Editing In vitro fertilisation (section)

== Method ==
{{more citations needed section|date=July 2020}}
[[File:In Vitro Fertilization (IVF) - English.png|thumb|412x412px|A graphic explaining the details of IVF]]
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 [[Controlled ovarian hyperstimulation|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===
{{Main|Controlled ovarian hyperstimulation}}
Ovarian hyperstimulation is the stimulation to induce development of multiple follicles of the ovaries. It should start with response prediction based on factors such as age, [[antral follicle count]] and level of [[anti-Müllerian hormone]].<ref name=LaMarca2013>{{cite journal | vauthors = La Marca A, Sunkara SK | title = Individualization of controlled ovarian stimulation in IVF using ovarian reserve markers: from theory to practice | journal = Human Reproduction Update | volume = 20 | issue = 1 | pages = 124–140 | year = 2014 | pmid = 24077980 | doi = 10.1093/humupd/dmt037 | doi-access = free }}</ref> The resulting prediction (e.g. poor or hyper-response to ovarian hyperstimulation) determines the protocol and dosage for ovarian hyperstimulation.<ref name=LaMarca2013/>

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.<ref name=LaMarca2013/> 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.<ref name=LaMarca2013/>

For the ovarian hyperstimulation in itself, injectable [[gonadotropin]]s (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.

When stimulating ovulation after suppressing endogenous secretion, it is necessary to supply exogenous gonadotropines. The most common one is the [[Menotropin|human menopausal gonadotropin]] (hMG), which is obtained by donation of menopausal women. Other [[Gonadotropin preparations|pharmacological preparations]] are FSH+LH or coripholitropine alpha.

===Natural IVF===
{{Main|Natural cycle in vitro fertilisation}}
There are several methods termed natural cycle IVF:<ref name="Allersma2013">{{cite journal | vauthors = Allersma T, Farquhar C, Cantineau AE | title = Natural cycle in vitro fertilisation (IVF) for subfertile couples | journal = The Cochrane Database of Systematic Reviews | volume = 2013 | issue = 8 | pages = CD010550 | date = August 2013 | pmid = 23990351 | pmc = 7390465 | doi = 10.1002/14651858.CD010550.pub2 }}</ref>
* IVF using no drugs for [[Controlled ovarian hyperstimulation|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 [[Controlled ovarian hyperstimulation|ovarian hyperstimulation]], followed by [[embryo cryopreservation]], followed by [[embryo transfer]] in a later, natural, cycle.<ref name="EvansHannan2014">{{cite journal | vauthors = Evans J, Hannan NJ, Edgell TA, Vollenhoven BJ, Lutjen PJ, Osianlis T, Salamonsen LA, Rombauts LJ | display-authors = 6 | title = Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence | journal = Human Reproduction Update | volume = 20 | issue = 6 | pages = 808–821 | year = 2014 | pmid = 24916455 | doi = 10.1093/humupd/dmu027 | doi-access = free }}</ref>

IVF using no drugs for ovarian hyperstimulation was the method for the conception of [[Louise Brown]]. This method can be successfully used when people 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.<ref>{{cite web | url = http://www.hfea.gov.uk/natural-cycle-ivf.html | title = Natural cycle IVF | archive-url = https://web.archive.org/web/20120512083412/http://www.hfea.gov.uk/natural-cycle-ivf.html | archive-date=12 May 2012 | work = [[Human Fertilisation and Embryology Authority]] }}</ref>

