In-vitro fertilization

In vitro fertilisation (IVF) is a process of fertilisation where an egg is combined with sperm in vitro ("in glass"). The process involves monitoring and stimulating an individual's ovulatory process, removing an ovum or ova (egg or eggs) from their ovaries and letting sperm fertilise them in a culture medium in a laboratory. After the fertilised egg (zygote) undergoes embryo culture for 2–6 days, it is transferred by catheter into the uterus, with the intention of establishing a successful pregnancy.

IVF is a type of assisted reproductive technology used for infertility treatment, gestational surrogacy, and, in combination with pre-implantation genetic testing, avoiding transmission of genetic conditions. A fertilised egg from a donor may implant into a surrogate's uterus, and the resulting child is genetically unrelated to the surrogate. Some countries have banned or otherwise regulate the availability of IVF treatment, giving rise to fertility tourism. Restrictions on the availability of IVF include costs and age, in order for a woman to carry a healthy pregnancy to term. Children born through IVF are commonly called test tube babies.

In July 1978, Louise Brown was the first child successfully born after her mother received IVF treatment. Brown was born as a result of natural-cycle IVF, where no stimulation was made. The procedure took place at Dr Kershaw's Cottage Hospital (now Dr Kershaw's Hospice) in Royton, Oldham, England. Robert G. Edwards was awarded the Nobel Prize in Physiology or Medicine in 2010. The physiologist co-developed the treatment together with Patrick Steptoe and embryologist Jean Purdy but the latter two were not eligible for consideration as they had died and the Nobel Prize is not awarded posthumously.

With egg donation and IVF, women who are past their reproductive years, have infertile partners, have idiopathic female-fertility issues, or have reached menopause can still become pregnant. After the IVF treatment, some couples get pregnant without any fertility treatments. In 2018, it was estimated that eight million children had been born worldwide using IVF and other assisted reproduction techniques. A 2019 study that explores 10 adjuncts with IVF (screening hysteroscopy, DHEA, testosterone, GH, aspirin, heparin, antioxidants in males and females, seminal plasma, and PRP) suggests that until more evidence is done to show that these adjuncts are safe and effective, they should be avoided.

Terminology

The Latin term in vitro, meaning "in glass", is used because early biological experiments involving cultivation of tissues outside the living organism were carried out in glass containers, such as beakers, test tubes, or Petri dishes. Today, the scientific term "in vitro" is used to refer to any biological procedure that is performed outside the organism in which it would normally have occurred, to distinguish it from an in vivo procedure (such as in vivo fertilisation), where the tissue remains inside the living organism in which it is normally found.

A colloquial term for babies conceived as the result of IVF, "test tube babies", refers to the tube-shaped containers of glass or plastic resin, called test tubes, that are commonly used in chemistry and biology labs. However, IVF is usually performed in Petri dishes, which are both wider and shallower and often used to cultivate cultures.

In a broader sense, IVF is a form of assisted reproductive technology (ART).

Medical uses

Indications

IVF may be used to overcome female infertility when it is due to problems with the fallopian tubes, making in vivo fertilisation difficult. It can also assist in male infertility, in those cases where there is a defect in sperm quality; in such situations intracytoplasmic sperm injection (ICSI) may be used, where a sperm cell is injected directly into the egg cell. This is used when sperm has difficulty penetrating the egg. In these cases the partner's or a donor's sperm may be used. ICSI is also used when sperm numbers are very low. When indicated, the use of ICSI has been found to increase the success rates of IVF.

According to UK's National Institute for Health and Care Excellence (NICE) guidelines, IVF treatment is appropriate in cases of unexplained infertility for women who have not conceived after 2 years of regular unprotected sexual intercourse.Fertility: assessment and treatment for people with fertility problems
NICE clinical guideline;– Issued: February 2013

In women with anovulation, it may be an alternative after 7–12 attempted cycles of ovulation induction, since the latter is expensive and more easy to control.

Success rates

IVF success rates are the percentage of all IVF procedures that result in favourable outcomes. Depending on the type of calculation used, this outcome may represent the number of confirmed pregnancies, called the pregnancy rate, or the number of live births, called the live birth rate. The success rate depends on variable factors such as maternal age, cause of infertility, embryo status, reproductive history, and lifestyle factors.

