What is the average length of a woman's period?

Menstruation, also known as a period or monthly, is the regular discharge of blood and mucosal tissue (known as menses) from the inner lining of the uterus through the vagina. The first period usually begins between twelve and fifteen years of age, a point in time known as menarche. However, periods may occasionally start as young as eight years old and still be considered normal. The average age of the first period is generally later in the developing world, and earlier in the developed world. The typical length of time between the first day of one period and the first day of the next is 21 to 45 days in young women, and 21 to 31 days in adults (an average of 28 days). Bleeding usually lasts around 2 to 7 days. Menstruation stops occurring after menopause, which usually occurs between 45 and 55 years of age. Periods also stop during pregnancy and typically do not resume during the initial months of breastfeeding.
Up to 80% of women report having some symptoms prior to menstruation. Common signs and symptoms include acne, tender breasts, bloating, feeling tired, irritability, and mood changes. These may interfere with normal life, therefore qualifying as premenstrual syndrome, in 20 to 30% of women. In 3 to 8%, symptoms are severe.
A lack of periods, known as amenorrhea, is when periods do not occur by age 15 or have not occurred in 90 days. Other problems with the menstrual cycle include painful periods and abnormal bleeding such as bleeding between periods or heavy bleeding. Menstruation in other animals occur in primates (apes and monkeys).
The menstrual cycle occurs due to the rise and fall of hormones. This cycle results in the thickening of the lining of the uterus, and the growth of an egg, (which is required for pregnancy). The egg is released from an ovary around day fourteen in the cycle; the thickened lining of the uterus provides nutrients to an embryo after implantation. If pregnancy does not occur, the lining is released in what is known as menstruation.

Menstruation

The first menstrual period occurs after the onset of pubertal growth, and is called menarche. The average age of menarche is 12 to 15. However, it may start as early as eight. The average age of the first period is generally later in the developing world, and earlier in the developed world. The average age of menarche has changed little in the United States since the 1950s.

Menstruation is the most visible phase of the menstrual cycle and its beginning is used as the marker between cycles. The first day of menstrual bleeding is the date used for the last menstrual period (LMP). The typical length of time between the first day of one period and the first day of the next is 21 to 45 days in young women, and 21 to 31 days in adults (an average of 28 days).

Perimenopause is when fertility in a female declines, and menstruation occurs less regularly in the years leading up to the final menstrual period, when a female stops menstruating completely and is no longer fertile. The medical definition of menopause is one year without a period and typically occurs between 45 and 55 in Western countries.

During pregnancy and for some time after childbirth, menstruation does not occur. The average length of postpartum amenorrhoea is longer when breastfeeding; this is termed lactational amenorrhoea.

In most women, various physical changes are brought about by fluctuations in hormone levels during the menstrual cycle. This includes muscle contractions of the uterus (menstrual cramping) that can precede or accompany menstruation. Some may notice water retention, changes in sex drive, fatigue, breast tenderness, or nausea. Breast swelling and discomfort may be caused by water retention during menstruation. Usually, such sensations are mild, and some females notice very few physical changes associated with menstruation. A healthy diet, reduced consumption of salt, caffeine and alcohol, and regular exercise may be effective for women in controlling some symptoms. Severe symptoms that disrupt daily activities and functioning may be diagnosed as premenstrual dysphoric disorder. Symptoms before menstruation are known as premenstrual molimina.

Many women experience painful cramps, also known as dysmenorrhea, during menstruation. Pain results from ischemia and muscle contractions. Spiral arteries in the secretory endometrium constrict, resulting in ischemia to the secretory endometrium. This allows the uterine lining to slough off. The myometrium contracts spasmodically in order to push the menstrual fluid through the cervix and out of the vagina. The contractions are mediated by a release of prostaglandins.

Painful menstrual cramps that result from an excess of prostaglandin release are referred to as primary dysmenorrhea. Primary dysmenorrhea usually begins within a year or two of menarche, typically with the onset of ovulatory cycles. Treatments that target the mechanism of pain include non-steroidal anti-inflammatory drugs (NSAIDs) and hormonal contraceptives. NSAIDs inhibit prostaglandin production. With long-term treatment, hormonal birth control reduces the amount of uterine fluid/tissue expelled from the uterus. Thus resulting in shorter, less painful menstruation. These drugs are typically more effective than treatments that do not target the source of the pain (e.g. acetaminophen). Risk factors for primary dysmenorrhea include: early age at menarche, long or heavy menstrual periods, smoking, and a family history of dysmenorrhea. Regular physical activity may limit the severity of uterine cramps.

For many women, primary dysmenorrhea gradually subsides in late second generation. Pregnancy has also been demonstrated to lessen the severity of dysmenorrhea, when menstruation resumes. However, dysmenorrhea can continue until menopause. 5–15% of women with dysmenorrhea experience symptoms severe enough to interfere with daily activities.

Secondary dysmenorrhea is the diagnosis given when menstruation pain is a secondary cause to another disorder. Conditions causing secondary dysmenorrhea include endometriosis, uterine fibroids, and uterine adenomyosis. Rarely, congenital malformations, intrauterine devices, certain cancers, and pelvic infections cause secondary dysmenorrhea. Symptoms include pain spreading to hips, lower back and thighs, nausea, and frequent diarrhea or constipation. If the pain occurs between menstrual periods, lasts longer than the first few days of the period, or is not adequately relieved by the use of non-steroidal anti-inflammatory drugs (NSAIDs) or hormonal contraceptives, women should be evaluated for secondary causes of dysmenorrhea.

When severe pelvic pain and bleeding suddenly occur or worsen during a cycle, the woman or girl should be evaluated for ectopic pregnancy and spontaneous abortion. This evaluation begins with a pregnancy test and should be done as soon as unusual pain begins, because ectopic pregnancies can be life‑threatening.

In some cases, stronger physical and emotional or psychological sensations may interfere with normal activities, and include menstrual pain (dysmenorrhea), migraine headaches, and depression. Dysmenorrhea, or severe uterine pain, is particularly common for young females (one study found that 67.2% of girls aged 13–19 have it).

The medications in the non-steroidal anti-inflammatory drugs (NSAIDs) class are commonly used to relieve menstrual cramps. Some herbs are also claimed to help.

Some women experience emotional disturbances starting one or two weeks before their period, and stopping soon after the period has started. Symptoms may include mental tension, irritability, mood swings, and crying spells. Problems with concentration and memory may occur. There may also be depression or anxiety.

This is part of premenstrual syndrome (PMS) and is estimated to occur in 20 to 30% of women. In 3 to 8% it is severe.

More severe symptoms of anxiety or depression may be signs of premenstrual dysphoric disorder (PMDD). Rarely, in individuals who are susceptible, menstruation may be a trigger for menstrual psychosis.

Extreme psychological stress can also result in periods stopping.

The average volume of menstrual fluid during a monthly menstrual period is 35 milliliters (2.4 tablespoons of menstrual fluid) with 10–80 milliliters (1–6 tablespoons of menstrual fluid) considered typical. Menstrual fluid is the correct name for the flow, although many people prefer to refer to it as menstrual blood. Menstrual fluid contains some blood, as well as cervical mucus, vaginal secretions, and endometrial tissue. Menstrual fluid is reddish-brown, a slightly darker color than venous blood.

About half of menstrual fluid is blood. This blood contains sodium, calcium, phosphate, iron, and chloride, the extent of which depends on the woman. As well as blood, the fluid consists of cervical mucus, vaginal secretions, and endometrial tissue. Vaginal fluids in menses mainly contribute water, common electrolytes, organ moieties, and at least 14 proteins, including glycoproteins.

