Artificial insemination

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https://en.wikipedia.org/wiki/Artificial_insemination 

Artificial insemination is the deliberate introduction of sperm into the cervix or uterine cavity for the purpose of achieving pregnancy through in vivo fertilization by means other than sexual intercourse. It is a fertility treatment for humans, and is a common practice in animal breeding, including cattle (see frozen bovine semen) and pigs.

Artificial insemination may employ assisted reproductive technology, sperm donation and animal husbandry techniques. Artificial insemination techniques available include intracervical insemination (ICI) and intrauterine insemination (IUI). Where gametes from a third party are used, the procedure may be known as 'assisted insemination'.

Humans

History

The first recorded case of artificial insemination was by Lazzaro Spallanzani in 1784, who performed it on a dog. It was followed in 1790 by John Hunter, who helped impregnate a linen draper's wife. The first reported case of artificial insemination by donor occurred in 1884: William H. Pancoast, a professor in Philadelphia, took sperm from his "best looking" student to inseminate an anesthetized woman without her knowledge. The case was reported 25 years later in a medical journal. The sperm bank was developed in Iowa starting in the 1950s in research conducted by University of Iowa medical school researchers Jerome K. Sherman and Raymond Bunge.

In 1916, Australian eugenicist Marion Louisa Piddington published a pseudonymous tract titled Via Nuova or Science & Maternity in which she called for a programme of mass artificial insemination for the sweethearts of soldiers who had been killed in World War I. She described this as a "conscription of the virgins" – comparable to the conscription of men for military service – who would receive "artificial insemination from a eugenically-desirable donor". Piddington promoted her scheme for several years in Australia, Britain and the United States under the name of "scientific motherhood", but it was poorly received.

In the United Kingdom, British obstetrician Mary Barton founded one of the first fertility clinics to offer donor insemination in the 1930s, with her husband Bertold Wiesner fathering hundreds of offspring.

In the 1980s, direct intraperitoneal insemination (DIPI) was occasionally used, where doctors injected sperm into the lower abdomen through a surgical hole or incision, with the intention of letting them find the oocyte at the ovary or after entering the genital tract through the ostium of the fallopian tube.

Patients and gamete donors

Artificial insemination (AI) is a medical procedure in which sperm is introduced into a woman's reproductive system to achieve pregnancy without sexual intercourse. The sperm used may come from the recipient's partner or from a donor, whose identity may be known or anonymous. Various methods exist to obtain sperm for use in artificial insemination.

In 2016, an article was published in Seventeen magazine that highlighted the story of Kacie Saxer-Taulbee, a teenager conceived from a sperm donor father. Using her donor's cryobank number in the Donor Sibling Registry, she managed to find other siblings conceived from the same donor. They became known as the "5010ers" and formed a Facebook group to keep in touch.

Barriers for patients and donors

Some countries have laws which restrict and regulate who can donate sperm and who is able to receive artificial insemination.

Preparations

Timing is critical, as the window and opportunity for fertilization is little more than twelve hours from the release of the ovum. To increase the chance of success, the woman's menstrual cycle is closely observed, often using ovulation kits, ultrasounds or blood tests, such as basal body temperature tests over, noting the color and texture of the vaginal mucus, and the softness of the nose of her cervix. To improve the success rate of artificial insemination, drugs to create a stimulated cycle may be used, but the use of such drugs also results in an increased chance of a multiple birth.

Sperm can be provided fresh or washed.

Techniques

The human female reproductive system. The cervix is part of the uterus. The cervical canal connects the interiors of the uterus and vagina.

Semen used is either fresh, raw, or frozen.

Intracervical

Intracervical insemination (ICI) is the method of artificial insemination which most closely mimics the natural ejaculation of semen by the penis into the vagina during sexual intercourse. It is painless and is the simplest and most common method of artificial insemination involving the introduction of unwashed or raw semen into the vagina at the entrance to the cervix, usually by means of a needle-less syringe. The vagina acts as a filter to separate out the sperm from other chemicals in the ejaculate, as with intercourse, so that only sperm pass through the cervix on their way to the uterus.

Although ICI is the simplest method of artificial insemination, a meta-analysis has shown no difference in live birth rates compared with IUI.

During ICI, air is expelled from a needleless syringe which is then filled with semen which has been allowed to liquify. A specially designed syringe, wider and with a more rounded end, may be used for this purpose. Any further enclosed air is removed by gently pressing the plunger forward. The woman lies on her back and the syringe is inserted into the vagina. Care is optimal when inserting the syringe, so that the tip is as close to the entrance to the cervix as possible. A vaginal speculum may be used for this purpose and a catheter may be attached to the tip of the syringe to ensure delivery of the semen as close to the entrance to the cervix as possible. The plunger is then slowly pushed forward and the semen in the syringe is gently emptied deep into the vagina. It is important that the syringe is emptied slowly for safety and for the best results, bearing in mind that the purpose of the procedure is to replicate as closely as possible a natural deposit of the semen in the vagina. The syringe (and catheter if used) may be left in place for several minutes before removal. The woman can bring herself to orgasm so that the cervix 'dips down' into the pool of semen, again replicating closely vaginal intercourse, and this may improve the success rate.

When performed at home without the presence of a professional, aiming the sperm in the vagina at the neck of the cervix may be more difficult to achieve and the effect may be to 'flood' the vagina with semen, rather than to target it specifically at the entrance to the cervix. This procedure is sometimes referred to as 'intravaginal insemination' (IVI).

A conception cap, which is a form of conception device, may be inserted into the vagina following insemination and may be left in place for several hours

Intrauterine

"IUI" redirects here. For other uses, see IUI (disambiguation).

Intrauterine insemination (IUI) involves injection of 'washed' sperm directly into the uterus with a catheter. Washing involves the removal of chemicals other than sperm which are in the natural ejaculate. In forms of vaginal insemination, including artificial vaginal insemination and ICI, these chemicals will be filtered out by the vagina. Insemination in this way also means that the sperm do not have to swim through the cervix which is coated with a mucus layer. This layer of mucus can slow down the passage of sperm and can result in many sperm perishing before they can enter the uterus. Donor sperm is sometimes tested for mucus penetration if it is to be used for ICI inseminations but partner sperm may or may not be able to pass through the cervix. In these cases, the use of IUI can provide a more efficient delivery of the sperm. In general terms, IUI is usually regarded as more efficient than ICI or IVI. It is therefore the method of choice for single and lesbian women wishing to conceive using donor sperm since this group of recipients usually require artificial insemination because they do not have a male partner, not because they have medical problems. Owing to the high number of these recipients using donor sperm services, IUI is therefore the most popular method of insemination today at a fertility clinic. The term 'artificial insemination' has, in many cases, come to mean IUI insemination.

It is important that washed sperm is used because unwashed sperm may elicit uterine cramping, expelling the semen and causing pain, due to content of prostaglandins. (Prostaglandins are also the compounds responsible for causing the myometrium to contract and expel the menses from the uterus, during menstruation.) Resting on the table for fifteen minutes after an IUI is optimal for the woman to increase the pregnancy rate.

Using this technique, as with ICI, fertilization takes place naturally in the external part of the fallopian tubes in the same way that occurs following intercourse.

