Sperm sorting

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Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. Several conventional techniques of 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.

Contents

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- (female) and 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. [1] [2] 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. [1] Conventional techniques are routinely used in assisted reproductive technology. [3]

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. [4] [5]

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. [6] 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. [7] 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. [8]

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 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. [9] MACS obviates the need for fluorescent DNA binding molecules.

Other techniques

DNA damage in sperm cells may be detected by using Raman spectroscopy. [10] It is not specific enough to detect individual traits, however. [10] The sperm cells having least DNA damage may subsequently be injected into the egg cell by intracytoplasmic sperm injection (ICSI). [10] Many other methods for sperm sorting have been proposed or are currently tested. [1] [3]

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, [11] Z method [12] and MACS (Magnetic Activating Cell Sorting), which in combination with density gradient centrifugation in single sperm preparation protocols results in spermatozoa with superior quality. [13]

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 fertilize it. The sperm is selected not only by its highest motility but also by other factors such as DNA integrity, production of reactive oxygen species and viability. This selection is largely circumvented in case of in-vitro fertilization which leads to higher incidence of birth defects associated with assisted reproductive techniques. Egg cells are often fertilized by sperm which would have low chance of fertilizing it in natural conditions. [1] 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. [14] [15] 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 apoptotic and dead spermatozoa from the viable ones. [2] For example, annexin V staining followed by MACS can significantly improve pregnancy rates in couples with previous assisted reproduction failure. [9]

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 calves (using sperm sorting is less common in other species of farm animals, however artificial insemination is common). [16] 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 milk production. [16]

In humans

Choosing the sex of children might help prevent sex-associated heritable diseases such as 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 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). [17]

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. [18] In the US, for humans, the application of sperm sorting in sex selection is tightly regulated by the 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). [19] 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). [20]

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. [19]

See also

Related Research Articles

is the process of male gamete formation in animals This process also involves

<span class="mw-page-title-main">Sex</span> Trait that determines an organisms sexually reproductive function

Sex is the trait that determines whether a sexually reproducing organism produces male or female gametes. A male organism produces small mobile gametes, while a female organism produces larger, non-mobile gametes. An organism that produces both types of gamete is called a hermaphrodite. During sexual reproduction, a male and a female gamete fuse to form a zygote, which develops into an offspring that inherits traits from each parent.

<span class="mw-page-title-main">Spermatozoon</span> Motile sperm cell

A spermatozoon is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote.

Reproductive technology encompasses all current and anticipated uses of technology in human and animal reproduction, including assisted reproductive technology (ART), contraception and others. It is also termed Assisted Reproductive Technology, where it entails an array of appliances and procedures that enable the realization of safe, improved and healthier reproduction. While this is not true of all people, for an array of married couples, the ability to have children is vital. But through the technology, infertile couples have been provided with options that would allow them to conceive children.

<span class="mw-page-title-main">Intracytoplasmic sperm injection</span> In vitro fertilization procedure

Intracytoplasmic sperm injection is an in vitro fertilization (IVF) procedure in which a single sperm cell is injected directly into the cytoplasm of an egg. This technique is used in order to prepare the gametes for the obtention of embryos that may be transferred to a maternal uterus. With this method, the acrosome reaction is skipped.

Infertility is the inability of an animal or plant to reproduce by natural means. It is usually not the natural state of a healthy adult, except notably among certain eusocial species. It is the normal state of a human child or other young offspring, because they have not undergone puberty, which is the body's start of reproductive capacity.

<span class="mw-page-title-main">Acrosome reaction</span> Sperm-meets-egg process

During fertilization, a sperm must first fuse with the plasma membrane and then penetrate the female egg cell to fertilize it. Fusing to the egg cell usually causes little problem, whereas penetrating through the egg's hard shell or extracellular matrix can be more difficult. Therefore, sperm cells go through a process known as the acrosome reaction, which is the reaction that occurs in the acrosome of the sperm as it approaches the egg.