Many mature females notice blood clots during menstruation. These appear as clumps of blood that may look like tissue. If there are questions (for example, was there a miscarriage?), examination under a microscope can confirm if it was endometrial tissue or pregnancy tissue (products of conception) that was shed. Sometimes menstrual clots or shed endometrial tissue is incorrectly thought to indicate an early-term miscarriage of an embryo. An enzyme called plasmin – contained in the endometrium – tends to inhibit the blood from clotting.

The amount of iron lost in menstrual fluid is relatively small for most women. In one study, premenopausal women who exhibited symptoms of iron deficiency were given endoscopies. 86% of them actually had gastrointestinal disease and were at risk of being misdiagnosed simply because they were menstruating. Heavy menstrual bleeding, occurring monthly, can result in anemia.

There is a wide spectrum of differences in how women experience menstruation. There are several ways that someone's menstrual cycle can differ from the norm, any of which should be discussed with a doctor to identify the underlying cause:
There is a movement among gynecologists to discard the terms noted above, which although they are widely used, do not have precise definitions. Many now argue to describe menstruation in simple terminology, including:

Dysfunctional uterine bleeding is a hormonally caused bleeding abnormality. Dysfunctional uterine bleeding typically occurs in premenopausal women who do not ovulate normally (i.e. are anovulatory). All these bleeding abnormalities need medical attention; they may indicate hormone imbalances, uterine fibroids, or other problems. As pregnant women may bleed, a pregnancy test forms part of the evaluation of abnormal bleeding.

Women who had undergone female genital mutilation (particularly type III- infibulation) a practice common in parts of Africa, may experience menstrual problems, such as slow and painful menstruation, that is caused by the near-complete sealing off of the vagina.

Premature or delayed menarche should be investigated if menarche begins before 9 years, if menarche has not begun by age 15, if there is no breast development by age 13, or if there is no period by 3 years after the onset of breast development.

Sexual intercourse during menstruation does not cause damage in and of itself, but the woman's body is more vulnerable during this time. Vaginal pH is higher and thus less acidic than normal, the cervix is lower in its position, the cervical opening is more dilated, and the uterine endometrial lining is absent, thus allowing organisms direct access to the bloodstream through the numerous blood vessels that nourish the uterus. All these conditions increase the chance of infection during menstruation.

Hormonal contraception affects the frequency, duration, severity, volume, and regularity of menstruation and menstrual symptoms.

The most common form of hormonal contraception is the combined birth control pill, which contains both estrogen and progestogen. It is typically taken in 28-day cycles, 21 hormonal pills with either a 7-day break from pills, or 7 placebo pills during which the woman menstruates. Although the primary function of the pill is to prevent pregnancy, it may be used to treat some menstrual symptoms and syndromes which affect menstruation, such as polycystic ovary syndrome (PCOS), endometriosis, adenomyosis, amenorrhea, menstrual cramps, menstrual migraines, menorrhagia (excessive menstrual bleeding), menstruation-related or fibroid-related anemia and dysmenorrhea (painful menstruation). Using the combined birth control pill, it is also possible for a woman to delay or completely eliminate menstrual periods, a practice called menstrual suppression. Some women do this simply for convenience in the short-term, while others prefer to eliminate periods altogether when possible. This can be done either by skipping the placebo pills, or using an extended cycle combined oral contraceptive pill, which were first marketed in the U.S. in the early 2000s.

Injections such as depo-provera became available in the 1960s. When using progestogen implants, menstruation may be reduced to 3 or 4 menstrual periods per year.

Progestogen-only contraceptive pills (sometimes called the'mini pill') are taken continuously without a 7-day span of using placebo pills, and therefore menstrual periods are less likely to occur than with the combined pill with placebo pills. However, disturbance of the menstrual cycle is common with the mini-pill; 1/3-1/2 of women taking it will experience prolonged periods, and up to 70% experience break-through bleeding (metrorrhagia). Irregular and prolonged bleeding is the most common reason that women discontinue using the mini pill.

When the first birth control pill was being developed, the researchers were aware that they could use the contraceptive to space menstrual periods up to 90 days apart, but they settled on a 28-day cycle that would mimic a natural menstrual cycle and produce monthly periods. The intention behind this decision was the hope of the inventor, John Rock, to win approval for his invention from the Roman Catholic Church. That attempt failed, but the 28-day cycle remained the standard when the pill became available to the public.

There is debate among medical researchers about the potential long-term impacts of these practices upon female health. Some researchers point to the fact that historically, females have had far fewer menstrual periods throughout their lifetimes, a result of shorter life expectancies, as well as a greater length of time spent pregnant or breast-feeding, which reduced the number of periods experienced by females. These researchers believe that the higher number of menstrual periods by females in modern societies may have a negative impact upon their health. On the other hand, some researchers believe there is a greater potential for negative impacts from exposing females perhaps unnecessarily to regular low doses of synthetic hormones over their reproductive years.

Breastfeeding causes negative feedback to occur on pulse secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). Depending on the strength of the negative feedback, breastfeeding women may experience complete suppression of follicular development, follicular development but no ovulation, or normal menstrual cycles may resume. Suppression of ovulation is more likely when suckling occurs more frequently. The production of prolactin in response to suckling is important to maintaining lactational amenorrhea. On average, women who are fully breastfeeding whose infants suckle frequently experience a return of menstruation at fourteen and a half months postpartum. There is a wide range of response among individual breastfeeding women, however, with some experiencing return of menstruation at two months and others remaining amenorrheic for up to 42 months postpartum.

Menstruation is managed by menstruating women to avoid damage to clothing or to accord with norms of public life. Menstrual management practices range from medical suppression of menstruation, through wearing special garments or other items, washing or avoidance of washing, disposal and laundry of stained materials, to separation of menstruators to particular places or activities.

Menstrual products (also called "feminine hygiene" products) are made to absorb or catch menstrual blood. A number of different products are available - some are disposable, some are reusable. Where women can afford it, items used to absorb or catch menses are usually commercially manufactured products.

There are disposable products:

Reusable products include:

In developing countries, many poor women cannot afford commercial feminine hygiene products. Instead, they use materials found in the environment or other improvised materials. Absorption materials that may be used by women who cannot afford anything else include: sand, ash, small hole in earth, cloth, whole leaf, leaf fibre (such as water hyacinth, banana, papyrus, cotton fibre), paper (toilet paper, re-used newspaper, pulped and dried paper), animal pelt e.g. goat skin, double layer of underwear, skirt or sari.

Many religions have menstruation-related traditions, for example: Islam prohibits sexual contact with women during menstruation in the 2nd chapter of the Quran. Some scholars argue that menstruating women are in a state in which they are unable to maintain wudhu, and are therefore prohibited from touching the Arabic version of the Qur'an. Other biological and involuntary functions such as vomiting, bleeding, sexual intercourse, and going to the bathroom also invalidate one's wudhu. In Judaism, a woman during menstruation is called Niddah and may be banned from certain actions. For example, the Jewish Torah prohibits sexual intercourse with a menstruating woman. In Hinduism, menstruating women are traditionally considered ritually impure and given rules to follow. During menstruation, women are not allowed to “enter the kitchen and temples", wear flowers, have sex, touch other males or females.” Western civilization, which has been predominantly Christian, has a history of menstrual taboos.

Menstruation education is frequently taught in combination with sex education at school in Western countries, although girls may prefer their mothers to be the primary source of information about menstruation and puberty. Information about menstruation is often shared among friends and peers, which may promote a more positive outlook on puberty. The quality of menstrual education in a society determines the accuracy of people's understanding of the process. In many Western countries where menstruation is a taboo subject, girls tend to conceal the fact that they may be menstruating and struggle to ensure that they give no sign of menstruation. Effective educational programs are essential to providing children and adolescents with clear and accurate information about menstruation. Schools can be an appropriate place for menstrual education to take place. Programs led by peers or third-party agencies are another option.