For heterosexual couples, the indications to perform an intrauterine insemination are usually a moderate male factor, the incapability to ejaculate in vagina and an idiopathic infertility. A short period of ejaculatory abstinence before intrauterine insemination is associated with higher pregnancy rates. For the man, a TMS of more than 5 million per ml is optimal. In practice, donor sperm will satisfy these criteria and since IUI is a more efficient method of artificial insemination than ICI and, because of its generally higher success rate, IUI is usually the insemination procedure of choice for single women and lesbians using donor semen in a fertility centre. Lesbians and single women are less likely to have fertility issues of their own and enabling donor sperm to be inserted directly into the womb will often produce a better chance of conceiving. A 2019 showed that pregnancy rates were similar between lesbian women and heterosexual women undergoing IUI. However, it was found that there is a significantly higher multiple gestation rate among lesbian women undergoing ovulation induction (OI) when compared to lesbian women undergoing natural cycles.

Unlike ICI, intrauterine insemination normally requires a medical practitioner to perform the procedure. One of the requirements is to have at least one permeable tube, proved by hysterosalpingography. The infertility duration is also important. A female under 30 years of age has optimal chances with IUI; A promising cycle is one that offers two follicles measuring more than 16 mm, and estrogen of more than 500 pg/mL on the day of hCG administration. However, GnRH agonist administration at the time of implantation does not improve pregnancy outcome in intrauterine insemination cycles according to a randomized controlled trial. One of the prominent private clinic in Europe has published a data A multiple logistic regression model showed that sperm origin, maternal age, follicle count at hCG administration day, follicle rupture, and the number of uterine contractions observed after the second insemination procedure were associated with the live-birth rate The steps to follow in order to perform an intrauterine insemination are:

• Mild controlled ovarian stimulation (COS): there is no control of how many oocytes are at the same time when stimulating ovulation. For that reason, it is necessary to check the amount being ovulated via ultrasound (checking the amount of follicles developing at the same time) and administering the desired amount of hormones.
• Ovulation induction: using substances known as ovulation inductors.
• Semen capacitation: wash and centrifugation, swim-up, or gradient. The insemination should not be performed later than an hour after capacitation. 'Washed sperm' may be purchased directly from a sperm bank if donor semen is used, or 'unwashed semen' may be thawed and capacitated before performing IUI insemination, provided that the capacitation leaves a minimum of, usually, five million motile sperm.
• Luteal phase support: a lack of progesterone in the endometrium could end a pregnancy. To avoid that 200 mg/day of micronized progesterone are administered via vagina. If there is pregnancy, this hormone is kept administering until the tenth week of pregnancy.

The cost breakdown for Intrauterine Insemination (IUI) involves several components. The procedure itself typically ranges from $300 to $1,000 per cycle without insurance. The cost of the sperm may vary widely, with prices per vial ranging from $500 to $1,000 or more from a sperm bank. Additional expenses might include consultation fees, ovulation-inducing medication, ultrasounds, and blood tests.

The extent of insurance coverage for fertility treatments, including Intrauterine Insemination (IUI), varies considerably. Some insurance plans may cover some of the costs, while others may not provide any financial support for fertility treatments. Coverage depends on various factors, such as the insurance plan, state policies and regulations, and the underlying cause of infertility. Several states have mandated insurers to provide coverage for infertility services.

IUI can be used in conjunction with controlled ovarian hyperstimulation (COH). Clomiphene Citrate is the first line, Letrozole is second line, in order to stimulate ovaries before moving on to IVF. Still, advanced maternal age causes decreased success rates; women aged 38–39 years appear to have reasonable success during the first two cycles of ovarian hyperstimulation and IUI. However, for women aged over 40 years, there appears to be no benefit after a single cycle of COH/IUI. Medical experts therefore recommend considering in vitro fertilization after one failed COH/IUI cycle for women aged over 40 years.

A double intrauterine insemination theoretically increases pregnancy rates by decreasing the risk of missing the fertile window during ovulation. However, a randomized trial of insemination after ovarian hyperstimulation found no difference in live birth rate between single and double intrauterine insemination. A Cochrane found uncertain evidence about the effect of IUI compared with timed intercourse or expectant management on live birth rates but IUI with controlled ovarian hyperstimulation is probably better than expectant management.

Due to the lack of reliable evidence from controlled clinical trials, it is not certain which semen preparation techniques are more effective (wash and centrifugation; swim-up; or gradient) in terms of pregnancy and live birth rates.

Intrauterine insemination success factors

Intrauterine insemination (IUI) procedures have shown to be more successful and effective with certain factors taken into account. One major factor is the health of the sperm that is used. Sperm motility, which is improved by the sperm washing procedure, sperm density, and the sperm concentration index, all of which are found through washing and studying of the health of the specimen, are major indicators of a positive pregnancy test following IUI.

The age of both the male and female (egg and sperm donors) involved in the process are extremely important. Although age has typically been pinned on the women as a determining factor, research shows that both male and female age has about equal impact on the success of the procedure. Along with age, the duration of fertility is also found to be a factor in IUI success, the longer one faces infertility, the lower the chance of a positive pregnancy test occurring. When people talk about age as a risk factor, they are generally speaking to the way in which the DNA in the eggs and sperm have increased probabilities of mutations.

Lastly, the biological factors of the female's body can have some impact on the success of the IUI procedure. The endometrial thickness at time of insemination is moderately important, though less of a concern than some of the other factors. The number of follicles developed, grown, and retrieved from the ovaries during ovarian stimulation is particularly important and a major success factor in fertility treatments. And lastly, for the female partner, the estradiol concentration within the body on the day of HCG administration.

Who IUI can be used for

Because IUI is less expensive and less invasive than other fertility options (for example, in vitro fertilisation, or IVF), it is typically the first outlet for those looking for fertility treatments. For individuals or couples who struggle with getting pregnant, but haven't explored any fertility treatments yet, they would be good candidates for IUI. IUI provides those with a more affordable and accessible outlet for fertility treatments, however, IUI may not be the most successful option if it is determined to be female factor infertility. IUI is also a very good option for single individuals who are using donor sperm, as donor sperm undergoes regulations and checks which may not be the case for a partner sperm donation. IUI can additionally be a good fertility outlet for lesbian or queer couples as they most often do not face infertility, and would most likely be using regulated and checked donor sperm. Furthermore, surrogates can be artificially inseminated through IUI to help other individuals and/or couples become pregnant with their sperm.

Intrauterine tuboperitoneal

Intrauterine tuboperitoneal insemination (IUTPI) involves injection of washed sperm into both the uterus and fallopian tubes. The cervix is then clamped to prevent leakage to the vagina, best achieved with a specially designed double nut bivalve (DNB) speculum. The sperm is mixed to create a volume of 10 ml, sufficient to fill the uterine cavity, pass through the interstitial part of the tubes and the ampulla, finally reaching the peritoneal cavity and the Pouch of Douglas where it would be mixed with the peritoneal and follicular fluid. IUTPI can be useful in unexplained infertility, mild or moderate male infertility, and mild or moderate endometriosis. In non-tubal sub fertility, fallopian tube sperm perfusion may be the preferred technique over intrauterine insemination.

Intratubal

Intratubal insemination (ITI) involves injection of washed sperm into the fallopian tube, although this procedure is no longer generally regarded as having any beneficial effect compared with IUI. ITI however, should not be confused with gamete intrafallopian transfer, where both eggs and sperm are mixed outside the woman's body and then immediately inserted into the fallopian tube where fertilization takes place.