Mild IVF<ref>{{cite journal | vauthors = Nargund G | title = Natural/mild assisted reproductive technologies: reducing cost and increasing safety | journal = Women's Health | volume = 5 | issue = 4 | pages = 359–360 | date = July 2009 | pmid = 19586428 | doi = 10.2217/whe.09.32 | doi-access = free }}</ref> 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.<ref name="pmid17336650">{{cite journal | vauthors = Heijnen EM, Eijkemans MJ, De Klerk C, Polinder S, Beckers NG, Klinkert ER, Broekmans FJ, Passchier J, Te Velde ER, Macklon NS, Fauser BC | display-authors = 6 | title = A mild treatment strategy for in-vitro fertilisation: a randomised non-inferiority trial | journal = Lancet | volume = 369 | issue = 9563 | pages = 743–749 | date = March 2007 | pmid = 17336650 | doi = 10.1016/S0140-6736(07)60360-2 | s2cid = 25591825 }}</ref> Mild IVF can be cheaper than conventional IVF and with a significantly reduced risk of [[multiple birth|multiple gestation]] and [[ovarian hyperstimulation syndrome|OHSS]].<ref>{{cite journal | vauthors = Fauser BC, Nargund G, Andersen AN, Norman R, Tarlatzis B, Boivin J, Ledger W | title = Mild ovarian stimulation for IVF: 10 years later | journal = Human Reproduction | volume = 25 | issue = 11 | pages = 2678–2684 | date = November 2010 | pmid = 20858698 | doi = 10.1093/humrep/deq247 | doi-access = free }}</ref>

===Final maturation induction===
{{Further|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."<ref>{{cite web |url=http://infertility.about.com/od/infertilitytreatments/ss/ivf_treatment_5.htm |title=IVF Treatment Step Four: Final Oocyte |publisher=Infertility.about.com |access-date=22 May 2012 |archive-date=18 November 2012 |archive-url=https://web.archive.org/web/20121118205858/http://infertility.about.com/od/infertilitytreatments/ss/ivf_treatment_5.htm |url-status=dead }}</ref> hCG acts as an analogue of [[luteinising hormone]], and ovulation would occur between 38 and 40 hours after a single HCG injection,<ref>{{cite web | url = http://www.medscape.com/viewarticle/473515 | title = HCG Injection After Ovulation Induction With Clomiphene Citrate] | work = Medscape | vauthors = Kovacs P | date = 23 April 2004 }}</ref> 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.<ref name=Humaidan2011>{{cite journal | vauthors = Humaidan P, Kol S, Papanikolaou EG | title = GnRH agonist for triggering of final oocyte maturation: time for a change of practice? | journal = Human Reproduction Update | volume = 17 | issue = 4 | pages = 510–524 | year = 2011 | pmid = 21450755 | doi = 10.1093/humupd/dmr008 | collaboration = Copenhagen GnRH Agonist Triggering Workshop Group | doi-access = free }}</ref>  For this reason, many centers will freeze all oocytes or embryos following agonist trigger.

===Egg retrieval===
{{Further|Transvaginal oocyte retrieval}}
The eggs are retrieved from the patient using a transvaginal technique called [[transvaginal oocyte retrieval|transvaginal]] ultrasound aspiration involving an ultrasound-guided needle being injected through follicles upon collection. Through this needle, the oocyte and follicular fluid are aspirated and the follicular fluid is then passed to an embryologist to identify ova. It is common to remove between ten and thirty eggs. The retrieval process, which lasts approximately 20 to 40 minutes, is performed under [[conscious sedation]] or [[general anesthesia]] to ensure patient comfort. Following optimal follicular development, the eggs are meticulously retrieved using transvaginal ultrasound guidance with the aid of a specialised ultrasound probe and a fine needle aspiration technique. The follicular fluid, containing the retrieved eggs, is expeditiously transferred to the embryology laboratory for subsequent processing.<ref>IVF Process, Methods and Solutions https://techxplore.online/ivf-process-methods-and-solutions/ {{Webarchive|url=https://web.archive.org/web/20230609095407/https://techxplore.online/ivf-process-methods-and-solutions/ |date=9 June 2023 }}</ref>

===Egg and sperm preparation===
In the laboratory, for ICSI treatments, the identified eggs are stripped of surrounding cells (also known as [[cumulus cell]]s) 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.{{citation needed|date=July 2020}}