Maternal age: Younger candidates of IVF are more likely to get pregnant. Females older than 41 are more likely to get pregnant with a donor egg.

Reproductive history: women who have been previously pregnant are in many cases more successful with IVF treatments than those who have never been pregnant.

Due to advances in reproductive technology, live birth rates by cycle five of IVF have increased from 76% in 2005 to 80% in 2010 despite a reduction in the number of embryos being transferred (which decreased the multiple birth rate from 25% to 8%).

Live birth rate

The live birth rate is the percentage of all IVF cycles that lead to a live birth. This rate does not include miscarriage or stillbirth; multiple-order births, such as twins and triplets, are counted as one pregnancy. A 2019 summary compiled by the Society for Assisted Reproductive Technology (SART) which reports the average IVF success rates in the United States per age group using non-donor eggs compiled the following data:

In 2006, Canadian clinics reported a live birth rate of 27%. Birth rates in younger patients were slightly higher, with a success rate of 35.3% for those 21 and younger, the youngest group evaluated. Success rates for older patients were also lower and decrease with age, with 37-year-olds at 27.4% and no live births for those older than 48, the oldest group evaluated. Some clinics exceeded these rates, but it is impossible to determine if that is due to superior technique or patient selection, since it is possible to artificially increase success rates by refusing to accept the most difficult patients or by steering them into oocyte donation cycles (which are compiled separately). Further, pregnancy rates can be increased by the placement of several embryos at the risk of increasing the chance for multiples.

Because not each IVF cycle that is started will lead to oocyte retrieval or embryo transfer, reports of live birth rates need to specify the denominator, namely IVF cycles started, IVF retrievals, or embryo transfers. The SART summarised 2008–9 success rates for US clinics for fresh embryo cycles that did not involve donor eggs and gave live birth rates by the age of the prospective mother, with a peak at 41.3% per cycle started and 47.3% per embryo transfer for patients under 35 years of age.

IVF attempts in multiple cycles result in increased cumulative live birth rates. Depending on the demographic group, one study reported 45% to 53% for three attempts, and 51% to 71% to 80% for six attempts.

Effective from 15 February 2021 the majority of Australian IVF clinics publish their individual success rate online via YourIVFSuccess.com.au. This site also contains a predictor tool.

Pregnancy rate

Pregnancy rate may be defined in various ways. In the United States, SART and the Centers for Disease Control (and appearing in the table in the Success Rates section above) include statistics on positive pregnancy test and clinical pregnancy rate.

The 2019 summary compiled by the SART the following data for non-donor eggs (first embryo transfer) in the United States:

In 2006, Canadian clinics reported an average pregnancy rate of 35%.Branswell, Helen (15 December 2008
Success rate climbs for in vitro fertilisation
The Canadian Press. A French study estimated that 66% of patients starting IVF treatment finally succeed in having a child (40% during the IVF treatment at the centre and 26% after IVF discontinuation). Achievement of having a child after IVF discontinuation was mainly due to adoption (46%) or spontaneous pregnancy (42%).

Miscarriage rate

According to a study done by the Mayo Clinic miscarriage rates for IVF are somewhere between 15 and 25%.

Predictors of success

The main potential factors that influence pregnancy (and live birth) rates in IVF have been suggested to be maternal age, duration of infertility or subfertility, bFSH and number of oocytes, all reflecting ovarian function. Optimal woman's age is 23–39 years at time of treatment.