Most female mammals have an estrous cycle, yet only ten primates species, four bats species, the elephant shrew, and the spiny mouse have a menstrual cycle. The relatedness of these groups suggests that four distinct evolutionary events have caused menstruation to arise.

Some anthropologists have questioned the energy cost of rebuilding the endometrium every fertility cycle. It has proposed that the energy savings of not having to continuously maintain the uterine lining more than offsets energy cost of having to rebuild the lining in the next fertility cycle, even in species such as humans where much of the lining is lost through bleeding (overt menstruation) rather than reabsorbed (covert menstruation).

Many have questioned the evolution of overt menstruation in humans and related species, speculating on what advantage there could be to losing blood associated with dismantling the endometrium, rather than absorbing it, as most mammals do. Humans do, in fact, reabsorb about two-thirds of the endometrium each cycle. Some work asserts that overt menstruation occurs not because it is beneficial in itself. Rather, the fetal development of these species requires a more developed endometrium, one which is too thick to reabsorb completely. There is a correlation between species that have overt menstruation to those that have a large uterus relative to the adult female body size.

Recent reviews suggest that menstruation itself is not an evolved, adaptive trait. Rather, it is an inherent consequence of spontaneous decidualization evolving as a derived trait from non-spontaneous decidualization.

The grandmother hypothesis is a hypothesis to explain the existence of menopause in human life history by identifying the adaptive value of extended kin networking.

Beginning in 1971, some research suggested that menstrual cycles of cohabiting human females became synchronized (menstrual synchrony). However, there is currently significant dispute as to whether menstrual synchrony exists. A 2013 review concluded that menstrual synchrony likely does not exist.

The menstrual cycle is the regular natural change that occurs in the female reproductive system (specifically the uterus and ovaries) that makes pregnancy possible. The cycle is required for the production of oocytes, and for the preparation of the uterus for pregnancy. Up to 80% of women report having some symptoms during the one to two weeks prior to menstruation. Common symptoms include acne, tender breasts, bloating, feeling tired, irritability and mood changes. These symptoms interfere with normal life and therefore qualify as premenstrual syndrome in 20 to 30% of women. In 3 to 8%, they are severe.
The first period usually begins between twelve and fifteen years of age, a point in time known as menarche. They may occasionally start as early as eight, and this onset may still be normal. The average age of the first period is generally later in the developing world and earlier in developed world. The typical length of time between the first day of one period and the first day of the next is 21 to 45 days in young women and 21 to 35 days in adults (an average of 28 days). Menstruation stops occurring after menopause which usually occurs between 45 and 55 years of age. Bleeding usually lasts around 2 to 7 days.
The menstrual cycle is governed by hormonal changes. These changes can be altered by using hormonal birth control to prevent pregnancy. Each cycle can be divided into three phases based on events in the ovary (ovarian cycle) or in the uterus (uterine cycle). The ovarian cycle consists of the follicular phase, ovulation, and luteal phase whereas the uterine cycle is divided into menstruation, proliferative phase, and secretory phase.

Menstrual cycle

Stimulated by gradually increasing amounts of estrogen in the follicular phase, discharges of blood (menses) flow stop, and the lining of the uterus thickens. Follicles in the ovary begin developing under the influence of a complex interplay of hormones, and after several days one or occasionally two become dominant (non-dominant follicles shrink and die). Approximately mid-cycle, 24–36 hours after the luteinizing hormone (LH) surges, the dominant follicle releases an ovocyte, in an event called ovulation. After ovulation, the ovocyte only lives for 24 hours or less without fertilization while the remains of the dominant follicle in the ovary become a corpus luteum; this body has a primary function of producing large amounts of progesterone. Under the influence of progesterone, the uterine lining changes to prepare for potential implantation of an embryo to establish a pregnancy. If implantation does not occur within approximately two weeks, the corpus luteum will involute, causing a sharp drop in levels of both progesterone and estrogen. The hormone drop causes the uterus to shed its lining in a process termed menstruation. Menstruation also occurs in closely related primates (apes and monkeys).

The average age of menarche is 12–15. They may occasionally start as early as eight, and this onset may still be normal. This first period often occurs later in the developing world than the developed world.

The average age of menarche is approximately 12.5 years in the United States, 12.7 in Canada, 12.9 in the UK and 13.1 years in Iceland. Factors such as genetics, diet and overall health can affect timing.

The cessation of menstrual cycles at the end of a woman's reproductive period is termed menopause. The average age of menopause in women is 52 years, with anywhere between 45 and 55 being common. Menopause before age 45 is considered "premature" in industrialised countries. Like the age of menarche, the age of menopause is largely a result of cultural and biological factors; however, illnesses, certain surgeries, or medical treatments may cause menopause to occur earlier than it might have otherwise.

The length of a woman's menstrual cycle typically varies somewhat, with some shorter cycles and some longer cycles. A woman who experiences variations of less than eight days between her longest cycles and shortest cycles is considered to have regular menstrual cycles. It is unusual for a woman to experience cycle length variations of more than four days. Length variation between eight and 20 days is considered as moderately irregular cycles. Variation of 21 days or more between a woman's shortest and longest cycle lengths is considered very irregular.

The average menstrual cycle lasts 28 days. The variability of menstrual cycle lengths is highest for women under 25 years of age and is lowest, that is, most regular, for ages 25 to 39. Subsequently, the variability increases slightly for women aged 40 to 44.

The luteal phase of the menstrual cycle is about the same length in most individuals (mean 14.13 days, standard deviation 1.41 days) whereas the follicular phase tends to show much more variability (log-normally distributed with 95% of individuals having follicular phases between 10.3 and 16.3 days). The follicular phase also seems to get significantly shorter with age (geometric mean 14.2 days in women aged 18–24 vs. 10.4 days in women aged 40–44).

Some women with neurological conditions experience increased activity of their conditions at about the same time during each menstrual cycle. For example, drops in estrogen levels have been known to trigger migraines, especially when the woman who suffers migraines is also taking the birth control pill. Many women with epilepsy have more seizures in a pattern linked to the menstrual cycle; this is called "catamenial epilepsy". Different patterns seem to exist (such as seizures coinciding with the time of menstruation, or coinciding with the time of ovulation), and the frequency with which they occur has not been firmly established. Using one particular definition, one group of scientists found that around one-third of women with intractable partial epilepsy has catamenial epilepsy. An effect of hormones has been proposed, in which progesterone declines and estrogen increases would trigger seizures. Recently, studies have shown that high doses of estrogen can cause or worsen seizures, whereas high doses of progesterone can act like an antiepileptic drug. Studies by medical journals have found that women experiencing menses are 1.68 times more likely to attempt suicide.

Mice have been used as an experimental system to investigate possible mechanisms by which levels of sex steroid hormones might regulate nervous system function. During the part of the mouse estrous cycle when progesterone is highest, the level of nerve-cell GABA receptor subtype delta was high. Since these GABA receptors are inhibitory, nerve cells with more delta receptors are less likely to fire than cells with lower numbers of delta receptors. During the part of the mouse estrous cycle when estrogen levels are higher than progesterone levels, the number of delta receptors decrease, increasing nerve cell activity, in turn increasing anxiety and seizure susceptibility.

Estrogen levels may affect thyroid behavior. For example, during the luteal phase (when estrogen levels are lower), the velocity of blood flow in the thyroid is lower than during the follicular phase (when estrogen levels are higher).

Among women living closely together, it was once thought that the onsets of menstruation tend to synchronize. This effect was first described in 1971, and possibly explained by the action of pheromones in 1998. Subsequent research has called this hypothesis into question.

Research indicates that women have a significantly higher likelihood of anterior cruciate ligament injuries in the pre-ovulatory stage, than post-ovulatory stage.

