Embryo transfer refers to a step in the process of assisted reproduction in which embryos are placed into the uterus of a female with the intent to establish a pregnancy. This technique (which is often used in connection with in vitro fertilization (IVF)), may be used in humans or in animals, in which situations the goals may vary.
8-cell embryo for transfer 3 days after fertilization
Fresh versus frozenEdit
Embryos can be either “fresh” from fertilized egg cells of the same menstrual cycle, or “frozen”, that is they have been generated in a preceding cycle and undergone embryo cryopreservation, and are thawed just prior to the transfer, which is then termed "frozen embryo transfer" (FET). The outcome from using cryopreserved embryos has uniformly been positive with no increase in birth defects or development abnormalities, also between fresh versus frozen eggs used for intracytoplasmic sperm injection (ICSI). In fact, pregnancy rates are increased following FET, and perinatal outcomes are less affected, compared to embryo transfer in the same cycle as ovarian hyperstimulation was performed. The endometrium is believed to not be optimally prepared for implantation following ovarian hyperstimulation, and therefore frozen embryo transfer avails for a separate cycle to focus on optimizing the chances of successful implantation. Children born from vitrified blastocysts have significantly higher birthweight than those born from non-frozen blastocysts. When transferring a frozen-thawed oocyte, the chance of pregnancy is essentially the same whether it is transferred in a natural cycle or one with ovulation induction.
In the human, the uterine lining (endometrium) needs to be appropriately prepared so that the embryo(s) can implant. In a natural cycle the embryo transfer takes place in the luteal phase at a time where the lining is appropriately undeveloped in relation to the status of the present Luteinizing Hormone. In a stimulated or a cycle where a "frozen" embryo is transferred, the recipient woman could be given first estrogen preparations (about 2 weeks), then a combination of oestrogen and progesterone so that the lining becomes receptive for the embryo. The time of receptivity is the implantation window. A scientific review in 2013 came to the conclusion that it is not possible to identify one method of endometrium preparation in frozen embryo transfer as being more effective than another.
Embryo transfer can be performed after various durations of embryo culture, conferring different stages in embryogenesis. The main stages at which embryo transfer is performed are cleavage stage (day 2 to 4 after co-incubation) or the blastocyst stage (day 5 or 6 after co-incubation).
Embryos who reach the day 3 cell stage can be tested for chromosomal or specific genetic defects prior to possible transfer by preimplantation genetic diagnosis (PGD). Transferring at the blastocyst stage confers a significant increase in live birth rate per 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.[needs update] Transfer day 2 instead of day 3 after fertilization has no differences in live birth rate.
There is a significantly higher odds of preterm birth (odds ratio 1.3) and congenital anomalies (odds ratio 1.3) among births having reached the blastocyst stage compared with cleavage stage. Because of increased female embryo mortality due to epigenetic modifications induced by extended culture, blastocyst transfer leads to more male births (56.1% male) versus 2 or 3 day transfer (a normal sex ratio of 51.5% male).
Laboratories have developed grading methods to judge oocyte 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.[needs update] A small prospectively randomized study in 2016 reported poorer embryo quality and more staff time in an automated time-lapse embryo imaging device compared to conventional embryology.
The embryo transfer procedure starts by placing a speculum in the vagina to visualize the cervix, which is cleansed with saline solution or culture media. A soft transfer catheter is loaded with the embryos and handed to the clinician after confirmation of the patient’s identity. The catheter is inserted through the cervical canal and advanced into the uterine cavity.
There is good and consistent evidence of benefit in ultrasound guidance, that is, making an abdominal ultrasound to ensure correct placement, which is 1–2 cm from the uterine fundus. There is evidence of a significant increase in clinical pregnancy using ultrasound guidance compared with only "clinical touch". Anesthesia is generally not required. Single embryo transfers in particular require accuracy and precision in placement within the uterine cavity. The optimal target for embryo placement, known as the maximal implantation potential (MIP) point, is identified using 3D/4D ultrasound. However, there is limited evidence that supports deposition of embryos in the midportion of the uterus.
After insertion of the catheter, the contents are expelled and the embryos are deposited. Limited evidence supports making trial transfers before performing the procedure with embryos. After expulsion, the duration that the catheter remains inside the uterus has no effect on pregnancy rates. Limited evidence suggests avoiding negative pressure from the catheter after expulsion. After withdrawal, the catheter is handed to the embryologist, who inspects it for retained embryos.