Biomarkers that affect the pregnancy chances of IVF include:
* Antral follicle count, with higher count giving higher success rates.
* Anti-Müllerian hormone levels, with higher levels indicating higher chances of pregnancy, as well as of live birth after IVF, even after adjusting for age.
* Level of DNA fragmentation as measured, e.g. by Comet assay, advanced maternal age.
* Women with ovary-specific FMR1 genotypes including ''het-norm/low'' have significantly decreased pregnancy chances in IVF.
* Progesterone elevation on the day of induction of final maturation is associated with lower pregnancy rates in IVF cycles in women undergoing ovarian stimulation using GnRH analogues and gonadotrophins. At this time, compared to a progesterone level below 0.8 ng/ml, a level between 0.8 and 1.1 ng/ml confers an odds ratio of pregnancy of approximately 0.8, and a level between 1.2 and 3.0 ng/ml confers an odds ratio of pregnancy of between 0.6 and 0.7. On the other hand, progesterone elevation does not seem to confer a decreased chance of pregnancy in frozen–thawed cycles and cycles with egg donation.
* Characteristics of cells from the cumulus oophorus and the membrana granulosa, which are easily aspirated during oocyte retrieval. These cells are closely associated with the oocyte and share the same microenvironment, and the rate of expression of certain genes in such cells are associated with higher or lower pregnancy rate.
* An endometrial thickness (EMT) of less than 7 mm decreases the pregnancy rate by an odds ratio of approximately 0.4 compared to an EMT of over 7 mm. However, such low thickness rarely occurs, and any routine use of this parameter is regarded as not justified.

Other determinants of outcome of IVF include:
* As the maternal age increases, the likelihood of conception decreases and the chance of miscarriage increases.
*With increasing paternal age, especially 50 years and older, the rate of blastocyst formation decreases.
* Tobacco smoking reduces the chances of IVF producing a live birth by 34% and increases the risk of an IVF pregnancy miscarrying by 30%.Regulated fertility services: a commissioning aid – June 2009
Department of Health UK. 18 June 2009
* A body mass index (BMI) over 27 causes a 33% decrease in likelihood to have a live birth after the first cycle of IVF, compared to those with a BMI between 20 and 27. Also, pregnant females who are obese have higher rates of miscarriage, gestational diabetes, hypertension, thromboembolism and problems during delivery, as well as leading to an increased risk of fetal congenital abnormality. Ideal body mass index is 19–30.
* Salpingectomy or laparoscopic tubal occlusion before IVF treatment increases chances for women with hydrosalpinges.Nice.org

* Success with previous pregnancy and/or live birth increases chances
* Low alcohol/caffeine intake increases success rate
* The number of embryos transferred in the treatment cycleFactors affecting IVF success – February 2011
from IVF-infertility.com
* Embryo quality
* Some studies also suggest that autoimmune disease may also play a role in decreasing IVF success rates by interfering with the proper implantation of the embryo after transfer.

Aspirin is sometimes prescribed to women for the purpose of increasing the chances of conception by IVF, but there was no evidence to show that it is safe and effective.

A 2013 review and meta analysis of randomised controlled trials of acupuncture as an adjuvant therapy in IVF found no overall benefit, and concluded that an apparent benefit detected in a subset of published trials where the control group (those not using acupuncture) experienced a lower than average rate of pregnancy requires further study, due to the possibility of publication bias and other factors.

A Cochrane review came to the result that endometrial injury performed in the month prior to ovarian induction appeared to increase both the live birth rate and clinical pregnancy rate in IVF compared with no endometrial injury. There was no evidence of a difference between the groups in miscarriage, multiple pregnancy or bleeding rates. Evidence suggested that endometrial injury on the day of oocyte retrieval was associated with a lower live birth or ongoing pregnancy rate.

Intake of antioxidants (such as N-acetyl-cysteine, melatonin, vitamin A, vitamin C, vitamin E, folic acid, myo-inositol, zinc or selenium) has not been associated with a significantly increased live birth rate or clinical pregnancy rate in IVF according to Cochrane reviews. The review found that oral antioxidants given to men in couples with male factor or unexplained subfertility may improve live birth rates, but more evidence is needed.

A Cochrane review in 2015 came to the result that there is no evidence identified regarding the effect of preconception lifestyle advice on the chance of a live birth outcome.

Complications

Multiple births

The major complication of IVF is the risk of multiple births. This is directly related to the practice of transferring multiple embryos at embryo transfer. Multiple births are related to increased risk of pregnancy loss, obstetrical complications, prematurity, and neonatal morbidity with the potential for long term damage. Strict limits on the number of embryos that may be transferred have been enacted in some countries (e.g. Britain, Belgium) to reduce the risk of high-order multiples (triplets or more), but are not universally followed or accepted. Spontaneous splitting of embryos in the womb after transfer can occur, but this is rare and would lead to identical twins. A double blind, randomised study followed IVF pregnancies that resulted in 73 infants (33 boys and 40 girls) and reported that 8.7% of singleton infants and 54.2% of twins had a birth weight of less than . There is some evidence that making a double embryo transfer during one cycle achieves a higher live birth rate than a single embryo transfer; but making two single embryo transfers in two cycles has the same live birth rate and would avoid multiple pregnancies.