===Co-incubation===
[[File:Ivf.png|thumb|317x317px|Demonstration of ICSI]]
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 rate]]s than 16 to 24 hours.<ref>{{cite journal | vauthors = Zhang XD, Liu JX, Liu WW, Gao Y, Han W, Xiong S, Wu LH, Huang GN | display-authors = 6 | title = Time of insemination culture and outcomes of in vitro fertilization: a systematic review and meta-analysis | journal = Human Reproduction Update | volume = 19 | issue = 6 | pages = 685–695 | year = 2013 | pmid = 23912477 | doi = 10.1093/humupd/dmt036 | doi-access = free }}</ref> 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 embryo 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.<ref>{{cite journal | vauthors = Abyholm T, Tanbo T, Dale PO, Magnus O | title = In vivo fertilization procedures in infertile women with patent fallopian tubes: a comparison of gamete intrafallopian transfer, combined intrauterine and intraperitoneal insemination, and controlled ovarian hyperstimulation alone | journal = Journal of Assisted Reproduction and Genetics | volume = 9 | issue = 1 | pages = 19–23 | date = February 1992 | pmid = 1617244 | doi = 10.1007/BF01204109 | s2cid = 25057205 }}</ref><ref>{{cite journal | vauthors = Wetscher F, Havlicek V, Huber T, Gilles M, Tesfaye D, Griese J, Wimmers K, Schellander K, Müller M, Brem G, Besenfelder U | display-authors = 6 | title = Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle | journal = Theriogenology | volume = 64 | issue = 1 | pages = 30–40 | date = July 2005 | pmid = 15935840 | doi = 10.1016/j.theriogenology.2004.11.018 }}</ref>

===Embryo culture===
{{Main|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]]).<ref name="DarLazer2014">{{cite journal | vauthors = Dar S, Lazer T, Shah PS, Librach CL | title = Neonatal outcomes among singleton births after blastocyst versus cleavage stage embryo transfer: a systematic review and meta-analysis | journal = Human Reproduction Update | volume = 20 | issue = 3 | pages = 439–448 | year = 2014 | pmid = 24480786 | doi = 10.1093/humupd/dmu001 | doi-access = free }}</ref> 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.<ref name=":0" /> Transfer day two instead of day three after fertilisation has no differences in [[live birth rate]].<ref name=":0" /> 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.<ref name="DarLazer2014"/>