The most fertile period (the time with the highest likelihood of pregnancy resulting from sexual intercourse) covers the time from some 5 days before until 1 to 2 days after ovulation. In a 28‑day cycle with a 14‑day luteal phase, this corresponds to the second and the beginning of the third week. A variety of methods have been developed to help individual women estimate the relatively fertile and the relatively infertile days in the cycle; these systems are called fertility awareness.

There are many fertility testing methods, including urine test kits that detect the LH surge that occurs 24 to 36 hours before ovulation; these are known as ovulation predictor kits (OPKs). Computerized devices that interpret basal body temperatures, urinary test results, or changes in saliva are called fertility monitors. Fertility awareness methods that rely on cycle length records alone are called calendar-based methods. Methods that require observation of one or more of the three primary fertility signs (basal body temperature, cervical mucus, and cervical position) are known as symptoms-based methods.

A woman's fertility is also affected by her age. As a woman's total egg supply is formed in fetal life, to be ovulated decades later, it has been suggested that this long lifetime may make the chromatin of eggs more vulnerable to division problems, breakage, and mutation than the chromatin of sperm, which are produced continuously during a man's reproductive life. However, despite this hypothesis, a similar paternal age effect has also been observed.

As measured on women undergoing in vitro fertilization, a longer menstrual cycle length is associated with higher pregnancy and delivery rates, even after age adjustment. Delivery rates after IVF have been estimated to be almost doubled for women with a menstrual cycle length of more than 34 days compared with women with a menstrual cycle length shorter than 26 days. A longer menstrual cycle length is also significantly associated with better ovarian response to gonadotropin stimulation and embryo quality.

The different phases of the menstrual cycle correlate with women's moods. In some cases, hormones released during the menstrual cycle can cause behavioral changes in females; mild to severe mood changes can occur. The menstrual cycle phase and ovarian hormones may contribute to increased empathy in women. The natural shift of hormone levels during the different phases of the menstrual cycle has been studied in conjunction with test scores. When completing empathy exercises, women in the follicular stage of their menstrual cycle performed better than women in their midluteal phase. A significant correlation between progesterone levels and the ability to accurately recognize emotion was found. Performances on emotion recognition tasks were better when women had lower progesterone levels. Women in the follicular stage showed higher emotion recognition accuracy than their midluteal phase counterparts. Women were found to react more to negative stimuli when in midluteal stage over the women in the follicular stage, perhaps indicating more reactivity to social stress during that menstrual cycle phase. Overall, it has been found that women in the follicular phase demonstrated better performance in tasks that contain empathetic traits.

Fear response in women during two different points in the menstrual cycle has been examined. When estrogen is highest in the preovulatory stage, women are significantly better at identifying expressions of fear than women who were menstruating, which is when estrogen levels are lowest. The women were equally able to identify happy faces, demonstrating that the fear response was a more powerful response. To summarize, menstrual cycle phase and the estrogen levels correlates with women’s fear processing.

However, the examination of daily moods in women with measuring ovarian hormones may indicate a less powerful connection. In comparison to levels of stress or physical health, the ovarian hormones had less of an impact on overall mood. This indicates that while changes of ovarian hormones may influence mood, on a day-to-day level it does not influence mood more than other stressors do.

Sexual feelings and behaviors change during the menstrual cycle. Before and during ovulation, high levels of estrogen and androgens result in women having an increased interest in sexual activity. Unlike other animal species, women show interest in sex across all days of the menstrual cycle, regardless of fertility.

Behavior towards potential mating partners changes during different phases of the menstrual cycle. Near ovulation, women may have increased physical attraction and interest in attending social gatherings with men. During the fertile phase of the cycle, women appear to prefer males who are more masculine. The intensity of mate guarding differs across the phases of the cycle, with increased mate guarding occurring when women are fertile.

During the fertile phase, many women experience more attraction, fantasies and sexual interest for extra pair men but not for the primary partner. They also engage in extra-pair flirtations and demonstrate a preference for extra pair copulation.

Preferences for voice pitch change across the cycle. When seeking a short term mating partner, women may prefer a male with a low voice pitch, particularly during the fertile phase. During the late follicular phase, it is common for women demonstrate a preference for mates with a masculine, deep voice. Research has also been conducted on the attractiveness of the female voice throughout the cycle. During their most fertile phase of the menstrual cycle, there is some evidence that female voices are rated as significantly more attractive. This effect is not found with women on the birth control pill.

Women's preference for male's body odor can change across the menstrual cycle. Males who score highly on dominance have been rated as sexier by females during the fertile phase of the menstrual cycle. Additionally, during their most fertile phase of the menstrual cycle, women may show preference for the odor of symmetrical men. This effect is not found for women on the birth control pill. Also, during the late follicular and ovulatory phases, women prefer the scent of masculine men. The scent of androsterone (responsible for testosterone levels) is highly preferred by women during the peak of their fertility in the menstrual cycle. Moreover, women may demonstrate preference for men with a scent that indicates developmental stability.

With regard to women's smell across the cycle, some evidence indicates that men use olfactory cues in order to know if a woman is ovulating. Using a rating of women's odors, women who are ovulating have been rated as more attractive by men. Men demonstrate preferences for the scent of fertile women.

Preferences for facial features in mates can also change across the cycle. There has been no difference found in preference for long-term mating partners during the menstrual cycle; however, those seeking a short-term relationship were more likely to choose a partner with more masculine features than usual. This was found to be the case especially during the woman's high conception risk stage and when salivary testosterone was high. However, when women are in the luteal (non-fertile) phase, they tend to prefer men (and females) with more feminine faces. A preference is also shown for self-resembling faces and apparent health in faces during the luteal phase of the cycle. Apparent health preferences were found to be strongest when progesterone levels were high. Additionally, during the fertile phase, many women show a preference for men with darker skin pigmentation. Research on facial symmetry is mixed.

Preferences for body features can change during the fertile phase of the cycle. Women seeking a short-term partner demonstrate a preference for taller and muscular males. Women also show preferences of males with masculine bodies at peak fertility. Mixed research has been found regarding body symmetry preferences throughout different phases of the cycle.

In short term mates, during the fertile phase, women may show more attraction to dominant men who display social presence. For long-term mates, shifts in desired trait preferences do not occur throughout the cycle.

Females have been found to experience different eating habits at different stages of their menstrual cycle, with food intake being higher during the luteal phase than the follicular phase. Food intake increases by approximately 10% during the luteal phase compared to the follicular phase.

Various studies have shown that during the luteal phase woman consume more carbohydrates, proteins and fats and that 24-hour energy expenditure shows increases between 2.5-11.5%. The increasing intake during the luteal phase may be related to higher preferences for sweet and fatty foods, which occurs naturally and is enhanced during the luteal phases of the menstrual cycle. This is due to the higher metabolic demand during this phase. In particular, women tend to show a cravings for chocolate, with higher cravings during the luteal phase.

Females with premenstrual syndrome (PMS) report changes in appetite across the menstrual cycle more than non-sufferers of PMS, possibly due to their oversensitivity to changes in hormone levels. In women with PMS, food intake is higher in the luteal phase than follicular. The remaining symptoms of PMS, including mood changes and physical symptoms, also occur during the luteal phase. No difference for preference of food types has been found between PMS sufferers and non-sufferers.

The different levels of ovarian hormones at different stages of the cycle have been used to explain eating behaviour changes. Progesterone has been shown to promote fat storage, causing a higher intake of fatty foods during the luteal phase when progesterone levels are higher. Additionally, with a high estrogen level dopamine is ineffective in converting to noradrenaline, a hormone which promotes eating, therefore decreasing appetite. In humans, the level of these ovarian hormones during the menstrual cycle have been found to influence binge eating.