Sex ratio distortions

Certain kinds of IVF, in particular ICSI (first applied in 1991) and blastocyst transfer (first applied in 1984) have been shown to lead to distortions in the sex ratio at birth. ICSI leads to slightly more female births (51.3% female) while blastocyst transfer leads to significantly more male births (56.1% male). Standard IVF done at the second or third day leads to a normal sex ratio.

Epigenetic modifications caused by extended culture leading to the death of more female embryos has been theorised as the reason why blastocyst transfer leads to a higher male sex ratio, however adding retinoic acid to the culture can bring this ratio back to normal.

Spread of infectious disease

By sperm washing, the risk that a chronic disease in the individual providing the sperm would infect the female or offspring can be brought to negligible levels.

In males with hepatitis B, The Practice Committee of the American Society for Reproductive Medicine advises that sperm washing is not necessary in IVF to prevent transmission, unless the female partner has not been effectively vaccinated. In females with hepatitis B, the risk of vertical transmission during IVF is no different from the risk in spontaneous conception. However, there is not enough evidence to say that ICSI procedures are safe in females with hepatitis B in regard to vertical transmission to the offspring.

Regarding potential spread of HIV/AIDS, Japan's government prohibited the use of IVF procedures for couples in which both partners are infected with HIV. Despite the fact that the ethics committees previously allowed the Ogikubo, Tokyo Hospital, located in Tokyo, to use IVF for couples with HIV, the Ministry of Health, Labour and Welfare of Japan decided to block the practice. Hideji Hanabusa, the vice president of the Ogikubo Hospital, states that together with his colleagues, he managed to develop a method through which scientists are able to remove HIV from sperm.

Other risks to the egg provider/retriever

A risk of ovarian stimulation is the development of ovarian hyperstimulation syndrome, particularly if hCG is used for inducing final oocyte maturation. This results in swollen, painful ovaries. It occurs in 30% of patients. Mild cases can be treated with over the counter medications and cases can be resolved in the absence of pregnancy. In moderate cases, ovaries swell and fluid accumulated in the abdominal cavities and may have symptoms of heartburn, gas, nausea or loss of appetite. In severe cases patients have sudden excess abdominal pain, nausea, vomiting and will result in hospitalisation.

During egg retrieval, there exists a small chance of bleeding, infection, and damage to surrounding structures such as bowel and bladder (transvaginal ultrasound aspiration) as well as difficulty in breathing, chest infection, allergic reactions to medication, or nerve damage (laparoscopy).

Ectopic pregnancy may also occur if a fertilised egg develops outside the uterus, usually in the fallopian tubes and requires immediate destruction of the fetus.

IVF does not seem to be associated with an elevated risk of cervical cancer, nor with ovarian cancer or endometrial cancer when neutralising the confounder of infertility itself. Nor does it seem to impart any increased risk for breast cancer.

Regardless of pregnancy result, IVF treatment is usually stressful for patients. Neuroticism and the use of escapist coping strategies are associated with a higher degree of distress, while the presence of social support has a relieving effect. A negative pregnancy test after IVF is associated with an increased risk for depression in women, but not with any increased risk of developing anxiety disorders. Pregnancy test results do not seem to be a risk factor for depression or anxiety among men.

Studies show that there is an increased risk of venous thrombosis or pulmonary embolism during the first trimester of IVF. When looking at long-term studies comparing women who received or did not receive IVF, there seems to be no correlation with increased risk of cardiac events. There are more ongoing studies to solidify this.

Spontaneous pregnancy has occurred after successful and unsuccessful IVF treatments. Within 2 years of delivering an infant conceived through IVF, subfertile couples had a conception rate of 18%.