===Embryo selection===
{{Further|Embryo quality}}
Laboratories have developed grading methods to judge ovocyte and [[embryo]] quality. In order to optimise [[pregnancy rate]]s, there is significant evidence that a morphological scoring system is the best strategy for the selection of embryos.<ref name=Rebmann>{{cite journal | vauthors = Rebmann V, Switala M, Eue I, Grosse-Wilde H | title = Soluble HLA-G is an independent factor for the prediction of pregnancy outcome after ART: a German multi-centre study | journal = Human Reproduction | volume = 25|issue = 7 | pages = 1691–1698 | date = July 2010 | pmid = 20488801 | doi = 10.1093/humrep/deq120 | doi-access = free }}</ref> 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 rate]]s further.<ref name="Meseguer">{{cite journal | vauthors = Meseguer M, Rubio I, Cruz M, Basile N, Marcos J, Requena A | title = Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study | journal = Fertility and Sterility|volume=98|issue=6|pages=1481–9.e10|date=December 2012 | pmid = 22975113 | doi = 10.1016/j.fertnstert.2012.08.016 | doi-access = free }}</ref> 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.<ref>{{cite journal|vauthors=Armstrong S, Bhide P, Jordan V, Pacey A, Marjoribanks J, Farquhar C|title=Time-lapse systems for embryo incubation and assessment in assisted reproduction | journal = The Cochrane Database of Systematic Reviews|volume=5|issue=5|pages=CD011320|date=May 2019|pmid=31140578|pmc=6539473|doi = 10.1002/14651858.CD011320.pub4 }}</ref> Active efforts to develop a more accurate embryo selection analysis based on Artificial Intelligence and Deep Learning are underway. Embryo Ranking Intelligent Classification Assistant ([[Embryo Ranking Intelligent Classification Algorithm|ERICA]]),<ref>{{Cite web|url=https://embryoranking.com/ |title = ERICA Embryo Ranking &#124; Artificial Intelligence for Assisted Reproduction}}</ref> 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.<ref>{{Cite journal | doi=10.1016/j.fertnstert.2019.07.715| title=Artificial vision and machine learning designed to predict PGT-A results|journal=Fertility and Sterility| volume=112| issue=3| pages=e231| year=2019| vauthors = Chavez-Badiola A, Farias AF, Mendizabal-Ruiz G, Drakeley AJ, Garcia-Sánchez R, Zhang JJ | doi-access=free}}</ref> Studies on this area are still pending and current feasibility studies support its potential.<ref>{{cite journal|vauthors = Chavez-Badiola A, Flores-Saiffe Farias A, Mendizabal-Ruiz G, Garcia-Sanchez R, Drakeley AJ, Garcia-Sandoval JP|title=Predicting pregnancy test results after embryo transfer by image feature extraction and analysis using machine learning|journal = Scientific Reports|volume = 10|issue=1|pages=4394|date=March 2020|pmid=32157183|pmc=7064494|doi = 10.1038/s41598-020-61357-9|bibcode = 2020NatSR..10.4394C}}</ref>

===Embryo transfer===
{{Main|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 their [[vagina]] and cervix. Several embryos may be passed into the uterus to improve chances of [[Implantation (human embryo)|implantation]] and pregnancy.<ref>{{cite journal | vauthors = Timeva T, Shterev A, Kyurkchiev S | title = Recurrent implantation failure: the role of the endometrium | journal = Journal of Reproduction & Infertility | volume = 15 | issue = 4 | pages = 173–183 | date = October 2014 | pmid = 25473625 | pmc = 4227974 }}</ref><ref>{{Cite web|title=In vitro fertilization (IVF) – Mayo Clinic|url=https://www.mayoclinic.org/tests-procedures/in-vitro-fertilization/about/pac-20384716|access-date=31 August 2020|website=www.mayoclinic.org}}</ref>

===Luteal support===
{{Main|Luteal support}}
[[Luteal support]] is the administration of medication, generally [[progesterone]], [[progestin]]s, hCG, or [[GnRH agonist]]s, 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.<ref name="van-der-Linden2011">{{cite journal | vauthors = van der Linden M, Buckingham K, Farquhar C, Kremer JA, Metwally M | title = Luteal phase support for assisted reproduction cycles | journal = The Cochrane Database of Systematic Reviews | volume = 2015 | issue = 7 | pages = CD009154 | date = July 2015 | pmid = 26148507 | pmc = 6461197 | doi = 10.1002/14651858.CD009154.pub3 | hdl = 2066/98072 }}</ref> Co-treatment with [[GnRH agonist]]s appears to improve outcomes,<ref name=van-der-Linden2011/> by a live birth rate [[risk difference|RD]] of +16% (95% [[confidence interval]] +10 to +22%).<ref>{{cite journal | vauthors = Kyrou D, Kolibianakis EM, Fatemi HM, Tarlatzi TB, Devroey P, Tarlatzis BC | title = Increased live birth rates with GnRH agonist addition for luteal support in ICSI/IVF cycles: a systematic review and meta-analysis | journal = Human Reproduction Update | volume = 17 | issue = 6 | pages = 734–740 | year = 2011 | pmid = 21733980 | doi = 10.1093/humupd/dmr029 | doi-access = free }}</ref> On the other hand, [[growth hormone]] or [[aspirin]] as adjunctive medication in IVF have no evidence of overall benefit.<ref name=":0" />