In the process of zygote intrafallopian transfer (ZIFT), eggs are removed from the woman, fertilised, and then placed in the woman's fallopian tubes rather than the uterus.
In 2015, the American Society for Reproductive Medicine developed a medical simulation of the Embryo Transfer procedure with Swiss company VirtaMed, designed for the education and training of clinicians. The virtual reality simulator, which includes real-time simulation of ultrasound guidance, was launched at the annual conference of the American Society for Reproductive Medicine.
A major issue is how many embryos should be transferred. Placement of multiple embryos carries the risk of multiple pregnancy. In the past, physicians have often placed too many embryos in the hope to establish a pregnancy. However, the rise in multiple pregnancies has led to a reassessment of this approach. Professional societies and in many countries, the legislature, have issued guidelines or laws to curtail a practice of placing too many embryos in an attempt to reduce multiple pregnancies. The appropriate number of embryos to be transferred depends on the age of the woman, whether it is the first, second or third full IVF cycle attempt and whether there are top-quality embryos available. According to a guideline from The National Institute for Health and Care Excellence (NICE) in 2013, the number of embryos transferred in a cycle should be chosen as in following table:
|Age||Attempt No.||Embryos transferred|
|2nd||1 if top-quality|
|3rd||No more than 2|
|37–39 years||1st & 2nd||1 if top-quality|
|2 if no top-quality|
|3rd||No more than 2|
The technique of selecting only one embryo to transfer to the woman is called elective-single embryo transfer (e-SET) or, when embryos are at the blastocyst stage, it can also be called elective single blastocyst transfer (eSBT). It significantly lowers the risk of multiple pregnancies, compared with e.g. Double Embryo Transfer (DET) or double blastocyst transfer (2BT), with a twinning rate of approximately 3.5% in sET compared with approximately 38% in DET, or 2% in eSBT compared with approximately 25% in 2BT. At the same time, pregnancy rates is not significantly less with eSBT than with 2BT. That is, the cumulative live birth rate associated with single fresh embryo transfer followed by a single frozen and thawed embryo transfer is comparable with that after one cycle of double fresh embryo transfer. Furthermore, SET has better outcomes in terms of mean gestational age at delivery, mode of delivery, birthweight, and risk of neonatal intensive care unit necessity than DET. e-SET of embryos at the cleavage stage reduces the likelihood of live birth by 38% and multiple birth by 94%. Evidence from randomized, controlled trials suggests that increasing the number of e-SET attempts (fresh and/or frozen) results in a cumulative live birth rate similar to that of DET.
The usage of single embryo transfer is highest in Sweden (69.4%), but as low as 2.8% in the USA. Access to public funding for ART, availability of good cryopreservation facilities, effective education about the risks of multiple pregnancy, and legislation appear to be the most important factors for regional usage of single embryo transfer. Also, personal choice plays a significant role as many subfertile couples have a strong preference for twins.
There is limited evidence to support the use of mechanical closure of the cervical canal following embryo transfer.
There is insufficient evidence to support a certain amount of time for women to remain recumbent following embryo transfer.
There is no evidence of benefit in terms of live birth rate of using hyaluronic acid as adherence medium for the embryo. Neither is there any evidence of benefit of having a full bladder, removal of cervical mucus, or flushing of the endometrial or endocervical cavity at the time of embryo transfer. Adjunctive antibiotics in the form of amoxicillin plus clavulanic acid does not increase the clinical pregnancy rate compared with no antibiotics.
For frozen-thawed embryo transfer or transfer of embryo from egg donation, no previous ovarian hyperstimulation is required for the recipient before transfer, which can be performed in spontaneous ovulatory cycles. Still, various protocols exist for frozen-thawed embryo transfers as well, such as protocols with ovarian hyperstimulation, protocols in which the endometrium is artificially prepared by estrogen and/or progesterone. A Cochrane review in 2010 of randomized studies came to the result that there generally is insufficient evidence to support the use of one intervention in preference to another, but with some evidence that in cycles where the endometrium is artificially prepared by estrogen or progesterone, it is beneficial to administer an additional drug that suppresses hormone production by the ovaries such as continuous administration of a gonadotropin releasing hormone agonist (GnRHa). For egg donation, there is evidence of a lower pregnancy rate and a higher cycle cancellation rate when the progesterone supplementation in the recipient is commenced prior to oocyte retrieval from the donor, as compared to commenced day of oocyte retrieval or the day after.