LGBTQ+ concerns

Although many fertilization procedures, like IUI are typically carried out in a medical setting, society is increasingly recognizing the important role that this plays in the lives of individuals who might otherwise not conceive through heterosexual penetrative sexual intercourse. Artificial insemination using a sperm donor for LGBTQ+ individuals and couples is one of the more cost-effective avenues to parenting. While clinic based IUI may be open to many, it typically still includes hetero-reproductive narratives which dates from the early days of fertilization procedures when these were often exclusively for married couples and when there was a resistance in many societies to extend these services to the LGBTQ+ community. Indeed, in the early days, there were very few fertility clinics which would provide services to single women and lesbian couples. In the UK, notable pioneers in this respect were the British Pregnancy Advisory Service (BPAS) and the Pregnancy Advisory Service (PAS), both of which operated before statutory control of fertility services in 1992, and the London Women's Clinic (LWC) which provided artificial insemination to single women and lesbians from 1998. Most donor insemination procedures undertaken in many countries today are for lesbian couples or single mainly lesbian women, yet much of their rhetoric and advertising is directed at heterosexual couples. Indeed, many sperm banks seem reluctant to inform donors that most of their donations will be used for lesbians and single women. To improve the way society talks about and carries out donor insemination inclusive language may be used. One way to do this is to bring LGBTQ narratives into this process, with a particular emphasis on this being a family-centered process. Even in a medical setting, it is important to bring intimacy and family-centeredness into this process, as this promotes connectedness and inclusiveness in what can be seen as a hostile and discriminatory environment. LGBTQ couples or individuals typically have to navigate more complexities and barriers than heterosexual couples when undergoing fertility treatment, such as stigma and carrier decisions, so allowing room for intimacy and connectedness in the process can improve the experience for individuals, reduce stress, and minimize barriers that target marginalized individuals.

Lesbian couples may either select a friend or family member as their sperm donor or choose an anonymous donor. After a sperm donor is selected, a couple can proceed with donor sperm IUI. IUI is an economic option for same-sex couples and can be done without the use of medication. According to a study from 2021, lesbian women undergoing IUI had an average clinical pregnancy rate of 13.2% per cycle and 42.2% success rate giving the average number of cycles at 3.6.

Pregnancy rate

Approximate pregnancy rate as a function of total sperm count (may be twice as large as total motile sperm count). Values are for intrauterine insemination. (Old data, rates are likely higher these days)

Main article: Pregnancy rate

The rates of successful pregnancy for artificial insemination are 10-15% per menstrual cycle using ICI, and 15–20% per cycle for IUI. In IUI, about 60 to 70% have achieved pregnancy after 6 cycles.

However, these pregnancy rates may be very misleading, since many factors have to be included to give a meaningful answer, e.g. definition of success and calculation of the total population. These rates can be influenced by age, overall reproductive health, and if the patient had an orgasm during the insemination. The literature is conflicting on immobilization after insemination has increasing the chances of pregnancy. Previous data suggests that it is statistically significant for the patient to remain immobile for 15 minutes after insemination, while another review article claims that it is not. A point of consideration, is that it does cost the patient or healthcare system to remain immobile for 15 minutes if it does increase the chances. For couples with unexplained infertility, unstimulated IUI is no more effective than natural means of conception.

The pregnancy rate also depends on the total sperm count, or, more specifically, the total motile sperm count (TMSC), used in a cycle. The success rate increases with increasing TMSC, but only up to a certain count, when other factors become limiting to success. The summed pregnancy rate of two cycles using a TMSC of 5 million (may be a TSC of ~10 million on graph) in each cycle is substantially higher than one single cycle using a TMSC of 10 million. However, although more cost-efficient, using a lower TMSC also increases the average time taken to achieve pregnancy. Women whose age is becoming a major factor in fertility may not want to spend that extra time.

Samples per child

The number of samples (ejaculates) required to give rise to a child varies substantially from person to person, as well as from clinic to clinic. However, the following equations generalize the main factors involved:

For intracervical insemination:

${\displaystyle N=\frac{V_s\times c\times r_s}{n_r}}$

• N is how many children a single sample can give rise to.
• V_s is the volume of a sample (ejaculate), usually between 1.0 mL and 6.5 mL
• c is the concentration of motile sperm in a sample after freezing and thawing, approximately 5–20 million per ml but varies substantially
• r_s is the pregnancy rate per cycle, between 10% and 35%
• n_r is the total motile sperm count recommended for vaginal insemination (VI) or intra-cervical insemination (ICI), approximately 20 million pr. ml.

The pregnancy rate increases with increasing number of motile sperm used, but only up to a certain degree, when other factors become limiting instead.

Derivation of the equation (click at right to view)

Approximate live birth rate (r_s) among infertile couples as a function of total motile sperm count (n_r). Values are for intrauterine insemination.

With these numbers, one sample would on average help giving rise to 0.1–0.6 children, that is, it actually takes on average 2–5 samples to make a child.

For intrauterine insemination, a centrifugation fraction (f_c) may be added to the equation:

f_c is the fraction of the volume that remains after centrifugation of the sample, which may be about half (0.5) to a third (0.33).

${\displaystyle N=\frac{V_s\times f_c\times c\times r_s}{n_r}}$

On the other hand, only 5 million motile sperm may be needed per cycle with IUI (n_r=5 million)

Thus, only 1–3 samples may be needed for a child if used for IUI.

Social implications

One of the key issues arising from the rise of dependency on assisted reproductive technology (ARTs) is the pressure placed on couples to conceive, "where children are highly desired, parenthood is culturally mandatory, and childlessness socially unacceptable".

The medicalization of infertility creates a framework in which individuals are encouraged to think of infertility quite negatively. In many cultures donor insemination is religiously and culturally prohibited, often meaning that less accessible "high tech" and expensive ARTs, like IVF, are the only solution.

An over-reliance on reproductive technologies in dealing with infertility prevents many – especially, for example, in the "infertility belt" of central and southern Africa – from dealing with many of the key causes of infertility treatable by artificial insemination techniques; namely preventable infections, dietary and lifestyle influences.

If good records are not kept, the offspring when grown up risk accidental incest.

Risk factors

The risk factors of artificial insemination are comparatively low to other forms of fertility treatment. The most prominent risk factor would be infection after the procedure, with other risk factors including a higher risk of having twins or triplets, and minor vaginal bleeding during the procedure.

Although these risk factors are minor and generally manageable, there is a significant knowledge gap between identity groups around risk factors for fertility treatments in general. For instance, it was found that LGBTQ+ individuals had "had significant knowledge gaps of risk factors associated with reproductive outcomes when compared to heterosexual female peers." Therefore, it is imperative that providers take extra care in educating their LGBTQ+ patients on potential risk factors of artificial insemination. The implications of this knowledge gap between LGTBQ+ individuals and their heterosexual counterparts are serious and worth noting. Lack of access to proper information and risk factors around procedures like these may dissuade someone from pursuing these procedures altogether. As a result, there will be less normalization of LGBTQ+ family making and reproduction, which only perpetuates this cycle of lack of information among LGBTQ+ folks.

Legal restrictions

Some countries restrict artificial insemination in a variety of ways. For example, some countries do not permit AI for single women, and other countries do not permit the use of donor sperm.

Europe

As of May 2013, the following European countries permit medically assisted AI for single women:

• Armenia
• Belarus
• Belgium
• Bulgaria
• Cyprus
• Denmark
• Estonia
• Finland
• Germany
• Greece
• Hungary
• Iceland
• Ireland
• Latvia
• Moldova
• Montenegro
• Netherlands
• North Macedonia
• Romania
• Russia
• Spain
• Ukraine
• United Kingdom

Law in the United States

History of Law Around Artificial Insemination

Artificial insemination used to be seen as adultery and was illegal until the 1960s when states started recognizing the child born from artificial insemination as legitimate. Once the children began to be recognized as legitimate, legal questions around who the parents of the child are, how to handle surrogacy, paternity rights, and eventually artificial insemination and LGBT+ parents began to arise. Prior to the use of artificial insemination, the legal parents of a child were the two people who conceived the child or the person who birthed the child and their legal spouse, but artificial insemination complicates the legal process of becoming a parent as well as who is the parent of the child. Deciding who the parents of the child are is the largest legal predicament around artificial insemination. However, questions around surrogacy and donor's rights also appear as a side question to determining the parent(s). Some major cases that deal with artificial insemination and parental rights are, K.M v E.G, Johnson v Calvert, Matter of Baby M, and In Re K.M.H.

