tubes for subsequent processing.<ref>Seidel GE, Jr., Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002;124: 733-743.</ref>

Another cytometric technique used in sperm sorting is [[magnetic-activated cell sorting]] (MACS) which is routinely applied in assisted reproduction hospitals to sort out sperm with fragmented DNA. This is achieved using [[Antibody|antibodies]] to surface markers of programmed cell death ([[apoptosis]]) such as [[annexin V]], coupled with magnetic beads. Following the binding of these antibodies, spermatozoa which undergo apoptosis are sorted by applying [[magnetic field]] to the sperm suspension.<ref name=":4">{{Cite journal|last1=Gil|first1=Monica|last2=Sar-Shalom|first2=Valerie|last3=Sivira|first3=Yolisid Melendez|last4=Carreras|first4=Ramon|last5=Checa|first5=Miguel Angel|date=2013-03-07|title=Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: a systematic review and meta-analysis|journal=Journal of Assisted Reproduction and Genetics|language=en|volume=30|issue=4|pages=479–485|doi=10.1007/s10815-013-9962-8|pmid=23468098|issn=1058-0468|pmc=3644127}}</ref> MACS obviates the need for fluorescent DNA binding molecules.

=== Other techniques ===
[[DNA damage]] in sperm cells may be detected by using [[Raman spectroscopy]].<ref name="MIT">[http://www.technologyreview.com/biomedicine/21982/page1/ Ranking sperm cells could improve the odds of in vitro fertilization.] By Courtney Humphries. TechnologyReview. Wednesday, January 21, 2009</ref> It is not specific enough to detect individual traits, however.<ref name="MIT" /> The sperm cells having least DNA damage may subsequently be injected into the egg cell by [[intracytoplasmic sperm injection]] (ICSI).<ref name="MIT" /> Many other methods for sperm sorting have been proposed or are currently tested.<ref name=":0" /><ref name=":2" />

To select spermatozoa with low [[DNA damage]] index the population of sperm could be enriched with spermatozoa with non-fragmented DNA, with techniques like [[electrophoresis]],<ref>{{Cite journal|last1=Fleming|first1=S.D.|last2=Ilad|first2=R.S.|last3=Griffin|first3=A-M.G.|last4=Wu|first4=Y.|last5=Ong|first5=K.J.|last6=Smith|first6=H.C.|last7=Aitken|first7=R.J.|date=2008-09-01|title=Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: comparison with density gradient centrifugation|journal=Human Reproduction|language=en|volume=23|issue=12|pages=2646–2651|doi=10.1093/humrep/den330|pmid=18765400|issn=0268-1161|doi-access=free}}</ref> [[Z method]]<ref>{{Cite journal|last1=Kheirollahi-Kouhestani|first1=M.|last2=Razavi|first2=S.|last3=Tavalaee|first3=M.|last4=Deemeh|first4=M.R.|last5=Mardani|first5=M.|last6=Moshtaghian|first6=J.|last7=Nasr-Esfahani|first7=M.H.|date=2009-10-01|title=Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome|journal=Human Reproduction|language=en|volume=24|issue=10|pages=2409–2416|doi=10.1093/humrep/dep088|pmid=19553239|issn=0268-1161|doi-access=free}}</ref> and MACS (Magnetic Activating Cell Sorting), which in combination with density gradient centrifugation in single sperm preparation protocols results in spermatozoa with superior quality.<ref>{{Cite journal|last1=Said|first1=Tamer|last2=Agarwal|first2=Ashok|last3=Grunewald|first3=Sonja|last4=Rasch|first4=Manja|last5=Baumann|first5=Thomas|last6=Kriegel|first6=Christian|last7=Li|first7=Liang|last8=Glander|first8=Hans-Juergen|last9=Thomas|first9=Anthony J.|last10=Paasch|first10=Uwe|date=2006-03-01|title=Selection of Nonapoptotic Spermatozoa As a New Tool for Enhancing Assisted Reproduction Outcomes: An In Vitro Model1|journal=Biology of Reproduction|language=en|volume=74|issue=3|pages=530–537|doi=10.1095/biolreprod.105.046607|pmid=16306419|issn=0006-3363|doi-access=free}}</ref>