Seminal fluid contains several proteins that interact with epithelial cells of the cervix and uterus, inducing active gestational immune tolerance. There are significantly improved outcomes when women are exposed to seminal plasma around the time of embryo transfer, with statistical significance for clinical pregnancy, but not for ongoing pregnancy or live birth rates with the limited data available.
Patients usually start progesterone medication after egg (also called oocyte) retrieval. While daily intramuscular injections of progesterone-in-oil (PIO) have been the standard route of administration, PIO injections are not FDA-approved for use in pregnancy. A recent meta-analysis showed that the intravaginal route with an appropriate dose and dosing frequency is equivalent to daily intramuscular injections. In addition, a recent case-matched study comparing vaginal progesterone with PIO injections showed that live birth rates were nearly identical with both methods. A duration of progesterone administration of 11 days results in almost the same birth rates as longer durations.
Patients are also given estrogen medication in some cases after the embryo transfer. Pregnancy testing is done typically two weeks after egg retrieval.
It is not necessary that the embryo transfer be performed on the female who provided the eggs. Thus another female whose uterus is appropriately prepared can receive the embryo and become pregnant. Embryo transfer may be used where a woman who has eggs but no uterus and wants to have a biological baby; she would require the help of a gestational carrier or surrogate to carry the pregnancy. Also, a woman who has no eggs but a uterus may utilize egg donor IVF, in which case another woman would provide eggs for fertilization and the resulting embryos are placed into the uterus of the patient. Fertilization may be performed using the woman's partner's sperm or by using donor sperm. 'Spare' embryos which are created for another couple undergoing IVF treatment but which are then surplus to that couple's needs may also be transferred (called embryo donation). Embryos may be specifically created by using eggs and sperm from donors and these can then be transferred into the uterus of another woman. A surrogate may carry a baby produced by embryo transfer for another couple, even though neither she nor the 'commissioning' couple is biologically related to the child. Third party reproduction is controversial and regulated in many countries. Persons entering gestational surrogacy arrangements must make sense of an entirely new type of relationship that does not fit any of the traditional scripts we use to categorize relations as kinship, friendship, romantic partnership or market relations. Surrogates have the experience of carrying a baby that they conceptualize as not of their own kin, while intended mothers have the experience of waiting through nine months of pregnancy and transitioning to motherhood from outside of the pregnant body. This can lead to new conceptualizations of body and self.
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The first transfer of an embryo from one human to another resulting in pregnancy was reported in July 1983 and subsequently led to the announcement of the first human birth 3 February 1984. This procedure was performed at the Harbor UCLA Medical Center  under the direction of Dr. John Buster and the University of California at Los Angeles School of Medicine.
In the procedure, an embryo that was just beginning to develop was transferred from one woman in whom it had been conceived by artificial insemination to another woman who gave birth to the infant 38 weeks later. The sperm used in the artificial insemination came from the husband of the woman who bore the baby.
This scientific breakthrough established standards and became an agent of change for women suffering from the afflictions of infertility and for women who did not want to pass on genetic disorders to their children. Donor embryo transfer has given women a mechanism to become pregnant and give birth to a child that will contain their husband’s genetic makeup. Although donor embryo transfer as practiced today has evolved from the original non-surgical method, it now accounts for approximately 5% of in vitro fertilization recorded births.
Prior to this, thousands of women who were infertile, had adoption as the only path to parenthood. This set the stage to allow open and candid discussion of embryo donation and transfer. This breakthrough has given way to the donation of human embryos as a common practice similar to other donations such as blood and major organ donations. At the time of this announcement the event was captured by major news carriers and fueled healthy debate and discussion on this practice which impacted the future of reproductive medicine by creating a platform for further advancements in woman's health.
This work established the technical foundation and legal-ethical framework surrounding the clinical use of human oocyte and embryo donation, a mainstream clinical practice, which has evolved over the past 25 years.
A Cochrane systematic review updated in 2012 showed that blastocyst stage transfer is more effective than cleavage (day 2 or 3) stage transfer in assisted reproductive technologies. It showed a small improvement in live birth rate per couple for blastocyst transfers. This would mean that for a typical rate of 31% in clinics that use early cleavage stage cycles, the rate would increase to 32% to 42% live births if clinics used blastocyst transfer.