<span class="mw-page-title-main">Preimplantation genetic diagnosis</span> Genetic profiling of embryos prior to implantation

Preimplantation genetic diagnosis is the genetic profiling of embryos prior to implantation, and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal diagnosis. When used to screen for a specific genetic disease, its main advantage is that it avoids selective abortion, as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology, and requires in vitro fertilization (IVF) to obtain oocytes or embryos for evaluation. Embryos are generally obtained through blastomere or blastocyst biopsy. The latter technique has proved to be less deleterious for the embryo, therefore it is advisable to perform the biopsy around day 5 or 6 of development.

Capacitation is the penultimate step in the maturation of mammalian spermatozoa and is required to render them competent to fertilize an oocyte. This step is a biochemical event; the sperm move normally and look mature prior to capacitation. In vivo, capacitation occurs after ejaculation, when the spermatozoa leave the vagina and enter the upper female reproductive tract. The uterus aids in the steps of capacitation by secreting sterol-binding albumin, lipoproteins, and proteolytic and glycosidasic enzymes such as heparin.

Sex selection is the attempt to control the sex of the offspring to achieve a desired sex. It can be accomplished in several ways, both pre- and post-implantation of an embryo, as well as at childbirth. It has been marketed under the title family balancing.

<span class="mw-page-title-main">Assisted reproductive technology</span> Methods to achieve pregnancy by artificial or partially artificial means

Assisted reproductive technology (ART) includes medical procedures used primarily to address infertility. This subject involves procedures such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), cryopreservation of gametes or embryos, and/or the use of fertility medication. When used to address infertility, ART may also be referred to as fertility treatment. ART mainly belongs to the field of reproductive endocrinology and infertility. Some forms of ART may be used with regard to fertile couples for genetic purpose. ART may also be used in surrogacy arrangements, although not all surrogacy arrangements involve ART. The existence of sterility will not always require ART to be the first option to consider, as there are occasions when its cause is a mild disorder that can be solved with more conventional treatments or with behaviors based on promoting health and reproductive habits.

<span class="mw-page-title-main">Sperm</span> Male reproductive cell in anisogamous forms of sexual reproduction

Sperm is the male reproductive cell, or gamete, in anisogamous forms of sexual reproduction. Animals produce motile sperm with a tail known as a flagellum, which are known as spermatozoa, while some red algae and fungi produce non-motile sperm cells, known as spermatia. Flowering plants contain non-motile sperm inside pollen, while some more basal plants like ferns and some gymnosperms have motile sperm.

Sperm washing is the process in which individual sperms are separated from the semen. Washed sperm is used in artificial insemination using the intrauterine insemination (IUI) technique and in in vitro fertilization (IVF). It may also be used to decrease the risk of HIV transmission by an HIV-positive male, in which case the washed sperm is injected into a female using an artificial insemination technique.

<span class="mw-page-title-main">Human fertilization</span> Union of a human egg and sperm

Human fertilization is the union of an egg and sperm, occurring primarily in the ampulla of the fallopian tube. The result of this union leads to the production of a fertilized egg called a zygote, initiating embryonic development. Scientists discovered the dynamics of human fertilization in the 19th century.

<span class="mw-page-title-main">Human reproduction</span> Procreative biological processes of humanity

Human reproduction is sexual reproduction that results in human fertilization to produce a human offspring. It typically involves sexual intercourse between a sexually mature human male and female. During sexual intercourse, the interaction between the male and female reproductive systems results in fertilization of the ovum by the sperm to form a zygote. While normal cells contain 46 chromosomes, gamete cells only contain 23 single chromosomes, and it is when these two cells merge into one zygote cell that genetic recombination occurs and the new zygote contains 23 chromosomes from each parent, giving it 46 chromosomes. The zygote then undergoes a defined development process that is known as human embryogenesis, and this starts the typical 9-month gestation period that is followed by childbirth. The fertilization of the ovum may be achieved by artificial insemination methods, which do not involve sexual intercourse. Assisted reproductive technology also exists.