It is theorized that the use of birth control pills should affect eating behaviour as they minimise or remove the fluctuations in hormone levels. The neurotransmitter serotonin is also thought to play a role in food intake. Serotonin is responsible for inhibiting eating and controlling meal size, among other things, and is modulated in part by ovarian hormones.

A number of factors affect whether dieting will affect these menstrual processes: age, weight loss and the diet itself. First, younger women are likely to experience menstrual irregularities due to their diet. Second, menstrual abnormalities are more likely with more weight loss. For example, anovulatory cycles can occur as a result of adopting a restricted diet, as well as engaging in a high amount of exercise. Finally, the cycle is affected more by a vegetarian diet compared to a non-vegetarian diet.

Studies investigating effects of the menstrual cycle on alcohol consumption have found mixed evidence. However, some evidence suggests that individuals consume more alcohol during the luteal stage, especially if these individuals are heavy drinkers or have a family history of alcohol abuse.

The level of substance abuse increases with PMS, mostly with addictive substances such as nicotine, tobacco and cocaine. One theory behind this suggests this higher level of substance abuse is due to decreased self-control as a result of the higher metabolic demands during the luteal phase.

Infrequent or irregular ovulation is called "oligoovulation". The absence of ovulation is called "anovulation". Normal menstrual flow can occur without ovulation preceding it: an anovulatory cycle. In some cycles, follicular development may start but not be completed; nevertheless, estrogens will be formed and stimulate the uterine lining. Anovulatory flow resulting from a very thick endometrium caused by prolonged, continued high estrogen levels is called "estrogen breakthrough bleeding". Anovulatory bleeding triggered by a sudden drop in estrogen levels is called withdrawal bleeding. Anovulatory cycles commonly occur before menopause (perimenopause) and in women with polycystic ovary syndrome.

Very little flow (less than 10 ml) is called "hypomenorrhea". Regular cycles with intervals of 21 days or fewer are "polymenorrhea"; frequent but irregular menstruation is known as "metrorrhagia". Sudden heavy flows or amounts greater than 80 ml are termed "menorrhagia". Heavy menstruation that occurs frequently and irregularly is "menometrorrhagia". The term for cycles with intervals exceeding 35 days is "oligomenorrhea". Amenorrhea refers to more than three to six months without menses (while not being pregnant) during a woman's reproductive years.
The term for painful periods is "Dysmenorrhea".

The menstrual cycle can be described by the ovarian or uterine cycle. The ovarian cycle describes changes that occur in the follicles of the ovary whereas the uterine cycle describes changes in the endometrial lining of the uterus. Both cycles can be divided into three phases. The ovarian cycle consists of the follicular phase, ovulation, and the luteal phase, whereas the uterine cycle consists of menstruation, proliferative phase, and secretory phase.

The follicular phase is the first part of the ovarian cycle. During this phase, the ovarian follicles mature and get ready to release an egg. The latter part of this phase overlaps with the proliferative phase of the uterine cycle.

Through the influence of a rise in follicle stimulating hormone (FSH) during the first days of the cycle, a few ovarian follicles are stimulated. These follicles, which were present at birth and have been developing for the better part of a year in a process known as folliculogenesis, compete with each other for dominance. Under the influence of several hormones, all but one of these follicles will stop growing, while one dominant follicle in the ovary will continue to maturity. The follicle that reaches maturity is called a tertiary or Graafian follicle, and it contains the ovum.

Ovulation is the second phase of the ovarian cycle in which a mature egg is released from the ovarian follicles into the oviduct. During the follicular phase, estradiol suppresses release of luteinizing hormone (LH) from the anterior pituitary gland. When the egg has nearly matured, levels of estradiol reach a threshold above which this effect is reversed and estrogen stimulates the production of a large amount of LH. This process, known as the LH surge, starts around day 12 of the average cycle and may last 48 hours.

The exact mechanism of these opposite responses of LH levels to estradiol is not well understood. In animals, a gonadotropin-releasing hormone (GnRH) surge has been shown to precede the LH surge, suggesting that estrogen's main effect is on the hypothalamus, which controls GnRH secretion. This may be enabled by the presence of two different estrogen receptors in the hypothalamus: estrogen receptor alpha, which is responsible for the negative feedback estradiol-LH loop, and estrogen receptor beta, which is responsible for the positive estradiol-LH relationship. However, in humans it has been shown that high levels of estradiol can provoke 32 increases in LH, even when GnRH levels and pulse frequencies are held constant, suggesting that estrogen acts directly on the pituitary to provoke the LH surge.

The release of LH matures the egg and weakens the wall of the follicle in the ovary, causing the fully developed follicle to release its secondary oocyte. If it is fertilized by a sperm, the secondary oocyte promptly matures into an ootid and then becomes a mature ovum. If it is not fertilized by a sperm, the secondary oocyte will degenerate. The mature ovum has a diameter of about 0.2 mm.

Which of the two ovaries—left or right—ovulates appears essentially random; no known left and right co-ordination exists. Occasionally, both ovaries will release an egg; if both eggs are fertilized, the result is fraternal twins.

After being released from the ovary, the egg is swept into the fallopian tube by the fimbria, which is a fringe of tissue at the end of each fallopian tube. After about a day, an unfertilized egg will disintegrate or dissolve in the fallopian tube.

Fertilization by a spermatozoon, when it occurs, usually takes place in the ampulla, the widest section of the fallopian tubes. A fertilized egg immediately begins the process of embryogenesis, or development. The developing embryo takes about three days to reach the uterus and another three days to implant into the endometrium. It has usually reached the blastocyst stage at the time of implantation.

In some women, ovulation features a characteristic pain called "mittelschmerz" (German term meaning "middle pain"). The sudden change in hormones at the time of ovulation sometimes also causes light mid-cycle blood flow.

The luteal phase is the final phase of the ovarian cycle and it corresponds to the secretory phase of the uterine cycle. During the luteal phase, the pituitary hormones FSH and LH cause the remaining parts of the dominant follicle to transform into the corpus luteum, which produces progesterone. The increased progesterone in the adrenals starts to induce the production of estrogen. The hormones produced by the corpus luteum also suppress production of the FSH and LH that the corpus luteum needs to maintain itself. Consequently, the level of FSH and LH fall quickly over time, and the corpus luteum subsequently atrophies. Falling levels of progesterone trigger menstruation and the beginning of the next cycle. From the time of ovulation until progesterone withdrawal has caused menstruation to begin, the process typically takes about two weeks, with 14 days considered normal. For an individual woman, the follicular phase often varies in length from cycle to cycle; by contrast, the length of her luteal phase will be fairly consistent from cycle to cycle.

The loss of the corpus luteum is prevented by fertilization of the egg. The syncytiotrophoblast, which is the outer layer of the resulting embryo-containing structure (the blastocyst) and later also becomes the outer layer of the placenta, produces human chorionic gonadotropin (hCG), which is very similar to LH and which preserves the corpus luteum. The corpus luteum can then continue to secrete progesterone to maintain the new pregnancy. Most pregnancy tests look for the presence of hCG.

The uterine cycle has three phases: menses, proliferative, secretory.

Menstruation (also called menstrual bleeding, menses, catamenia or a period) is the first phase of the uterine cycle. The flow of menses normally serves as a sign that a woman has not become pregnant. (However, this cannot be taken as certainty, as a number of factors can cause bleeding during pregnancy; some factors are specific to early pregnancy, and some can cause heavy flow.)

"Eumenorrhea" denotes normal, regular menstruation that lasts for a few days (usually 3 to 5 days, but anywhere from 2 to 7 days is considered normal). The average blood loss during menstruation is 35 milliliters with 10–80 ml considered normal. Women who experience Menorrhagia are more susceptible to iron deficiency than the average person. An enzyme called plasmin inhibits clotting in the menstrual fluid.