Legal Parental Relations and Artificial Insemination

When children are conceived the traditional way, there is little discrepancy around who the legal parents of the child are. However, because children conceived using artificial insemination may not be genetically related to one or more of their parents, who the legal parents of the child are can come into question. Prior to the passage of the Uniform Parentage Act in 1973, children conceived via artificial insemination were deemed as "illegitimate" children. The Uniform Parentage Act then recognized the children born from artificial insemination as legal and laid precedent for how the legal parents of the child were decided. However, this act applied only to the children of those married couples. It established that the person who birthed the child was the mother and the father would be the husband of the woman. In 2002, the Uniform Parentage Act, which is adopted individually on a state by state basis, was revised to address non married couples and states that an unmarried couple has the same rights to the child that a married couple would. This extended who has the right to be a parent to a man who would supposedly fill in the social role as a "father." There were now numerous ways to establish parental rights for both the mother and the father depending on if the child was born using a sperm donor or a surrogate. Currently, a revised version of the Uniform Parentage Act is starting to be passed in a few states that expands how parental relations can be determined. This bill includes expanding "father" to mean any person who would fill the role of a father, regardless of their gender and "mother" is expanded to anyone who gives birth to the child regardless of gender. In addition, this act would also change any language of "husband" or "wife" to "spouse."

Paternity rights

There is no federal law that applies to all fifty states when it comes to artificial insemination and paternity rights, but the Uniform Parentage Act is a model which many states have adopted. Under the 1973 UPA, married heterosexual couples making use of artificial insemination through a licensed physician could list the husband as the natural father of the child, rather than the sperm donor. Since then a revised version of the Act has been introduced, though to less widespread adoption

Generally paternity is not an issue when artificial insemination is between a married woman and an anonymous donor. Most states provide that anonymous donors' paternity claims are not recognized, and most sperm donation centers make use of contracts that require donors to sign away their paternity rights before they can participate. When the mother knows the donor, however, or engages in artificial insemination while unmarried, complications may arise. In cases of private sperm donation, paternity rights and responsibilities are often conferred onto sperm donors when: the donor and recipient did not comply with state laws regarding artificial insemination, the sperm donor and recipient know one another, or the donor had the intent of being a father to the child. When one or a number of these things is true, courts have at times found written agreements relinquishing parental rights to be unenforceable.

Opposition and criticism

Religious opposition

Main article: Religious response to assisted reproductive technology

Some theologically buttressed arguments reject the moral validity of this practice, such as Pope John XXIII. However, according to a document of the USCCB, the intrauterine insemination (IUI) of "licitly obtained" (normal intercourse with a silastic sheath i.e. a perforated condom) but technologically prepared semen sample (washed, etc.) has been neither approved nor disapproved by Church authority and its moral validity remains under discussion. Some religious groups, such as the Catholic Church, and individuals have also criticized artificial insemination because acquiring sperm for the procedure is seen as "a form of adultery promoting the vice of masturbation."

Other morality-based opposition

There are critics of artificial insemination who voice concerns regarding the potential for AI to encourage eugenicist practices through selection of particular traits. The line of reasoning follows the history of artificial insemination in breeding livestock and other domesticated animals wherein preferred traits are encouraged through human-controlled selection.

Other animals

A man performing artificial insemination of a cow.

A breeding mount with built-in artificial vagina used in semen collection from horses for use in artificial insemination

A breeder performing artificial insemination of a dog.

Artificial insemination is used for pets, livestock, endangered species, and animals in zoos or marine parks difficult to transport.

Reasons and techniques

See also: Captive breeding

It may be used for many reasons, including to allow a male to inseminate a much larger number of females, to allow the use of genetic material from males separated by distance or time, to overcome physical breeding difficulties, to control the paternity of offspring, to synchronize births, to avoid injury incurred during natural mating, and to avoid the need to keep a male at all (such as for small numbers of females or in species whose fertile males may be difficult to manage).

Artificial insemination is much more common than natural mating, as it allows several female animals to be impregnated from a single male. For instance, up to 30-40 female pigs can be impregnated from a single boar. Workers collect the semen by masturbating the boars, then insert it into the sows via a raised catheter known as a pork stork. Boars are still physically used to excite the females prior to insemination, but are prevented from actually mating.

Semen is collected, extended, then cooled or frozen. It can be used on-site or shipped to the female's location. If frozen, the small plastic tube holding the semen is referred to as a straw. To allow the sperm to remain viable during the time before and after it is frozen, the semen is mixed with a solution containing glycerol or other cryoprotectants. An extender is a solution that allows the semen from a donor to impregnate more females by making insemination possible with fewer sperm. Antibiotics, such as streptomycin, are sometimes added to the sperm to control some bacterial venereal diseases. Before the actual insemination, estrus may be induced through the use of progestogen and another hormone (usually PMSG or Prostaglandin F2α).

History

Artificial insemination tools brought from the USSR by Luis Thomasset in 1935 to work at Cambridge Laboratories and South America.

The first viviparous animal to be artificially fertilized was a dog. The experiment was conducted with success by the Italian Lazzaro Spallanzani in 1780. Another pioneer was the Russian Ilya Ivanov in 1899. In 1935, diluted semen from Suffolk sheep was flown from Cambridge in Britain to Kraków, Poland, as part of an international research project. The participants included Prawochenki (Poland), Milovanoff (USSR), Hammond and Walton (UK), and Thomasset (Uruguay).

Modern artificial insemination was pioneered by John O. Almquist of Pennsylvania State University. He improved breeding efficiency by the use of antibiotics (first proven with penicillin in 1946) to control bacterial growth, decreasing embryonic mortality, and increase fertility. This, and various new techniques for processing, freezing, and thawing of frozen semen significantly enhanced the practical utilization of artificial insemination in the livestock industry and earned him the 1981 Wolf Foundation Prize in Agriculture. Many techniques developed by him have since been applied to other species, including humans.

Species

Artificial insemination is used in many non-human animals, including sheep, horses, cattle, pigs, dogs, pedigree animals generally, zoo animals, turkeys and creatures as tiny as honeybees and as massive as orcas (killer whales).

Artificial insemination of farm animals is common in the developed world, especially for breeding dairy cattle (75% of all inseminations). Swine are also bred using this method (up to 85% of all inseminations). It is an economical means for a livestock breeder to improve their herds utilizing males having desirable traits.

Although common with cattle and swine, artificial insemination is not as widely practiced in the breeding of horses. A small number of equine associations in North America accept only horses that have been conceived by "natural cover" or "natural service" – the actual physical mating of a mare to a stallion – the Jockey Club being the most notable of these, as no artificial insemination is allowed in Thoroughbred breeding. Other registries such as the AQHA and warmblood registries allow registration of foals created through artificial insemination, and the process is widely used allowing the breeding of mares to stallions not resident at the same facility – or even in the same country – through the use of transported frozen or cooled semen.