<span class="mw-page-title-main">Semen analysis</span> Scientific analysis of semen

A semen analysis, also called seminogram or spermiogram, evaluates certain characteristics of a male's semen and the sperm contained therein. It is done to help evaluate male fertility, whether for those seeking pregnancy or verifying the success of vasectomy. Depending on the measurement method, just a few characteristics may be evaluated or many characteristics may be evaluated. Collection techniques and precise measurement method may influence results.

Female sperm can refer to either:

  1. A sperm which contains an X chromosome, produced in the usual way in the testicles, referring to the occurrence of such a sperm fertilizing an egg and giving birth to a female.
  2. A sperm which artificially contains genetic material from a female.
<span class="mw-page-title-main">Sexual reproduction</span> Biological process

Sexual reproduction is a type of reproduction that involves a complex life cycle in which a gamete with a single set of chromosomes combines with another gamete to produce a zygote that develops into an organism composed of cells with two sets of chromosomes (diploid). This is typical in animals, though the number of chromosome sets and how that number changes in sexual reproduction varies, especially among plants, fungi, and other eukaryotes.

Religious response to assisted reproductive technology deals with the new challenges for traditional social and religious communities raised by modern assisted reproductive technology. Because many religious communities have strong opinions and religious legislation regarding marriage, sex and reproduction, modern fertility technology has forced religions to respond.

<span class="mw-page-title-main">Sperm Chromatin Structure Assay</span>

Sperm Chromatin Structure Assay (SCSA) is a diagnostic approach that detects sperm abnormality with a large extent of DNA fragmentation. First described by Evenson in 1980, the assay is a flow cytometric test that detects the vulnerability of sperm DNA to acid-induced denaturation DNA in situ. SCSA measures sperm DNA fragmentation attributed to intrinsic and extrinsic factors and reports the degree of fragmentation in terms of DNA Fragmentation Index (DFI). The use of SCSA expands from evaluation of male infertility and subfertility, toxicology studies and evaluation of quality of laboratory semen samples. Notably, SCSA outcompetes other convention sperm DNA fragmentation (sDF) assays such as TUNEL and COMET in terms of efficiency, objectivity, and repeatability.