Birth defects

A review in 2013 came to the result that infants resulting from IVF (with or without ICSI) have a relative risk of birth defects of 1.32 (95% confidence interval 1.24–1.42) compared to naturally conceived infants. In 2008, an analysis of the data of the National Birth Defects Study in the US found that certain birth defects were significantly more common in infants conceived through IVF, notably septal heart defects, cleft lip with or without cleft palate, esophageal atresia, and anorectal atresia; the mechanism of causality is unclear. However, in a population-wide cohort study of 308,974 births (with 6,163 using assisted reproductive technology and following children from birth to age five) researchers found: "The increased risk of birth defects associated with IVF was no longer significant after adjustment for parental factors." Parental factors included known independent risks for birth defects such as maternal age, smoking status, etc. Multivariate correction did not remove the significance of the association of birth defects and ICSI (corrected odds ratio 1.57), although the authors speculate that underlying male infertility factors (which would be associated with the use of ICSI) may contribute to this observation and were not able to correct for these confounders. The authors also found that a history of infertility elevated risk itself in the absence of any treatment (odds ratio 1.29), consistent with a Danish national registry study and "implicates patient factors in this increased risk." The authors of the Danish national registry study speculate: "our results suggest that the reported increased prevalence of congenital malformations seen in singletons born after assisted reproductive technology is partly due to the underlying infertility or its determinants."

Other risks to the offspring

If the underlying infertility is related to abnormalities in spermatogenesis, it is plausible, but too early to examine that male offspring are at higher risk for sperm abnormalities.

IVF does not seem to confer any risks regarding cognitive development, school performance, social functioning, and behaviour. Also, IVF infants are known to be as securely attached to their parents as those who were naturally conceived, and IVF adolescents are as well-adjusted as those who have been naturally conceived.

Limited long-term follow-up data suggest that IVF may be associated with an increased incidence of hypertension, impaired fasting glucose, increase in total body fat composition, advancement of bone age, subclinical thyroid disorder, early adulthood clinical depression and binge drinking in the offspring. It is not known, however, whether these potential associations are caused by the IVF procedure in itself, by adverse obstetric outcomes associated with IVF, by the genetic origin of the children or by yet unknown IVF-associated causes. Increases in embryo manipulation during IVF result in more deviant fetal growth curves, but birth weight does not seem to be a reliable marker of fetal stress.

IVF, including ICSI, is associated with an increased risk of imprinting disorders (including Prader-Willi syndrome and Angelman syndrome), with an odds ratio of 3.7 (95% confidence interval 1.4 to 9.7).

An IVF-associated incidence of cerebral palsy and neurodevelopmental delay are believed to be related to the confounders of prematurity and low birthweight. Similarly, an IVF-associated incidence of autism and attention-deficit disorder are believed to be related to confounders of maternal and obstetric factors.

Overall, IVF does not cause an increased risk of childhood cancer. Studies have shown a decrease in the risk of certain cancers and an increased risks of certain others including retinoblastoma, hepatoblastoma and rhabdomyosarcoma.

Method

Theoretically, IVF could be performed by collecting the contents from the fallopian tubes or uterus after natural ovulation, mixing it with sperm, and reinserting the fertilised ova into the uterus. However, without additional techniques, the chances of pregnancy would be extremely small. The additional techniques that are routinely used in IVF include ovarian hyperstimulation to generate multiple eggs, ultrasound-guided transvaginal oocyte retrieval directly from the ovaries, co-incubation of eggs and sperm, as well as culture and selection of resultant embryos before embryo transfer into a uterus.

Ovarian hyperstimulation

Ovarian hyperstimulation is the stimulation to induce development of multiple follicles of the ovaries. It should start with response prediction by e.g. age, antral follicle count and level of anti-Müllerian hormone. The resulting prediction of e.g. poor or hyper-response to ovarian hyperstimulation determines the protocol and dosage for ovarian hyperstimulation.

Ovarian hyperstimulation also includes suppression of spontaneous ovulation, for which two main methods are available: Using a (usually longer) GnRH agonist protocol or a (usually shorter) GnRH antagonist protocol. In a standard long GnRH agonist protocol the day when hyperstimulation treatment is started and the expected day of later oocyte retrieval can be chosen to conform to personal choice, while in a GnRH antagonist protocol it must be adapted to the spontaneous onset of the previous menstruation. On the other hand, the GnRH antagonist protocol has a lower risk of ovarian hyperstimulation syndrome (OHSS), which is a life-threatening complication.