Painful cramping in the abdomen, back, or upper thighs is common during the first few days of menstruation. Severe uterine pain during menstruation is known as dysmenorrhea, and it is most common among adolescents and younger women (affecting about 67.2% of adolescent females). When menstruation begins, symptoms of premenstrual syndrome (PMS) such as breast tenderness and irritability generally decrease. Many sanitary products are marketed to women for use during their menstruation.

The proliferative phase is the second phase of the uterine cycle when estrogen causes the lining of the uterus to grow, or proliferate, during this time. As they mature, the ovarian follicles secrete increasing amounts of estradiol, and estrogen. The estrogens initiate the formation of a new layer of endometrium in the uterus, histologically identified as the proliferative endometrium. The estrogen also stimulates crypts in the cervix to produce fertile cervical mucus, which may be noticed by women practicing fertility awareness.

The secretory phase is the final phase of the uterine cycle and it corresponds to the luteal phase of the ovarian cycle. During the secretory phase, the corpus luteum produces progesterone, which plays a vital role in making the endometrium receptive to implantation of the blastocyst and supportive of the early pregnancy, by increasing blood flow and uterine secretions and reducing the contractility of the smooth muscle in the uterus; it also has the side effect of raising the woman's basal body temperature.

While some forms of birth control do not affect the menstrual cycle, hormonal contraceptives work by disrupting it. Progestogen negative feedback decreases the pulse frequency of gonadotropin-releasing hormone (GnRH) release by the hypothalamus, which decreases the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the anterior pituitary. Decreased levels of FSH inhibit follicular development, preventing an increase in estradiol levels. Progestogen negative feedback and the lack of estrogen positive feedback on LH release prevent a mid-cycle LH surge. Inhibition of follicular development and the absence of a LH surge prevent ovulation.

The degree of ovulation suppression in progestogen-only contraceptives depends on the progestogen activity and dose. Low dose progestogen-only contraceptives—traditional progestogen only pills, subdermal implants Norplant and Jadelle, and intrauterine system Mirena—inhibit ovulation in about 50% of cycles and rely mainly on other effects, such as thickening of cervical mucus, for their contraceptive effectiveness. Intermediate dose progestogen-only contraceptives—the progestogen-only pill Cerazette and the subdermal implant Nexplanon—allow some follicular development but more consistently inhibit ovulation in 97–99% of cycles. The same cervical mucus changes occur as with very low-dose progestogens. High-dose, progestogen-only contraceptives—the injectables Depo-Provera and Noristerat—completely inhibit follicular development and ovulation.

Combined hormonal contraceptives include both an estrogen and a progestogen. Estrogen negative feedback on the anterior pituitary greatly decreases the release of FSH, which makes combined hormonal contraceptives more effective at inhibiting follicular development and preventing ovulation. Estrogen also reduces the incidence of irregular breakthrough bleeding. Several combined hormonal contraceptives—the pill, NuvaRing, and the contraceptive patch—are usually used in a way that causes regular withdrawal bleeding. In a normal cycle, menstruation occurs when estrogen and progesterone levels drop rapidly. Temporarily discontinuing use of combined hormonal contraceptives (a placebo week, not using patch or ring for a week) has a similar effect of causing the uterine lining to shed. If withdrawal bleeding is not desired, combined hormonal contraceptives may be taken continuously, although this increases the risk of breakthrough bleeding.

Breastfeeding causes negative feedback to occur on pulse secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). Depending on the strength of the negative feedback, breastfeeding women may experience complete suppression of follicular development, but no ovulation, or normal menstrual cycle may resume. Suppression of ovulation is more likely when suckling occurs more frequently. The production of prolactin in response to suckling is important to maintaining lactational amenorrhea. On average, women who are fully breastfeeding whose infants suckle frequently experience a return of menstruation at fourteen and a half months postpartum. There is a wide range of response among individual breastfeeding women, however, with some experiencing return of menstruation at two months and others remaining amenorrheic for up to 42 months postpartum.

The word "menstruation" is etymologically related to "moon". The terms "menstruation" and "menses" are derived from the Latin "mensis" (month), which in turn relates to the Greek "mene" (moon) and to the roots of the English words "month" and "moon".

Even though the average length of the human menstrual cycle is similar to that of the lunar cycle, in modern humans there is no relation between the two. The relationship is believed to be a coincidence. Light exposure does not appear to affect the menstrual cycle in humans. A meta-analysis of studies from 1996 showed no correlation between the human menstrual cycle and the lunar cycle, nor did data analysed by period-tracking app Clue, submitted by 1.5m women, of 7.5m menstrual cycles.

Dogon villagers did not have electric lighting and spent most nights outdoors, talking and sleeping, so they were apparently an ideal population for detecting a lunar influence; none was found.

In a number of countries, mainly in Asia, legislation or corporate practice has introduced formal menstrual leave to provide women with either paid or unpaid leave of absence from their employment while they are menstruating. Countries with policies include Japan, Taiwan, Indonesia, and South Korea. The practice is controversial due to concerns that it bolsters the perception of women as weak, inefficient workers, as well as concerns that it is unfair to men.


Who first identified the process of oligonucleotide synthesis?

In the 1960s, groups led by R. Letsinger and C. Reese developed a phosphotriester approach. The defining difference from the phosphodiester approach was the protection of the phosphate moiety in the building block 1 (Scheme 4) and in the product 3 with 2-cyanoethyl group. This precluded the formation of oligonucleotides branched at the internucleosidic phosphate. The higher selectivity of the method allowed the use of more efficient coupling agents and catalysts, which dramatically reduced the length of the synthesis. The method, initially developed for the solution-phase synthesis, was also implemented on low-cross-linked "popcorn" polystyrene, and later on controlled pore glass (CPG, see "Solid support material" below), which initiated a massive research effort in solid-phase synthesis of oligonucleotides and eventually led to the automation of the oligonucleotide chain assembly.

Oligonucleotide synthesis

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA only in a 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is, most often, carried out in the opposite, 3' to 5' direction. Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. LNA or BNA.

To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product (see Synthetic cycle below). The process has been fully automated since the late 1970s. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized. Products are often isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15–25 bases in length.

Oligonucleotides find a variety of applications in molecular biology and medicine. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

The evolution of oligonucleotide synthesis saw four major methods of the formation of internucleosidic linkages and has been reviewed in the literature in great detail.

In the early 1950s, Alexander Todd’s group pioneered H-phosphonate and phosphate triester methods of oligonucleotide synthesis. The reaction of compounds 1 and 2 to form H-phosphonate diester 3 is an H-phosphonate coupling in solution while that of compounds 4 and 5 to give 6 is a phosphotriester coupling (see phosphotriester synthesis below).

Thirty years later, this work inspired, independently, two research groups to adopt the H-phosphonate chemistry to the solid-phase synthesis using nucleoside H-phosphonate monoesters 7 as building blocks and pivaloyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), and other compounds as activators. The practical implementation of H-phosphonate method resulted in a very short and simple synthetic cycle consisting of only two steps, detritylation and coupling (Scheme 2). Oxidation of internucleosidic H-phosphonate diester linkages in 8 to phosphodiester linkages in 9 with a solution of iodine in aqueous pyridine is carried out at the end of the chain assembly rather than as a step in the synthetic cycle. If desired, the oxidation may be carried out under anhydrous conditions. Alternatively, 8 can be converted to phosphorothioate 10 or phosphoroselenoate 11 (X = Se), or oxidized by CCl in the presence of primary or secondary amines to phosphoramidate analogs 12. The method is very convenient in that various types of phosphate modifications (phosphate/phosphorothioate/phosphoramidate) may be introduced to the same oligonucleotide for modulation of its properties.

Most often, H-phosphonate building blocks are protected at the 5'-hydroxy group and at the amino group of nucleic bases A, C, and G in the same manner as phosphoramidite building blocks (see below). However, protection at the amino group is not mandatory.