In modern species conservation, semen collection and artificial insemination are used also in birds. In 2013 scientist of the Justus-Liebig-University of Giessen, Germany, from the working group of Michael Lierz, Clinic for birds, reptiles, amphibians, and fish, developed a novel technique for semen collection and artificial insemination in parrots producing the world's first macaw by assisted reproduction.

Scientists working with captive orcas were able to pioneer the technique in the early 2000s, resulting in "the first successful conceptions, resulting in live offspring, using artificial insemination in any cetacean species". John Hargrove, a SeaWorld trainer, describes Kasatka as being the first orca to receive artificial insemination.

Violation of rights

Main article: Animal rights

Artificial insemination on animals has been criticised as a violation of animal rights, with animal rights advocates equating it with rape and arguing it constitutes institutionalized bestiality.^[69][70] Artificial insemination of farm animals is condemned by animal rights campaigners such as People for the Ethical Treatment of Animals (PETA) and Joey Carbstrong, who identify the practice as a form of rape due to its sexual, involuntary and perceived painful nature. Animal rights organizations such as PETA and Mercy for Animals frequently write against the practice in their articles. Much of the meat production in the United States depends on artificial insemination, resulting in an explosive growth of the procedure over the past three decades. The state of Kansas makes no exceptions for artificial insemination under its bestiality law, thus making the procedure illegal.

Criteria for benefiting from artificial insemination according to the 2021 Bioethics Law

According to the 2021 Bioethics Law, the criteria that must be met to benefit from artificial insemination are as follows:

1. Artificial insemination can be performed using sperm from the husband or frozen sperm from an anonymous donor.
2. Both spouses or the unmarried woman must consent in advance to artificial insemination or embryo transfer.
3. The parenting project must be validated through a series of interviews with professionals (doctors, psychologists, etc.).
4. Individuals benefiting from artificial insemination must be of reproductive age.

The 2021 Bioethics Law has expanded the scope of Medically Assisted Procreation (MAP).

Embryo

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Embryo

 

Embryo
A human embryo seven weeks after conception (nine weeks gestational age)

An embryo (/ˈɛmbrioʊ/ EM-bree-oh) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sperm cell. The resulting fusion of these two cells produces a single-celled zygote that undergoes many cell divisions that produce cells known as blastomeres. The blastomeres are arranged as a solid ball that when reaching a certain size, called a morula, takes in fluid to create a cavity called a blastocoel. The structure is then termed a blastula, or a blastocyst in mammals.

The mammalian blastocyst hatches before implantating into the endometrial lining of the womb. Once implanted the embryo will continue its development through the next stages of gastrulation, neurulation, and organogenesis. Gastrulation, possibly induced in part by paracrine signalling from the amnion, is the formation of the three germ layers that will form all of the different parts of the body. Neurulation forms the nervous system, and organogenesis is the development of all the various tissues and organs of the body.

A newly developing human is typically referred to as an embryo until the ninth week after conception, when it is then referred to as a fetus. In other multicellular organisms, the word embryo can be used more broadly to any early developmental or life cycle stage prior to birth or hatching.

Etymology

First attested in English in the mid-14th century, the word embryon derives from Medieval Latin embryo, itself from Greek ἔμβρυον (émbryon) 'young one', which is the neuter form of ἔμβρυος (émbryos) 'growing in'. Morphologically it is derived from ἐν (en) 'in' and βρύω (brýō) 'to swell, to be full'. The proper Latinized form of the Greek term would be embryum.

Development

Animal embryos

Main article: Animal embryonic development

Embryos (and one tadpole) of the wrinkled frog (Rana rugosa)

Mouse and snake embryos

In animals, fertilization begins the process of embryonic development with the creation of a zygote, a single cell resulting from the fusion of gametes (e.g. egg and sperm). The development of a zygote into a multicellular embryo proceeds through a series of recognizable stages, often divided into cleavage, blastula, gastrulation, and organogenesis.

Cleavage is the period of rapid mitotic cell divisions that occur after fertilization. During cleavage, the overall size of the embryo does not change, but the size of individual cells decrease rapidly, as they divide to increase the total number of cells. Cleavage results in a blastula.

Depending on the species, a blastula or blastocyst stage embryo can appear as a ball of cells on top of yolk, or as a hollow sphere of cells surrounding a middle cavity. The embryo's cells continue to divide and increase in number, while molecules within the cells such as RNAs and proteins actively promote key developmental processes such as gene expression, cell fate specification, and polarity. Before implanting into the uterine wall the embryo is sometimes known as the pre-implantation embryo or pre-implantation conceptus. Sometimes this is called the pre-embryo, a term employed to differentiate from an embryo proper in relation to embryonic stem cell discourses.

Gastrulation is the next phase of embryonic development, and involves the development of two or more layers of cells (germinal layers). Animals that form two layers (such as Cnidaria) are called diploblastic, and those that form three (most other animals, from flatworms to humans) are called triploblastic. During gastrulation of triploblastic animals, the three germinal layers that form are called the ectoderm, mesoderm, and endoderm. All tissues and organs of a mature animal can trace their origin back to one of these layers. For example, the ectoderm will give rise to the skin epidermis and the nervous system, the mesoderm will give rise to the vascular system, muscles, bone, and connective tissues, and the endoderm will give rise to organs of the digestive system and epithelium of the digestive system and respiratory system. Many visible changes in embryonic structure happen throughout gastrulation as the cells that make up the different germ layers migrate and cause the previously round embryo to fold or invaginate into a cup-like appearance.

Past gastrulation, an embryo continues to develop into a mature multicellular organism by forming structures necessary for life outside of the womb or egg. As the name suggests, organogenesis is the stage of embryonic development when organs form. During organogenesis, molecular and cellular interactions prompt certain populations of cells from the different germ layers to differentiate into organ-specific cell types. For example, in neurogenesis, a subpopulation of cells from the ectoderm segregate from other cells and further specialize to become the brain, spinal cord, or peripheral nerves.

The embryonic period varies from species to species. In human development, the term fetus is used instead of embryo after the ninth week after conception, whereas in zebrafish, embryonic development is considered finished when a bone called the cleithrum becomes visible. In animals that hatch from an egg, such as birds, a young animal is typically no longer referred to as an embryo once it has hatched. In viviparous animals (animals whose offspring spend at least some time developing within a parent's body), the offspring is typically referred to as an embryo while inside of the parent, and is no longer considered an embryo after birth or exit from the parent. However, the extent of development and growth accomplished while inside of an egg or parent varies significantly from species to species, so much so that the processes that take place after hatching or birth in one species may take place well before those events in another. Therefore, according to one textbook, it is common for scientists to interpret the scope of embryology broadly as the study of the development of animals.

Plant embryos

Main article: Plant embryonic development

Further information: Sporophyte

The inside of a Ginkgo seed, showing the embryo

Flowering plants (angiosperms) create embryos after the fertilization of a haploid ovule by pollen. The DNA from the ovule and pollen combine to form a diploid, single-cell zygote that will develop into an embryo. The zygote, which will divide multiple times as it progresses throughout embryonic development, is one part of a seed. Other seed components include the endosperm, which is tissue rich in nutrients that will help support the growing plant embryo, and the seed coat, which is a protective outer covering. The first cell division of a zygote is asymmetric, resulting in an embryo with one small cell (the apical cell) and one large cell (the basal cell). The small, apical cell will eventually give rise to most of the structures of the mature plant, such as the stem, leaves, and roots. The larger basal cell will give rise to the suspensor, which connects the embryo to the endosperm so that nutrients can pass between them. The plant embryo cells continue to divide and progress through developmental stages named for their general appearance: globular, heart, and torpedo. In the globular stage, three basic tissue types (dermal, ground, and vascular) can be recognized. The dermal tissue will give rise to the epidermis or outer covering of a plant, ground tissue will give rise to inner plant material that functions in photosynthesis, resource storage, and physical support, and vascular tissue will give rise to connective tissue like the xylem and phloem that transport fluid, nutrients, and minerals throughout the plant. In heart stage, one or two cotyledons (embryonic leaves) will form. Meristems (centers of stem cell activity) develop during the torpedo stage, and will eventually produce many of the mature tissues of the adult plant throughout its life. At the end of embryonic growth, the seed will usually go dormant until germination. Once the embryo begins to germinate (grow out from the seed) and forms its first true leaf, it is called a seedling or plantlet.