Embryo transfer in animalsEdit
Embryo transfer techniques allow top quality female livestock to have a greater influence on the genetic advancement of a herd or flock in much the same way that artificial insemination has allowed greater use of superior sires. ET also allows the continued use of animals such as competition mares to continue training and showing, while producing foals. The general epidemiological aspects of embryo transfer indicates that the transfer of embryos provides the opportunity to introduce genetic material into populations of livestock while greatly reducing the risk for transmission of infectious diseases. Recent developments in the sexing of embryos before transfer and implanting has great potential in the dairy and other livestock industries.
Embryo transfer is also used in laboratory mice. For example, embryos of genetically modified strains that are difficult to breed or expensive to maintain may be stored frozen, and only thawed and implanted into a pseudopregnant dam when needed.
Frozen embryo transfer in animalsEdit
The development of various methods of cryopreservation of bovine embryos improved embryo transfer technique considerably efficient technology, no longer depending on the immediate readiness of suitable recipients. Pregnancy rates are just slightly less than those achieved with fresh embryos. Recently, the use of cryoprotectants such as ethylene glycol has permitted the direct transfer of bovine embryos. The world's first live crossbred bovine calf produced under tropical conditions by Direct Transfer (DT) of embryo frozen in ethylene glycol freeze media was born on 23 June 1996. Dr. Binoy Sebastian Vettical of Kerala Livestock Development Board Ltd has produced the embryo stored frozen in Ethylene Glycol freeze media by slow programmable freezing (SPF) technique and transferred directly to recipient cattle immediately after thawing the frozen straw in water for the birth of this calf. In a study, in vivo produced crossbred bovine embryos stored frozen in ethylene glycol freeze media were transferred directly to recipients under tropical conditions and achieved a pregnancy rate of 50 percent. In a survey of the North American embryo transfer industry, embryo transfer success rates from direct transfer of embryos were as good as to those achieved with glycerol. Moreover, in 2011, more than 95% of frozen-thawed embryos were transferred by Direct Transfer.
- Cohen J, Simons RF, Fehilly CB, Fishel SB, Edwards RG, Hewitt J, Rowlant GF, Steptoe PC, Webster JM (March 1985). "Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage". Lancet. 1 (8429): 647. doi:10.1016/s0140-6736(85)92194-4. PMID 2857991.
- "Genetics & IVF Institute". Givf.com. Archived from the original on 6 December 2012. Retrieved 22 September 2016.
- Wennerholm UB, Söderström-Anttila V, Bergh C, Aittomäki K, Hazekamp J, Nygren KG, Selbing A, Loft A (September 2009). "Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data". Human Reproduction. 24 (9): 2158–72. doi:10.1093/humrep/dep125. PMID 19458318.
- Evans J, Hannan NJ, Edgell TA, Vollenhoven BJ, Lutjen PJ, Osianlis T, Salamonsen LA, Rombauts LJ (2014). "Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence". Human Reproduction Update. 20 (6): 808–21. doi:10.1093/humupd/dmu027. PMID 24916455.
- Wikland M, Hardarson T, Hillensjö T, Westin C, Westlander G, Wood M, Wennerholm UB (July 2010). "Obstetric outcomes after transfer of vitrified blastocysts". Human Reproduction. 25 (7): 1699–707. doi:10.1093/humrep/deq117. PMID 20472913.
- Farquhar C, Rishworth JR, Brown J, Nelen WL, Marjoribanks J (December 2014). "Assisted reproductive technology: an overview of Cochrane reviews". The Cochrane Database of Systematic Reviews (12): CD010537. doi:10.1002/14651858.CD010537.pub3. PMID 25532533.
- Groenewoud ER, Cantineau AE, Kollen BJ, Macklon NS, Cohlen BJ (2013). "What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review and meta-analysis". Human Reproduction Update. 19 (5): 458–70. doi:10.1093/humupd/dmt030. PMID 23820515.
- Mains L, Van Voorhis BJ (August 2010). "Optimizing the technique of embryo transfer". Fertility and Sterility. 94 (3): 785–90. doi:10.1016/j.fertnstert.2010.03.030. PMID 20409543.
- Dar S, Lazer T, Shah PS, Librach CL (2014). "Neonatal outcomes among singleton births after blastocyst versus cleavage stage embryo transfer: a systematic review and meta-analysis". Human Reproduction Update. 20 (3): 439–48. doi:10.1093/humupd/dmu001. PMID 24480786.