For the ovarian hyperstimulation in itself, injectable gonadotropins (usually Follicle stimulating hormone, FSH analogues) are generally used under close monitoring. Such monitoring frequently checks the estradiol level and, by means of gynecologic ultrasonography, follicular growth. Typically approximately 10 days of injections will be necessary.

Natural IVF

There are several methods termed ''natural cycle IVF'':
* IVF using no drugs for ovarian hyperstimulation, while drugs for ovulation suppression may still be used.
* IVF using ovarian hyperstimulation, including gonadotropins, but with a GnRH antagonist protocol so that the cycle initiates from natural mechanisms.
* Frozen embryo transfer; IVF using ovarian hyperstimulation, followed by embryo cryopreservation, followed by embryo transfer in a later, natural, cycle.

IVF using no drugs for ovarian hyperstimulation was the method for the conception of Louise Brown. This method can be successfully used when women want to avoid taking ovarian stimulating drugs with its associated side-effects. Human Fertilisation and Embryology Authority, HFEA has estimated the live birth rate to be approximately 1.3% per IVF cycle using no hyperstimulation drugs for women aged between 40 and 42.

Mild IVF is a method where a small dose of ovarian stimulating drugs are used for a short duration during a natural menstrual cycle aimed at producing 2–7 eggs and creating healthy embryos. This method appears to be an advance in the field to reduce complications and side-effects for women, and it is aimed at quality, and not quantity of eggs and embryos. One study comparing a mild treatment (mild ovarian stimulation with GnRH antagonist co-treatment combined with single embryo transfer) to a standard treatment (stimulation with a GnRH agonist long-protocol and transfer of two embryos) came to the result that the proportions of cumulative pregnancies that resulted in term Live birth (human), live birth after 1 year were 43.4% with mild treatment and 44.7% with standard treatment. Mild IVF can be cheaper than conventional IVF and with a significantly reduced risk of multiple birth, multiple gestation and ovarian hyperstimulation syndrome, OHSS.

Final maturation induction

When the ovarian follicles have reached a certain degree of development, Final maturation induction, induction of final oocyte maturation is performed, generally by an injection of human chorionic gonadotropin (hCG). Commonly, this is known as the "trigger shot." hCG acts as an analogue of luteinising hormone, and ovulation would occur between 38 and 40 hours after a single HCG injection, but the egg retrieval is performed at a time usually between 34 and 36 hours after hCG injection, that is, just prior to when the follicles would rupture. This avails for scheduling the egg retrieval procedure at a time where the eggs are fully mature. HCG injection confers a risk of ovarian hyperstimulation syndrome. Using a GnRH agonist instead of hCG eliminates most of the risk of ovarian hyperstimulation syndrome, but with a reduced delivery rate if the embryos are transferred fresh. For this reason, many centers will freeze all oocytes or embryos following agonist trigger.

Egg retrieval

The eggs are retrieved from the patient using a transvaginal technique called transvaginal oocyte retrieval, involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle follicles can be aspirated, and the follicular fluid is passed to an embryologist to identify ova. It is common to remove between ten and thirty eggs. The retrieval procedure usually takes between 20 and 40 minutes, depending on the number of mature follicles, and is usually done under conscious sedation or general anaesthesia.

Egg and sperm preparation

In the laboratory, for ICSI treatments, the identified eggs are stripped of surrounding cells (also known as cumulus cells) and prepared for fertilisation. An oocyte selection may be performed prior to fertilisation to select eggs that can be fertilised, as it is required they are in metaphase II. There are cases in which if oocytes are in the metaphase I stage, they can be kept being cultured so as to undergo a posterior sperm injection. In the meantime, semen is prepared for fertilisation by removing inactive cells and seminal fluid in a process called sperm washing. If semen is being provided by a sperm donor, it will usually have been prepared for treatment before being frozen and quarantined, and it will be thawed ready for use.

Co-incubation

The sperm and the egg are incubated together at a ratio of about 75,000:1 in a culture media in order for the actual Human fertilization, fertilisation to take place. A review in 2013 came to the result that a duration of this co-incubation of about 1 to 4 hours results in significantly higher pregnancy rates than 16 to 24 hours. In most cases, the egg will be fertilised during co-incubation and will show two pronuclei. In certain situations, such as low sperm count or motility, a single sperm may be injected directly into the egg using intracytoplasmic sperm injection (ICSI). The fertilised egg is passed to a special growth medium and left for about 48 hours until the egg consists of six to eight cells.