[[Hyaluronic acid]] (HA) binding sites on the sperm plasma membrane are an indicator of sperm maturity (Huszar et al., 2003, Yudin et al.,1999). There are two methods based on this fact: physiological intracytoplasmic sperm injection (PICSI), and a sperm slow procedure; both methods require sperm preparation via sperm washing or centrifugation.

== Applications ==
Sperm undergoes a process of [[natural selection]] when millions of sperm enter the vagina but only few reach the egg cell and then only one is usually allowed to [[Fertilisation|fertilize]] it. The sperm is selected not only by its highest [[Sperm motility|motility]] but also by other factors such as [[DNA integrity]], production of [[reactive oxygen species]] and [[Cell viability|viability]]. This selection is largely circumvented in case of [[in-vitro fertilization]] which leads to higher incidence of [[Congenital disorder|birth defects]] associated with [[Assisted reproductive technology|assisted reproductive techniques]]. Egg cells are often fertilized by sperm which would have low chance of fertilizing it in natural conditions.<ref name=":0" /> Sperm sorting could thus be used to decrease risks associated with assisted reproduction. Additionally, there is ongoing debate about using sperm sorting for choosing the child's sex.

=== For general health ===
Conventional methods of sperm sorting have been widely used to assess quality of sperm before subsequent [[artificial insemination]] or [[in-vitro fertilization]]. It has been verified that sperm sorted using these techniques is of superior quality than unsorted.<ref>{{Cite journal|last1=Aitken|first1=R. John|last2=Clarkson|first2=Jane S.|date=1988-11-12|title=Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques|journal=Journal of Andrology|language=en|volume=9|issue=6|pages=367–376|doi=10.1002/j.1939-4640.1988.tb01067.x|pmid=3215823|issn=1939-4640|doi-access=free}}</ref><ref>{{Cite journal|last1=De Geyter|first1=C.|last2=Bals-Pratsch|first2=M.|last3=Doeren|first3=M.|last4=Yeung|first4=C. H.|last5=Grunert|first5=J. H.|last6=Bordt|first6=J.|last7=Schneider|first7=H. P.|last8=Nieschlag|first8=E.|date=1988-11-01|title=Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization|pmid=3204150|journal=Human Reproduction (Oxford, England)|volume=3|issue=8|pages=948–954|issn=0268-1161|doi=10.1093/oxfordjournals.humrep.a136824}}</ref> However, important characteristics of sperm such as DNA integrity remain untested by these conventional methods. New flow-cytometry based techniques such as [[YO-PRO]] staining can discriminate [[Apoptosis|apoptotic]] and dead spermatozoa from the viable ones.<ref name=":3" /> For example, [[annexin V]] staining followed by [[Magnetic-activated cell sorting|MACS]] can significantly improve pregnancy rates in couples with previous assisted reproduction failure.<ref name=":4" />

=== For sex selection ===

==== In farming ====
Sperm sorting by flow cytometry is an established technique in veterinary practice, and in the dairy industry most female cows are artificially inseminated with sorted semen to increase the number of female [[Calf (animal)|calves]] (using sperm sorting is less common in other species of farm animals, however artificial insemination is common).<ref name=":1">{{Cite journal|last1=Rath|first1=D.|last2=Barcikowski|first2=S.|last3=de Graaf|first3=S.|last4=Garrels|first4=W.|last5=Grossfeld|first5=R.|last6=Klein|first6=S.|last7=Knabe|first7=W.|last8=Knorr|first8=C.|last9=Kues|first9=W.|date=2013-01-01|title=Sex selection of sperm in farm animals: status report and developmental prospects|journal=Reproduction|volume=145|issue=1|pages=R15–30|doi=10.1530/REP-12-0151|issn=1741-7899|pmid=23148085|doi-access=free}}</ref> Artificial insemination of farm animals with sorted sperm is recognized by the [[Food and Agriculture Organization]] (FAO) as a promising way of increasing efficiency of agriculture needed to produce enough food for the growing human population. Utilizing artificial insemination with sorted sperm is seen as a way to create an optimal ratio of male and female calves to increase dairy [[Dairy|milk production]].<ref name=":1" />