- Papanikolaou EG, Fatemi H, Venetis C, Donoso P, Kolibianakis E, Tournaye H, Tarlatzis B, Devroey P (February 2010). "Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer". Fertility and Sterility. 93 (2): 592–7. doi:10.1016/j.fertnstert.2008.12.088. PMID 19243755.
- Rebmann V, Switala M, Eue I, Grosse-Wilde H (July 2010). "Soluble HLA-G is an independent factor for the prediction of pregnancy outcome after ART: a German multi-centre study". Human Reproduction. 25 (7): 1691–8. doi:10.1093/humrep/deq120. PMID 20488801.
- "Unisense FertiliTech A/S Receives CE Mark of Approval for EmbryoScope(TM) Embryo Monitoring System".
- Meseguer M, Rubio I, Cruz M, Basile N, Marcos J, Requena A (December 2012). "Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study". Fertility and Sterility. 98 (6): 1481–9.e10. doi:10.1016/j.fertnstert.2012.08.016. PMID 22975113.
- Armstrong S, Arroll N, Cree LM, Jordan V, Farquhar C (February 2015). "Time-lapse systems for embryo incubation and assessment in assisted reproduction". The Cochrane Database of Systematic Reviews. 2 (2): CD011320. doi:10.1002/14651858.CD011320.pub2. PMID 25721906.
- Wu YG, Lazzaroni-Tealdi E, Wang Q, Zhang L, Barad DH, Kushnir VA, Darmon SK, Albertini DF, Gleicher N (August 2016). "Different effectiveness of closed embryo culture system with time-lapse imaging (EmbryoScope(TM)) in comparison to standard manual embryology in good and poor prognosis patients: a prospectively randomized pilot study". Reproductive Biology and Endocrinology. 14 (1): 49. doi:10.1186/s12958-016-0181-x. PMC 4995783. PMID 27553622.
- Jain, John (25 March 2015). "Embryo Transfer". Dr. John Jain on Youtube. Retrieved 17 December 2015.
- Gergely RZ, DeUgarte CM, Danzer H, Surrey M, Hill D, DeCherney AH (August 2005). "Three dimensional/four dimensional ultrasound-guided embryo transfer using the maximal implantation potential point". Fertility and Sterility. 84 (2): 500–3. doi:10.1016/j.fertnstert.2005.01.141. PMID 16084896..
- Sroga JM, Montville CP, Aubuchon M, Williams DB, Thomas MA (April 2010). "Effect of delayed versus immediate embryo transfer catheter removal on pregnancy outcomes during fresh cycles". Fertility and Sterility. 93 (6): 2088–90. doi:10.1016/j.fertnstert.2009.07.1664. PMID 20116786.
- "ASRM and VirtaMed to create embryo transfer simulator". PR Newswire. 7 April 2015.
- "Launch of Embryo Transfer Simulator". VirtaMed. 17 October 2015.
- "New Law regarding the number of embryos transferred in Greece". newlife-ivf.co.uk. 22 December 2014.
- Fertility: assessment and treatment for people with fertility problems. NICE clinical guideline CG156 - Issued: February 2013
- Mullin CM, Fino ME, Talebian S, Krey LC, Licciardi F, Grifo JA (April 2010). "Comparison of pregnancy outcomes in elective single blastocyst transfer versus double blastocyst transfer stratified by age". Fertility and Sterility. 93 (6): 1837–43. doi:10.1016/j.fertnstert.2008.12.137. PMID 19249756.
- Fauque P, Jouannet P, Davy C, Guibert J, Viallon V, Epelboin S, Kunstmann JM, Patrat C (August 2010). "Cumulative results including obstetrical and neonatal outcome of fresh and frozen-thawed cycles in elective single versus double fresh embryo transfers". Fertility and Sterility. 94 (3): 927–35. doi:10.1016/j.fertnstert.2009.03.105. PMID 19446806.
- Gelbaya TA, Tsoumpou I, Nardo LG (August 2010). "The likelihood of live birth and multiple birth after single versus double embryo transfer at the cleavage stage: a systematic review and meta-analysis". Fertility and Sterility. 94 (3): 936–45. doi:10.1016/j.fertnstert.2009.04.003. PMID 19446809.
- Maheshwari A, Griffiths S, Bhattacharya S (2010). "Global variations in the uptake of single embryo transfer". Human Reproduction Update. 17 (1): 107–20. doi:10.1093/humupd/dmq028. PMID 20634207.