In gamete intrafallopian transfer, eggs are removed from the woman and placed in one of the fallopian tubes, along with the man's sperm. This allows fertilisation to take place inside the woman's body. Therefore, this variation is actually an in vivo fertilisation, not in vitro.

Embryo culture

The main durations of embryo culture are until human embryogenesis, cleavage stage (day two to four after co-incubation) or the blastocyst stage (day five or six after co-incubation). Embryo culture until the blastocyst stage confers a significant increase in live birth rate per embryo transfer, but also confers a decreased number of embryos available for transfer and embryo cryopreservation, so the cumulative clinical pregnancy rates are increased with cleavage stage transfer. Transfer day two instead of day three after fertilisation has no differences in live birth rate. There are significantly higher odds of preterm birth (odds ratio 1.3) and congenital anomalies (odds ratio 1.3) among births having from embryos cultured until the blastocyst stage compared with cleavage stage.

Embryo selection

Laboratories have developed grading methods to judge ovocyte and embryo quality. In order to optimise pregnancy rates, there is significant evidence that a morphological scoring system is the best strategy for the selection of embryos. Since 2009 where the first time-lapse microscopy system for IVF was approved for clinical use, morphokinetic scoring systems has shown to improve to pregnancy rates further. However, when all different types of time-lapse embryo imaging devices, with or without morphokinetic scoring systems, are compared against conventional embryo assessment for IVF, there is insufficient evidence of a difference in live-birth, pregnancy, stillbirth or miscarriage to choose between them. Active efforts to develop a more accurate embryo selection analysis based on Artificial Intelligence and Deep Learning are underway. Embryo Ranking Intelligent Classification Assistant (ERICA), is a clear example. This Deep Learning software substitutes manual classifications with a ranking system based on an individual embryo's predicted genetic status in a non-invasive fashion. Studies on this area are still pending and current feasibility studies support its potential.

Embryo transfer

The number to be transferred depends on the number available, the age of the patient and other health and diagnostic factors. In countries such as Canada, the UK, Australia and New Zealand, a maximum of two embryos are transferred except in unusual circumstances. In the UK and according to HFEA regulations, a woman over 40 may have up to three embryos transferred, whereas in the US, there is no legal limit on the number of embryos which may be transferred, although medical associations have provided practice guidelines. Most clinics and country regulatory bodies seek to minimise the risk of multiple pregnancy, as it is not uncommon for multiple embryos to implant if multiple embryos are transferred. Embryos are transferred to the patient's uterus through a thin, plastic catheter, which goes through her vagina and cervix. Several embryos may be passed into the uterus to improve chances of Implantation (human embryo), implantation and pregnancy.

Luteal support

Luteal support is the administration of medication, generally progesterone, progestins, hCG, or GnRH agonists, and often accompanied by estradiol, to increase the success rate of Implantation (human embryo), implantation and early Human embryogenesis, embryogenesis, thereby complementing and/or supporting the function of the corpus luteum. A Cochrane review found that hCG or progesterone given during the luteal phase may be associated with higher rates of Live birth rate, live birth or ongoing pregnancy, but that the evidence is not conclusive. Co-treatment with GnRH agonists appears to improve outcomes, by a live birth rate risk difference, RD of +16% (95% confidence interval +10 to +22%). On the other hand, growth hormone or aspirin as adjunctive medication in IVF have no evidence of overall benefit.

Expansions

There are various expansions or additional techniques that can be applied in IVF, which are usually not necessary for the IVF procedure itself, but would be virtually impossible or technically difficult to perform without concomitantly performing methods of IVF.

Preimplantation genetic screening or diagnosis

Preimplantation genetic screening (PGS) or preimplantation genetic diagnosis (PGD) has been suggested to be able to be used in IVF to select an embryo that appears to have the greatest chances for successful pregnancy. However, a systematic review and meta-analysis of existing randomised controlled trials came to the result that there is no evidence of a beneficial effect of PGS with cleavage-stage biopsy as measured by live birth rate. On the contrary, for women of advanced maternal age