==== In humans ====
{{Further|Sex selection#Ethical concerns}}Choosing the sex of children might help prevent [[Sex-linked inheritance|sex-associated heritable diseases]] such as [[Duchenne muscular dystrophy|Duchene muscular dystrophy]] or [[haemophilia]] in families with a history of these diseases. On the other hand, sperm sorting in humans raises the ethical concerns implicit to the idea of [[sex selection]]. If applied large-scale, it has a potential to elicit a [[Human sex ratio|sex-ratio imbalance]]. It could also have implications on gender equality if parents consistently choose to have a boy as their first-born (first-borns were shown to be more likely to succeed in life).<ref>{{Cite journal|last1=Kalfoglou|first1=A.L.|last2=Kammersell|first2=M.|last3=Philpott|first3=S.|last4=Dahl|first4=E.|title=Ethical arguments for and against sperm sorting for non-medical sex selection: a review|journal=Reproductive BioMedicine Online|volume=26|issue=3|pages=231–239|doi=10.1016/j.rbmo.2012.11.007|pmid=23337421|date=March 2013|doi-access=free}}</ref>

There is no country in the world which explicitly permits sex selection for non-medical purposes. There were 31 countries in 2009 which allowed sex selection in case of sex-linked disease risk or other medical purpose.<ref>{{Cite web|url=http://geneticsandsociety.org/downloads/200904_sex_selection_memo.pdf|title=Countries with laws or policies on sex selection|last=Darnovsky|first=Marcy|date=2009|website=|publisher=geneticsandsociety.org|access-date=2016-10-06}}</ref> In the US, for humans, the application of sperm sorting in sex selection is tightly regulated by the [[U.S. Food and Drug Administration|FDA]]. After the establishment of the MicroSort technique, it was offered to parents as a part of a [[clinical trial]]. The procedure was made available to a limited number of participants each month, in addition to fulfilling certain criteria, such as having a disease with [[sex linkage]] or having at least one child (for family balancing).<ref name="institute">[http://www.microsort.net/ Genetics & IVF Institute: MicroSort Clinical Trial]</ref> There are currently MicroSort laboratories and collaborating physicians in several countries (some for general purposes, some only offering service in case of genetic disease risks associated with one sex).<ref>{{Cite web|url=http://www.microsort.com/?page_id=319|title=About Us {{!}} MicroSort International|last=ThemeFuse|website=www.microsort.com|access-date=2016-10-06}}</ref>

While highly accurate, sperm sorting by flow cytometry will not produce two completely separate populations. That is to say, there will always be some "male" sperm among the "female" sperm and vice versa. The exact percentage purity of each population is dependent on the species being sorted and the 'gates' which the operator places around the total population visible to the machine. In general, the larger the DNA difference between the X and Y chromosome of a species, the easier it is to produce a highly pure population. In sheep and cattle, purities for each sex will usually remain above 90% depending on 'gating', while for humans these may be reduced to 90% for "female" spermatozoa and 70% for "male" spermatozoa.<ref name="institute" />

== See also ==
* [[Sex selection]]
* [[Preimplantation genetic diagnosis]]

== References ==
{{reflist}}

[[Category:Family]]
[[Category:Fertility medicine]]
[[Category:Flow cytometry]]
[[Category:Obstetrics]]
[[Category:Human pregnancy]]
[[Category:Reproduction]]
[[Category:Semen]]