- Ghobara T, Vandekerckhove P (January 2008). Ghobara T, ed. "Cycle regimens for frozen-thawed embryo transfer". The Cochrane Database of Systematic Reviews (1): CD003414. doi:10.1002/14651858.CD003414.pub2.
- Crawford G, Ray A, Gudi A, Shah A, Homburg R (2014). "The role of seminal plasma for improved outcomes during in vitro fertilization treatment: review of the literature and meta-analysis". Human Reproduction Update. 21 (2): 275–84. doi:10.1093/humupd/dmu052. PMID 25281684.
- Zarutskiea PW, Phillips JA (2007). "Re-analysis of vaginal progesterone as luteal phase support (LPS) in assisted reproduction (ART) cycles". Fertility and Sterility. 88 (supplement 1): S113. doi:10.1016/j.fertnstert.2007.07.365.
- Khan N, Richter KS, Blake EJ, et al. Case-matched comparison of intramuscular versus vaginal progesterone for luteal phase support after in vitro fertilization and embryo transfer. Presented at: 55th Annual Meeting of the Pacific Coast Reproductive Society; 18–22 April 2007; Rancho Mirage, CA.
- Goudge CS, Nagel TC, Damario MA (August 2010). "Duration of progesterone-in-oil support after in vitro fertilization and embryo transfer: a randomized, controlled trial". Fertility and Sterility. 94 (3): 946–51. doi:10.1016/j.fertnstert.2009.05.003. PMID 19523613.
- Teman, Elly. 2010. Birthing a Mother: the Surrogate Body and the Pregnant Self. Berkeley: University of California Press.
- Blakeslee, Sandra (4 February 1984). "Infertile Woman Has Baby Through Embryo Transfer". The New York Times. Retrieved 1 May 2010.
- "HUMC - Celebrating 50 Years of Caring". humc.edu.
- Friedrich, Otto; Constable, Anne; Samghabadi, Raji (10 September 1984). "Medicine: A Legal, Moral, Social Nightmare". Time. Retrieved 1 May 2010.
- "The New Origins of Life". Time. 10 September 1984. Retrieved 1 May 2010.
- Glujovsky D, Blake D, Farquhar C, Bardach A. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology" Cochrane Database of Systematic Reviews 2012, Issue 7. Art. No.: CD002118. doi:10.1002/14651858.CD002118.pub4
- Embryo Transfer in Cattle Archived 14 May 2008 at the Wayback Machine. Retrieved 21 October 2008.
- Embryo Sexing Technology Archived 2 March 2009 at the Wayback Machine. Retrieved 21 October 2008.
- Wilmut I, (Rowson LEA) (1973). "Experiments on the low-temperature preservation of cow embryos". Vet Rec. 92: 686–690. doi:10.1136/vr.92.26.686.
- Leibo SP, Mazur P. 1978. Methods for the preservation of mammalian embryos by freezing. In: Mapletoft. History and bovine embryo transfer. Anim. Reprod., v.10, n.3, p.168-173, Jul./Sept. 2013 173
- Leibo SP, Mapletoft RJ. 1998. Direct transfer of cryopreserved cattle embryos in North America. In: Proceedings of the 17th Annual Convention of AETA, 1998, San Antonio, TX. San Antonio, TX: AETA. pp. 91-98.
- Voelkel SA, Hu YX (1992). "Direct transfer of frozen-thawed bovine embryos". Theriogenology. 37: 23–37. doi:10.1016/0093-691x(92)90245-m.
- Hasler JF, Hurtgen PG, Jin ZQ, Stokes JE (1997). "bovine embryos frozen". Theriogenology. 48: 563–579. doi:10.1016/s0093-691x(97)00274-4.
- Binoy Sebastian Vettical, Kuruvilla Varghese and K.Muraleedharan. Cryopreservation of Embryos in Ethylene Glycol Freeze Media and Direct Transfer in Crossbred Cattle in the Tropics, Compendium: 9th International Congress on Biotechnology in Animal Reproduction
- Leibo SP, Mapletoft RJ. 1998. Direct transfer of cryopreserved cattle embryos in North America. In: Proceedings of the 17th Annual Convention of AETA, 1998, San Antonio, TX. San Antonio, TX: AETA. pp. 91-98.
- Stroud B (2012). "The year 2011 worldwide statistics of embryo transfer in domestic farm animals". IETS Newslet. 50: 16–25.