In the 1950s, Har Gobind Khorana and co-workers developed a phosphodiester method where 3’-"O"-acetylnucleoside-5’-"O"-phosphate 2 (Scheme 3) was activated with "N","N"-dicyclohexylcarbodiimide (DCC) or 4-toluenesulfonyl chloride (Ts-Cl). The activated species were reacted with a 5’-"O"-protected nucleoside 1 to give a protected dinucleoside monophosphate 3. Upon the removal of 3’-"O"-acetyl group using base-catalyzed hydrolysis, further chain elongation was carried out. Following this methodology, sets of tri- and tetradeoxyribonucleotides were synthesized and were enzymatically converted to longer oligonucleotides, which allowed elucidation of the genetic code. The major limitation of the phosphodiester method consisted in the formation of pyrophosphate oligomers and oligonucleotides branched at the internucleosidic phosphate. The method seems to be a step back from the more selective chemistry described earlier; however, at that time, most phosphate-protecting groups available now had not yet been introduced. The lack of the convenient protection strategy necessitated taking a retreat to a slower and less selective chemistry to achieve the ultimate goal of the study.

In the 1970s, substantially more reactive P(III) derivatives of nucleosides, 3'-"O"-chlorophosphites, were successfully used for the formation of internucleosidic linkages. This led to the discovery of the phosphite triester methodology. The group led by M. Caruthers took the advantage of less aggressive and more selective 1"H"-tetrazolidophosphites and implemented the method on solid phase. Very shortly after, the workers from the same group further improved the method by using more stable nucleoside phosphoramidites as building blocks. The use of 2-cyanoethyl phosphite-protecting group in place of a less user-friendly methyl group led to the nucleoside phosphoramidites currently used in oligonucleotide synthesis (see Phosphoramidite building blocks below). Many later improvements to the manufacturing of building blocks, oligonucleotide synthesizers, and synthetic protocols made the phosphoramidite chemistry a very reliable and expedient method of choice for the preparation of synthetic oligonucleotides.

As mentioned above, the naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford an expeditious synthetic preparation of oligonucleotides in high yields. The selectivity and the rate of the formation of internucleosidic linkages is dramatically improved by using 3'-"O"-("N","N"-diisopropyl phosphoramidite) derivatives of nucleosides (nucleoside phosphoramidites) that serve as building blocks in phosphite triester methodology. To prevent undesired side reactions, all other functional groups present in nucleosides have to be rendered unreactive (protected) by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired oligonucleotides. Below, the protecting groups currently used in commercially available and most common nucleoside phosphoramidite building blocks are briefly reviewed:

The protection of the exocyclic amino groups has to be orthogonal to that of the 5'-hydroxy group because the latter is removed at the end of each synthetic cycle. The simplest to implement, and hence the most widely used, strategy is to install a base-labile protection group on the exocyclic amino groups. Most often, two protection schemes are used.Non-nucleoside phosphoramidites are the phosphoramidite reagents designed to introduce various functionalities at the termini of synthetic oligonucleotides or between nucleotide residues in the middle of the sequence. In order to be introduced inside the sequence, a non-nucleosidic modifier has to possess at least two hydroxy groups, one of which is often protected with the DMT group while the other bears the reactive phosphoramidite moiety.

Non-nucleosidic phosphoramidites are used to introduce desired groups that are not available in natural nucleosides or that can be introduced more readily using simpler chemical designs. A very short selection of commercial phosphoramidite reagents is shown in Scheme for the demonstration of the available structural and functional diversity. These reagents serve for the attachment of 5'-terminal phosphate (1), NH (2), SH (3), aldehydo (4), and carboxylic groups (5), CC triple bonds (6), non-radioactive labels and quenchers (exemplified by 6-FAM amidite 7 for the attachment of fluorescein and dabcyl amidite 8, respectively), hydrophilic and hydrophobic modifiers (exemplified by hexaethyleneglycol amidite 9 and cholesterol amidite 10, respectively), and biotin amidite 11.

Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues to the 5'-terminus of the growing chain until the desired sequence is assembled. Each addition is referred to as a synthetic cycle (Scheme 5) and consists of four chemical reactions:

The DMT group is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene). The orange-colored DMT cation formed is washed out; the step results in the solid support-bound oligonucleotide precursor bearing a free 5'-terminal hydroxyl group.
It is worth remembering that conducting detritylation for an extended time or with stronger than recommended solutions of acids leads to depurination of solid support-bound oligonucleotide and thus reduces the yield of the desired full-length product.

A 0.02–0.2 M solution of nucleoside phosphoramidite (or a mixture of several phosphoramidites) in acetonitrile is activated by a 0.2–0.7 M solution of an acidic azole catalyst, 1"H"-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. A more extensive information on the use of various coupling agents in oligonucleotide synthesis can be found in a recent review. The mixing is usually very brief and occurs in fluid lines of oligonucleotide synthesizers (see below) while the components are being delivered to the reactors containing solid support. The activated phosphoramidite in 1.5 – 20-fold excess over the support-bound material is then brought in contact with the starting solid support (first coupling) or a support-bound oligonucleotide precursor (following couplings) whose 5'-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage. The coupling of 2'-deoxynucleoside phosphoramidites is very rapid and requires, on small scale, about 20 s for its completion. In contrast, sterically hindered 2'-"O"-protected ribonucleoside phosphoramidites require 5-15 min to be coupled in high yields. The reaction is also highly sensitive to the presence of water, particularly when dilute solutions of phosphoramidites are used, and is commonly carried out in anhydrous acetonitrile. Generally, the larger the scale of the synthesis, the lower the excess and the higher the concentration of the phosphoramidites is used. In contrast, the concentration of the activator is primarily determined by its solubility in acetonitrile and is irrespective of the scale of the synthesis. Upon the completion of the coupling, any unbound reagents and by-products are removed by washing.

The capping step is performed by treating the solid support-bound material with a mixture of acetic anhydride and 1-methylimidazole or, less often, DMAP as catalysts and, in the phosphoramidite method, serves two purposes.The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. Oxidation may be carried out under anhydrous conditions using tert-Butyl hydroperoxide or, more efficiently, (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). The step of oxidation may be substituted with a sulfurization step to obtain oligonucleotide phosphorothioates (see "Oligonucleotide phosphorothioates and their synthesis" below). In the latter case, the sulfurization step is best carried out prior to capping.

In solid-phase synthesis, an oligonucleotide being assembled is covalently bound, via its 3'-terminal hydroxy group, to a solid support material and remains attached to it over the entire course of the chain assembly. The solid support is contained in columns whose dimensions depend on the scale of synthesis and may vary between 0.05 mL and several liters. The overwhelming majority of oligonucleotides are synthesized on small scale ranging from 10 nmol to 1 μmol. More recently, high-throughput oligonucleotide synthesis where the solid support is contained in the wells of multi-well plates (most often, 96 or 384 wells per plate) became a method of choice for parallel synthesis of oligonucleotides on small scale. At the end of the chain assembly, the oligonucleotide is released from the solid support and is eluted from the column or the well.

In contrast to organic solid-phase synthesis and peptide synthesis, the synthesis of oligonucleotides proceeds best on non-swellable or low-swellable solid supports. The two most often used solid-phase materials are controlled pore glass (CPG) and macroporous polystyrene (MPPS).To make the solid support material suitable for oligonucleotide synthesis, non-nucleosidic linkers or nucleoside succinates are covalently attached to the reactive amino groups in aminopropyl CPG, LCAA CPG, or aminomethyl MPPS. The remaining unreacted amino groups are capped with acetic anhydride. Typically, three conceptually different groups of solid supports are used.