Plants that produce spores instead of seeds, like bryophytes and ferns, also produce embryos. In these plants, the embryo begins its existence attached to the inside of the archegonium on a parental gametophyte from which the egg cell was generated. The inner wall of the archegonium lies in close contact with the "foot" of the developing embryo; this "foot" consists of a bulbous mass of cells at the base of the embryo which may receive nutrition from its parent gametophyte. The structure and development of the rest of the embryo varies by group of plants.

Since all land plants create embryos, they are collectively referred to as embryophytes (or by their scientific name, Embryophyta). This, along with other characteristics, distinguishes land plants from other types of plants, such as algae, which do not produce embryos.

Research and technology

Biological processes

Embryos from numerous plant and animal species are studied in biological research laboratories across the world to learn about topics such as stem cells, evolution and development, cell division, and gene expression. Examples of scientific discoveries made while studying embryos that were awarded the Nobel Prize in Physiology or Medicine include the Spemann-Mangold organizer, a group of cells originally discovered in amphibian embryos that give rise to neural tissues, and genes that give rise to body segments discovered in Drosophila fly embryos by Christiane Nüsslein-Volhard and Eric Wieschaus.

Assisted reproductive technology

Creating and/or manipulating embryos via assisted reproductive technology (ART) is used for addressing fertility concerns in humans and other animals, and for selective breeding in agricultural species. Between the years 1987 and 2015, ART techniques including in vitro fertilization (IVF) were responsible for an estimated one million human births in the United States alone. Other clinical technologies include preimplantation genetic diagnosis (PGD), which can identify certain serious genetic abnormalities, such as aneuploidy, prior to selecting embryos for use in IVF. Some have proposed (or even attempted—see He Jiankui affair) genetic editing of human embryos via CRISPR-Cas9 as a potential avenue for preventing disease; however, this has been met with widespread condemnation from the scientific community.

ART techniques are also used to improve the profitability of agricultural animal species such as cows and pigs by enabling selective breeding for desired traits and/or to increase numbers of offspring. For example, when allowed to breed naturally, cows typically produce one calf per year, whereas IVF increases offspring yield to 9–12 calves per year. IVF and other ART techniques, including cloning via interspecies somatic cell nuclear transfer (iSCNT), are also used in attempts to increase the numbers of endangered or vulnerable species, such as Northern white rhinos, cheetahs, and sturgeons.

Cryoconservation of plant and animal biodiversity

Cryoconservation of genetic resources involves collecting and storing the reproductive materials, such as embryos, seeds, or gametes, from animal or plant species at low temperatures in order to preserve them for future use. Some large-scale animal species cryoconservation efforts include "frozen zoos" in various places around the world, including in the UK's Frozen Ark, the Breeding Centre for Endangered Arabian Wildlife (BCEAW) in the United Arab Emirates, and the San Diego Zoo Institute for Conservation in the United States. As of 2018, there were approximately 1,700 seed banks used to store and protect plant biodiversity, particularly in the event of mass extinction or other global emergencies. The Svalbard Global Seed Vault in Norway maintains the largest collection of plant reproductive tissue, with more than a million samples stored at −18 °C (0 °F).

Fossilized embryos

Main article: Fossil embryos

Fossilized animal embryos are known from the Precambrian, and are found in great numbers during the Cambrian period. Even fossilized dinosaur embryos have been discovered.

Mutagenesis

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Mutagenesis

Mutagenesis (/mjuːtəˈdʒɛnɪsɪs/) is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. A mutagen is a mutation-causing agent, be it chemical or physical, which results in an increased rate of mutations in an organism's genetic code. In nature, mutagenesis can lead to cancer and various heritable diseases, and it is also a driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.

History

DNA may be modified, either naturally or artificially, by a number of physical, chemical and biological agents, resulting in mutations. Hermann Muller found that "high temperatures" have the ability to mutate genes in the early 1920s, and in 1927, demonstrated a causal link to mutation upon experimenting with an x-ray machine, noting phylogenetic changes when irradiating fruit flies with relatively high dose of X-rays. Muller observed a number of chromosome rearrangements in his experiments, and suggested mutation as a cause of cancer. The association of exposure to radiation and cancer had been observed as early as 1902, six years after the discovery of X-ray by Wilhelm Röntgen, and the discovery of radioactivity by Henri Becquerel. Lewis Stadler, Muller's contemporary, also showed the effect of X-rays on mutations in barley in 1928, and of ultraviolet (UV) radiation on maize in 1936. In 1940s, Charlotte Auerbach and J. M. Robson found that mustard gas can also cause mutations in fruit flies.

While changes to the chromosome caused by X-ray and mustard gas were readily observable to early researchers, other changes to the DNA induced by other mutagens were not so easily observable; the mechanism by which they occur may be complex, and take longer to unravel. For example, soot was suggested to be a cause of cancer as early as 1775, and coal tar was demonstrated to cause cancer in 1915. The chemicals involved in both were later shown to be polycyclic aromatic hydrocarbons (PAH). PAHs by themselves are not carcinogenic, and it was proposed in 1950 that the carcinogenic forms of PAHs are the oxides produced as metabolites from cellular processes. The metabolic process was identified in 1960s as catalysis by cytochrome P450, which produces reactive species that can interact with the DNA to form adducts, or product molecules resulting from the reaction of DNA and, in this case, cytochrome P450; the mechanism by which the PAH adducts give rise to mutation, however, is still under investigation.

Distinction between a mutation and DNA damage

DNA damage is an abnormal alteration in the structure of DNA that cannot, itself, be replicated when DNA replicates. In contrast, a mutation is a change in the nucleic acid sequence that can be replicated; hence, a mutation can be inherited from one generation to the next. Damage can occur from chemical addition (adduct), or structural disruption to a base of DNA (creating an abnormal nucleotide or nucleotide fragment), or a break in one or both DNA strands. Such DNA damage may result in mutation. When DNA containing damage is replicated, an incorrect base may be inserted in the new complementary strand as it is being synthesized (see DNA repair § Translesion synthesis). The incorrect insertion in the new strand will occur opposite the damaged site in the template strand, and this incorrect insertion can become a mutation (i.e. a changed base pair) in the next round of replication. Furthermore, double-strand breaks in DNA may be repaired by an inaccurate repair process, non-homologous end joining, which produces mutations. Mutations can ordinarily be avoided if accurate DNA repair systems recognize DNA damage and repair it prior to completion of the next round of replication. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. Of these, 83 are directly employed in the 5 types of DNA repair processes indicated in the chart shown in the article DNA repair.