References

  1. 1 2 3 4 Rappa, Kari L.; Rodriguez, Harold F.; Hakkarainen, Gloria C.; Anchan, Raymond M.; Mutter, George L.; Asghar, Waseem (2016-09-01). "Sperm processing for advanced reproductive technologies: Where are we today?". Biotechnology Advances. 34 (5): 578–587. doi:10.1016/j.biotechadv.2016.01.007. PMID   26845061. S2CID   41862891.
  2. 1 2 Ribeiro, Sofia C.; Sartorius, Gideon; Pletscher, Flurina; Geyter, Maria de; Zhang, Hong; Geyter, Christian de (September 2013). "Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting". Fertility and Sterility. 100 (3): 686–694.e4. doi: 10.1016/j.fertnstert.2013.05.030 . PMID   23809499.
  3. 1 2 Paasch, Uwe; Grunewald, Sonja; Glander, Hans-Juergen (2007-01-01). "Sperm selection in assisted reproductive techniques". Society of Reproduction and Fertility Supplement. 65: 515–525. ISSN   1747-3403. PMID   17644989.
  4. ↑ Caroppo, Ettore (September 2013). "Sperm sorting for selection of healthy sperm: is it safe and useful?". Fertility and Sterility. 100 (3): 695–696. doi: 10.1016/j.fertnstert.2013.06.006 . PMID   23809504.
  5. ↑ Ribeiro, Sofia; Sartorius, Gideon; Geyter, Christian de (September 2013). "Sorting of spermatozoa with flow cytometry". Fertility and Sterility. 100 (3): e15. doi: 10.1016/j.fertnstert.2013.06.039 . PMID   23876533.
  6. ↑ Johnson, L. A.; Welch, G. R.; Rens, W. (1999-01-01). "The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI". Journal of Animal Science. 77 Suppl 2: 213–220. doi:10.2527/1999.77suppl_2213x. ISSN   0021-8812. PMID   15526798.
  7. ↑ Garner DL, Seidel GE. History of commercializing sexed semen for cattle. Theriogenology 2008;69: 886-895.
  8. ↑ Seidel GE, Jr., Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002;124: 733-743.
  9. 1 2 Gil, Monica; Sar-Shalom, Valerie; Sivira, Yolisid Melendez; Carreras, Ramon; Checa, Miguel Angel (2013-03-07). "Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: a systematic review and meta-analysis". Journal of Assisted Reproduction and Genetics. 30 (4): 479–485. doi:10.1007/s10815-013-9962-8. ISSN   1058-0468. PMC   3644127 . PMID   23468098.
  10. 1 2 3 Ranking sperm cells could improve the odds of in vitro fertilization. By Courtney Humphries. TechnologyReview. Wednesday, January 21, 2009
  11. ↑ Fleming, S.D.; Ilad, R.S.; Griffin, A-M.G.; Wu, Y.; Ong, K.J.; Smith, H.C.; Aitken, R.J. (2008-09-01). "Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: comparison with density gradient centrifugation". Human Reproduction. 23 (12): 2646–2651. doi: 10.1093/humrep/den330 . ISSN   0268-1161. PMID   18765400.
  12. ↑ Kheirollahi-Kouhestani, M.; Razavi, S.; Tavalaee, M.; Deemeh, M.R.; Mardani, M.; Moshtaghian, J.; Nasr-Esfahani, M.H. (2009-10-01). "Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome". Human Reproduction. 24 (10): 2409–2416. doi: 10.1093/humrep/dep088 . ISSN   0268-1161. PMID   19553239.
  13. ↑ Said, Tamer; Agarwal, Ashok; Grunewald, Sonja; Rasch, Manja; Baumann, Thomas; Kriegel, Christian; Li, Liang; Glander, Hans-Juergen; Thomas, Anthony J.; Paasch, Uwe (2006-03-01). "Selection of Nonapoptotic Spermatozoa As a New Tool for Enhancing Assisted Reproduction Outcomes: An In Vitro Model1". Biology of Reproduction. 74 (3): 530–537. doi: 10.1095/biolreprod.105.046607 . ISSN   0006-3363. PMID   16306419.
  14. ↑ Aitken, R. John; Clarkson, Jane S. (1988-11-12). "Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques". Journal of Andrology. 9 (6): 367–376. doi: 10.1002/j.1939-4640.1988.tb01067.x . ISSN   1939-4640. PMID   3215823.
  15. ↑ De Geyter, C.; Bals-Pratsch, M.; Doeren, M.; Yeung, C. H.; Grunert, J. H.; Bordt, J.; Schneider, H. P.; Nieschlag, E. (1988-11-01). "Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization". Human Reproduction (Oxford, England). 3 (8): 948–954. doi:10.1093/oxfordjournals.humrep.a136824. ISSN   0268-1161. PMID   3204150.
  16. 1 2 Rath, D.; Barcikowski, S.; de Graaf, S.; Garrels, W.; Grossfeld, R.; Klein, S.; Knabe, W.; Knorr, C.; Kues, W. (2013-01-01). "Sex selection of sperm in farm animals: status report and developmental prospects". Reproduction. 145 (1): R15–30. doi: 10.1530/REP-12-0151 . ISSN   1741-7899. PMID   23148085.
  17. ↑ Kalfoglou, A.L.; Kammersell, M.; Philpott, S.; Dahl, E. (March 2013). "Ethical arguments for and against sperm sorting for non-medical sex selection: a review". Reproductive BioMedicine Online. 26 (3): 231–239. doi: 10.1016/j.rbmo.2012.11.007 . PMID   23337421.
  18. ↑ Darnovsky, Marcy (2009). "Countries with laws or policies on sex selection" (PDF). geneticsandsociety.org. Retrieved 2016-10-06.
  19. 1 2 Genetics & IVF Institute: MicroSort Clinical Trial
  20. ↑ ThemeFuse. "About Us | MicroSort International". www.microsort.com. Retrieved 2016-10-06.
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