Oligonucleotide phosphorothioates (OPS) are modified oligonucleotides where one of the oxygen atoms in the phosphate moiety is replaced by sulfur. Only the phosphorothioates having sulfur at a non-bridging position as shown in figure are widely used and are available commercially. The replacement of the non-bridging oxygen with sulfur creates a new center of chirality at phosphorus. In a simple case of a dinucleotide, this results in the formation of a diastereomeric pair of S- and R-dinucleoside monophosphorothioates whose structures are shown in Figure. In an "n"-mer oligonucleotide where all ("n" – 1) internucleosidic linkages are phosphorothioate linkages, the number of diastereomers "m" is calculated as "m" = 2. Being non-natural analogs of nucleic acids, OPS are substantially more stable towards hydrolysis by nucleases, the class of enzymes that destroy nucleic acids by breaking the bridging P-O bond of the phosphodiester moiety. This property determines the use of OPS as antisense oligonucleotides in "in vitro" and "in vivo" applications where the extensive exposure to nucleases is inevitable. Similarly, to improve the stability of siRNA, at least one phosphorothioate linkage is often introduced at the 3'-terminus of both sense and antisense strands. In chirally pure OPS, all-Sp diastereomers are more stable to enzymatic degradation than their all-Rp analogs. However, the preparation of chirally pure OPS remains a synthetic challenge. In laboratory practice, mixtures of diastereomers of OPS are commonly used.

Synthesis of OPS is very similar to that of natural oligonucleotides. The difference is that the oxidation step is replaced by sulfur transfer reaction (sulfurization) and that the capping step is performed after the sulfurization. Of many reported reagents capable of the efficient sulfur transfer, only three are commercially available:In the past, oligonucleotide synthesis was carried out manually in solution or on solid phase. The solid phase synthesis was implemented using, as containers for the solid phase, miniature glass columns similar in their shape to low-pressure chromatography columns or syringes equipped with porous filters.
Currently, solid-phase oligonucleotide synthesis is carried out automatically using computer-controlled instruments (oligonucleotide synthesizers) and is technically implemented in column, multi-well plate, and array formats. The column format is best suited for research and large scale applications where a high-throughput is not required. Multi-well plate format is designed specifically for high-throughput synthesis on small scale to satisfy the growing demand of industry and academia for synthetic oligonucleotides. A number of oligonucleotide synthesizers for small scale synthesis and medium to large scale synthesis are available commercially.

In March 1982 a practical course was hosted by the Department of Biochemistry, Technische Hochschule Darmstadt, Germany. M.H. Caruthers, M.J. Gait, H.G. Gassen, H.Koster, K. Itakura, and C. Birr among others attended. The program comprised practical work, lectures, and seminars on solid-phase chemical synthesis of oligonucleotides. A select group of 15 students attended and had an unprecedented opportunity to be instructed by the esteemed teaching staff.
Along with manual exercises, several prominent automation companies attended the course. Biosearch of Novato, CA, Genetic Design of Watertown, MA, were two of several companies to demonstrate automated synthesizers at the course. Biosearch presented their new SAM I synthesizer. The Genetic Design had developed their synthesizer from the design of its sister companies (Sequemat) solid phase peptide sequencer. The Genetic Design arranged with Dr Christian Birr (Max-Planck-Institute for Medical Research) a week before the event to convert his solid phase sequencer into the semi-automated synthesizer. The team led by Dr Alex Bonner and Rick Neves converted the unit and transported it to Darmstadt for the event and installed into the Biochemistry lab at the Technische Hochschule. As the system was semi-automatic, the user injected the next base to be added to the growing sequence during each cycle. The system worked well and produced a series of test tubes filled with bright red trityl color indicating complete coupling at each step. This system was later fully automated by inclusion of an auto injector and was designated the Model 25A.

Large scale oligonucleotide synthesizers were often developed by augmenting the capabilities of a preexisting instrument platform. One of the first mid scale synthesizers appeared in the late 1980s, manufactured by the Biosearch company in Novato, CA (The 8800). This platform was originally designed as a peptide synthesizer and made use of a fluidized bed reactor essential for accommodating the swelling characteristics of polystyrene supports used in the Merrifield methodology. Oligonucleotide synthesis involved the use of CPG (controlled pore glass) which is a rigid support and is more suited for column reactors as described above. The scale of the 8800 was limited to the flow rate required to fluidize the support. Some novel reactor designs as well as higher than normal pressures enabled the 8800 to achieve scales that would prepare 1 mmole of oligonucleotide. In the mid 1990s several companies developed platforms that were based on semi-preparative and preparative liquid chromatographs. These systems were well suited for a column reactor approach. In most cases all that was required was to augment the number of fluids that could be delivered to the column. Oligo synthesis requires a minimum of 10 and liquid chromatographs usually accommodate 4. This was an easy design task and some semi-automatic strategies worked without any modifications to the preexisting LC equipment. PerSeptive Biosystems as well as Pharmacia (GE) were two of several companies that developed synthesizers out of liquid chromatographs. Genomic Technologies, Inc. was one of the few companies to develop a large scale oligonucleotide synthesizer that was, from the ground up, an oligonucleotide synthesizer. The initial platform called the VLSS for very large scale synthesizer utilized large Pharmacia liquid chromatograph columns as reactors and could synthesize up to 75 millimoles of material. Many oligonucleotide synthesis factories designed and manufactured their own custom platforms and little is known due to the designs being proprietary. The VLSS design continued to be refined and is continued in the QMaster synthesizer which is a scaled down platform providing milligram to gram amounts of synthetic oligonucleotide.

The current practices of synthesis of chemically modified oligonucleotides on large scale have been recently reviewed.

One may visualize an oligonucleotide microarray as a miniature multi-well plate where physical dividers between the wells (plastic walls) are intentionally removed. With respect to the chemistry, synthesis of oligonucleotide microarrays is different from the conventional oligonucleotide synthesis in two respects:After the completion of the chain assembly, the solid support-bound oligonucleotide is fully protected:
To furnish a functional oligonucleotide, all the protecting groups have to be removed. The N-acyl base protection and the 2-cyanoethyl phosphate protection may be, and is often removed simultaneously by treatment with inorganic bases or amines. However, the applicability of this method is limited by the fact that the cleavage of 2-cyanoethyl phosphate protection gives rise to acrylonitrile as a side product. Under the strong basic conditions required for the removal of N-acyl protection, acrylonitrile is capable of alkylation of nucleic bases, primarily, at the N3-position of thymine and uracil residues to give the respective N3-(2-cyanoethyl) adducts via Michael reaction. The formation of these side products may be avoided by treating the solid support-bound oligonucleotides with solutions of bases in an organic solvent, for instance, with 50% triethylamine in acetonitrile or 10% diethylamine in acetonitrile. This treatment is strongly recommended for medium- and large scale preparations and is optional for syntheses on small scale where the concentration of acrylonitrile generated in the deprotection mixture is low.

Regardless of whether the phosphate protecting groups were removed first, the solid support-bound oligonucleotides are deprotected using one of the two general approaches.

As with any other organic compound, it is prudent to characterize synthetic oligonucleotides upon their preparation. In more complex cases (research and large scale syntheses) oligonucleotides are characterized after their deprotection and after purification. Although the ultimate approach to the characterization is sequencing, a relatively inexpensive and routine procedure, the considerations of the cost reduction preclude its use in routine manufacturing of oligonucleotides. In day-by-day practice, it is sufficient to obtain the molecular mass of an oligonucleotide by recording its mass spectrum. Two methods are currently widely used for characterization of oligonucleotides: electrospray mass spectrometry (ES MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). To obtain informative spectra, it is very important to exchange all metal ions that might be present in the sample for ammonium or trialkylammonium ["e.c." triethylammonium, (CH)NH] ions prior to submitting a sample to the analysis by either of the methods.