Mammalian nuclear DNA may sustain more than 60,000 damage episodes per cell per day, as listed with references in DNA damage (naturally occurring). If left uncorrected, these adducts, after misreplication past the damaged sites, can give rise to mutations. In nature, the mutations that arise may be beneficial or deleterious—this is the driving force of evolution. An organism may acquire new traits through genetic mutation, but mutation may also result in impaired function of the genes and, in severe cases, causes the death of the organism. Mutation is also a major source for acquisition of resistance to antibiotics in bacteria, and to antifungal agents in yeasts and molds. In a laboratory setting, mutagenesis is a useful technique for generating mutations that allows the functions of genes and gene products to be examined in detail, producing proteins with improved characteristics or novel functions, as well as mutant strains with useful properties. Initially, the ability of radiation and chemical mutagens to cause mutation was exploited to generate random mutations, but later techniques were developed to introduce specific mutations.

In humans, an average of 60 new mutations are transmitted from parent to offspring. Human males, however, tend to pass on more mutations depending on their age, transmitting an average of two new mutations to their progeny with every additional year of their age.

Mechanisms

Mutagenesis may occur endogenously (e.g. spontaneous hydrolysis), through normal cellular processes that can generate reactive oxygen species and DNA adducts, or through error in DNA replication and repair. Mutagenesis may also occur as a result of the presence of environmental mutagens that induce changes to an organism's DNA, like radiation and/or radioactivity. The mechanism by which mutation occurs varies according to the mutagen, or the causative agent, involved. Most mutagens act either directly, or indirectly via mutagenic metabolites, on an organism's DNA, producing lesions. Some mutagens, however, may affect the replication or chromosomal partition mechanism, and other cellular processes.

Mutagenesis may also be self-induced by unicellular organisms when environmental conditions are restrictive to the organism's growth, such as bacteria growing in the presence of antibiotics, yeast growing in the presence of an antifungal agent, or other unicellular organisms growing in an environment lacking in an essential nutrient.

Many chemical mutagens require biological activation to become mutagenic. An important group of enzymes involved in the generation of mutagenic metabolites is cytochrome P450. Other enzymes that may also produce mutagenic metabolites include glutathione S-transferase and microsomal epoxide hydrolase. Mutagens that are not mutagenic by themselves but require biological activation are called promutagens.

While most mutagens produce effects that ultimately result in errors in replication, for example creating adducts that interfere with replication, some mutagens may directly affect the replication process or reduce its fidelity. Base analog such as 5-bromouracil may substitute for thymine in replication. Metals such as cadmium, chromium, and nickel can increase mutagenesis in a number of ways in addition to direct DNA damage, for example reducing the ability to repair errors, as well as producing epigenetic changes.

Mutations often arise as a result of problems caused by DNA lesions during replication, resulting in errors in replication. In bacteria, extensive damage to DNA due to mutagens results in single-stranded DNA gaps during replication. This induces the SOS response, an emergency repair process that is also error-prone, thereby generating mutations. In mammalian cells, stalling of replication at damaged sites induces a number of rescue mechanisms that help bypass DNA lesions, however, this may also result in errors. The Y family of DNA polymerases specializes in DNA lesion bypass in a process termed translesion synthesis (TLS) whereby these lesion-bypass polymerases replace the stalled high-fidelity replicative DNA polymerase, transit the lesion and extend the DNA until the lesion has been passed so that normal replication can resume; these processes may be error-prone or error-free.

Endogenous DNA damage

Endogenous DNA damage is caused by internal cellular processes rather than external agents. Cellular processes can generate reactive oxygen species that can modify the DNA, and DNA can also undergo spontaneous hydrolysis, while errors in replication can result in mutations.

DNA damage and spontaneous mutation

The number of DNA damage episodes occurring in a mammalian cell per day is high (more than 60,000 per day). Frequent occurrence of DNA damage is likely a problem for all DNA- containing organisms, and the need to cope with DNA damage and minimize their deleterious effects is likely a fundamental problem for life.

Most spontaneous mutations likely arise from error-prone trans-lesion synthesis past a DNA damage site in the template strand during DNA replication. This process can overcome potentially lethal blockages, but at the cost of introducing inaccuracies in daughter DNA. The causal relationship of DNA damage to spontaneous mutation is illustrated by aerobically growing E. coli bacteria, in which 89% of spontaneously occurring base substitution mutations are caused by DNA damage induced by reactive oxygen species. In yeast, more than 60% of spontaneous single-base pair substitutions and deletions are likely caused by trans-lesion synthesis.

An additional significant source of mutations in eukaryotes is the inaccurate DNA repair process non-homologous end joining, that is often employed in repair of double strand breaks.

In general, it appears that the main underlying cause of spontaneous mutation is error-prone trans-lesion synthesis during DNA replication and that the error-prone non-homologous end-joining repair pathway may also be an important contributor in eukaryotes.

Reactive oxygen species and oxidative damages

Reactive oxygen species is the typical byproducts of the electron transport chain during cellular respiration. Low levels of reactive oxygen species can function in cellular signaling and immune defenses, but excessive levels of reactive oxygen species can damage bases and the sugar phosphate backbone of the DNA. 8-oxo-guanine, an oxidized formed of guanine mispair with Adenine during replication instead of cytosine, causing an G:C to T:A mutation if left unrepaired.

Spontaneous hydrolysis

Base deamination

Base deamination is a major source of spontaneous mutagenesis happening in the human cells, and it is the loss of amine groups from a DNA base such as cytosine (C), adenine (A), guanine(G), and 5-methylcytosine. It changes the base pairing behavior so that cytosine (C) becomes Uracil (U) , adenine(A) becomes hypoxanthine, guanine (G) becomes xanthine, and 5-methylcytosine becomes thymine (T).

Cytosine and 5-methylcytosine deamination is the most frequently deaminated DNA bases, with 5-methylcytosine being three to four times more deaminated than Cytosine. For cytosine deamination, in DNA, cytosine (C) is usually paired with guanine (G). But after the deamination has happened, it creates a uracil (U) and Guanine (G) mismatch, and if the mismatch is not properly repaired, uracil can pair with adenine, creating a C:G to T:A mutation. While the deamination of 5-methylcytosine generates thymine (T) instead of uracil (U), creating a G:T mismatch.

Depurination

DNA is not entirely stable in aqueous solution, and depurination of the DNA can occur. Under physiological conditions the glycosidic bond may be hydrolyzed spontaneously and 5000 purine sites in DNA are estimated to be depurinated each day in a cell. Numerous DNA repair pathways exist for DNA; however, if the apurinic site is not repaired, misincorporation of nucleotides may occur during replication. Adenine is preferentially incorporated by DNA polymerases in an apurinic site.

Tautomerism

Main article: Tautomer

Tautomerization is the process by which compounds spontaneously rearrange themselves to assume their structural isomer forms. For example, the keto (C=O) forms of guanine and thymine can rearrange into their rare enol (-OH) forms, while the amino (-NH₂ ) forms of adenine and cytosine can result in the rarer imino (=NH) forms. In DNA replication, tautomerization alters the base-pairing sites and can cause the improper pairing of nucleic acid bases.

Modification of bases

Bases may be modified endogenously by normal cellular molecules. For example, DNA may be methylated by S-adenosylmethionine, thus altering the expression of the marked gene without incurring a mutation to the DNA sequence itself. Histone modification is a related process in which the histone proteins around which DNA coils can be similarly modified via methylation, phosphorylation, or acetylation; these modifications may act to alter gene expression of the local DNA, and may also act to denote locations of damaged DNA in need of repair. DNA may also be glycosylated by reducing sugars.

Many compounds, such as PAHs, aromatic amines, aflatoxin and pyrrolizidine alkaloids, may form reactive oxygen species catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication, and the bulky aromatic adducts may form stable intercalation between bases and block replication. The adducts may also induce conformational changes in the DNA. Some adducts may also result in the depurination of the DNA; it is, however, uncertain how significant such depurination as caused by the adducts is in generating mutation.

Alkylation and arylation of bases can cause errors in replication. Some alkylating agents such as N-nitrosamines may require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkyl cation. N⁷ and O⁶ of guanine and the N³ and N⁷ of adenine are most susceptible to attack. N⁷-guanine adducts form the bulk of DNA adducts, but they appear to be non-mutagenic. Alkylation at O⁶ of guanine, however, is harmful because excision repair of O⁶-adduct of guanine may be poor in some tissues such as the brain. The O⁶ methylation of guanine can result in G to A transition, while O⁴-methylthymine can be mispaired with guanine. The type of the mutation generated, however, may be dependent on the size and type of the adduct as well as the DNA sequence.

Ionizing radiation and reactive oxygen species often oxidize guanine to produce 8-oxoguanine.

See also: Epigenetics

Exogenous DNA damage

Arrows indicates chromosomal breakages due to DNA damage.

Exogenous DNA damage is structural alteration of DNA caused by external environmental agents, including UV radiation, ionizing radiation, and chemical toxins.

Backbone damage

Ionizing radiation may produce highly reactive free radicals that can break the bonds in the DNA. Double-stranded breakages are especially damaging and hard to repair, producing translocation and deletion of part of a chromosome. Alkylating agents like mustard gas, diet, tobacco smoke may also cause breakages in the DNA backbone. Endogenous processes may also such as oxidative stress may also generate highly reactive oxygen species that can damage the DNA.

Crosslinking

Main article: Crosslinking of DNA

Covalent bonds between the bases of nucleotides in DNA, be they in the same strand or opposing strands, is referred to as crosslinking of DNA; crosslinking of DNA may affect both the replication and the transcription of DNA, and it may be caused by exposure to a variety of agents. Some naturally occurring chemicals may also promote crosslinking, such as psoralens after activation by UV radiation, and nitrous acid. Interstrand cross-linking (between two strands) causes more damage, as it blocks replication and transcription and can cause chromosomal breakages and rearrangements. Some crosslinkers such as cyclophosphamide, mitomycin C and cisplatin are used as anticancer chemotherapeutic because of their high degree of toxicity to proliferating cells.

Dimerization

Main article: Dimer

Dimerization consists of the bonding of two monomers to form an oligomer, such as the formation of pyrimidine dimers as a result of exposure to UV radiation, which promotes the formation of a cyclobutyl ring between adjacent thymines in DNA . These bulky bases would cause a distortion in the DNA helixes and can interfere with DNA replication and transcription. In human skin cells, thousands of dimers may be formed in a day due to normal exposure to sunlight. DNA polymerase η may help bypass these lesions in an error-free manner; however, individuals with defective DNA repair function, such as those with xeroderma pigmentosum, are sensitive to sunlight and may be prone to skin cancer.

Ethidium intercalated between two adenine-thymine base pairs

Clinically, whether a tumor has formed as a direct consequence of UV radiation is discernible via DNA sequencing analysis for the characteristic context-specific dimerization pattern that occurs due to excessive exposure to sunlight.

Intercalation between bases

Main article: Intercalation (biochemistry)

The planar structure of chemicals such as ethidium bromide and proflavine allows them to insert between bases in DNA. This insert causes the DNA's backbone to stretch and makes slippage in DNA during replication more likely to occur since the bonding between the strands is made less stable by the stretching. Forward slippage will result in deletion mutation, while reverse slippage will result in an insertion mutation. Also, the intercalation into DNA of anthracyclines such as daunorubicin and doxorubicin interferes with the functioning of the enzyme topoisomerase II, blocking replication as well as causing mitotic homologous recombination.^{[citation needed]}

Insertional mutagenesis

Main article: Insertional mutagenesis

Transposons and viruses or retrotransposons may insert DNA sequences into coding regions or functional elements of a gene and result in inactivation of the gene.^[40]

DNA repair pathway

DNA damage happens frequently, but DNA damage does not always become a mutation. Only after the repair mechanism has failed or inaccurate, allowing the damages to bypass then the mutation would happen, and potentially lead to diseases like cancer.^{[citation needed]}

Base excision repair

Base excision repair corrects the small, non-helix distorting lesions of DNA helix such as oxidative, deaminated, alkylation, as well as basic single abase damages.^[30] DNA glycosylase within the base excision repair mechanism recognizes those damages and cleaves the N-glycosidic bond, leave behind an abasic sites. DNA backbone would then be cut at that site by AP endonuclease, after that DNA polymerase fills the gap.

Nucleotide excision repair

Nucleotide excision repair removes bulky lesion such as CPDs^{[clarification needed]} and (6-4) pp from UV radiation, or damage from chemotherapeutic agents. There are two major branches of nucleotide excision repair: Globular genome nucleotide excision repair and transcription-coupled nucleotide excision repair. Globular Genome NER, the XPC, RAD23B and CETN2 protein complex scans for whole genome damage, once the damage is found, endonuclease such as XPF–ERCC1 and XPG will cut out the lesion from 5' to 3', and POL ε or XRCC1–LIG3 will carry out the gap filling synthesis and ligation.^[30]

Mismatch repair

Mismatch repair removes base mismatch that have arisen during replication and the insertion-deletion loop.^[41] Humans employ the MutSα heterodimer (MSH2/MSH6) to recognize the base mismatch.^[42] Once the mismatch is found, Exo1 carries out the 5' directed mismatch excision, which creates a gap later being filled by Polδ, RFC, and HMGB.^[43]

Adaptive mutagenesis mechanisms

Main article: Adaptive mutation

Adaptive mutagenesis has been defined as mutagenesis mechanisms that enable an organism to adapt to an environmental stress. Since the variety of environmental stresses is very broad, the mechanisms that enable it are also quite broad, as far as research on the field has shown. For instance, in bacteria, while modulation of the SOS response and endogenous prophage DNA synthesis has been shown to increase Acinetobacter baumannii resistance to ciprofloxacin. Resistance mechanisms are presumed to be linked to chromosomal mutation untransferable via horizontal gene transfer in some members of family Enterobacteriaceae, such as E. coli, Salmonella spp., Klebsiella spp., and Enterobacter spp. Chromosomal events, specially gene amplification, seem also to be relevant to this adaptive mutagenesis in bacteria.

Research in eukaryotic cells is much scarcer, but chromosomal events seem also to be rather relevant: while an ectopic intrachromosomal recombination has been reported to be involved in acquisition of resistance to 5-fluorocytosine in Saccharomyces cerevisiae, genome duplications have been found to confer resistance in S. cerevisiae to nutrient-poor environments.

Laboratory applications

Main article: Mutagenesis (molecular biology technique)

In the laboratory, mutagenesis is a technique by which DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organisms. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the function of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with altered properties, or enhanced or novel functions that may prove to be of use commercially. Mutant strains of organisms that have practical applications, or allow the molecular basis of particular cell function to be investigated, may also be produced.

Early methods of mutagenesis produced entirely random mutations; however, modern methods of mutagenesis are capable of producing site-specific mutations. Modern laboratory techniques used to generate these mutations include:

• Directed mutagenesis
• Site-directed mutagenesis/ PCR mutagenesis
• Insertional mutagenesis
• Signature tagged mutagenesis
• Transposon mutagenesis
• Sequence saturation mutagenesis