In Vitro Fertilization (IVF) (test-tube baby), According to Oxford dictionary, the term “In Vitro” means taking place in a test tube or culture dish or elsewhere outside the body of living organism.
Sometime in fertilization it happens that the sperm from the male body cannot reach the female ovum due to some complications or obstruction. In such case the technique of in vitro fertilization is carried out in which the ovum from the female body is taken and then this ovum is fertilized outside the female body by the sperm taken from a male (Elder, Dale, 2003).
The procedures of in vitro fertilization are carried out in a media specifically designed to mimic biochemical constituents of the uterus to promote sperm capacitation, and of the oviduct to simulate the environment needed for oocyte maturation and acquisition of developmental competence for fertilization (Elder, Dale, 2003).
In 1978 Sir Robert G. Edwards, a scientist from Cambridge University with long-standing research interests in animal and human embryology, and Patrick Steptoe, a gynecologist from Oldham in Lancashire who was able to visualize pelvic structures laparoscopically., for the first ever succeeded in vitro fertilization (Figure 1).
This came into the attention of medicine and general public as the birth of first test tube baby, Louise Brown (Figure 2), (The New York Times, 1978; Ledger, 2002; Elder, Dale. 2003; Johnson, 2011).
The birth of Louise Brown occurred after reports of an ectopic pregnancy (a pregnancy in which the fetus develops outside the womb, typically in a fallopian tube) conceived after in vitro fertilization in Professor Charlwood’s unit in Australia, and was soon followed by deliveries of further IVF babies in Australia and the USA (Ledger, 2002).
In 2010, at the age of 85 years, Dr. Edwards received the Nobel Prize in Physiology or Medicine (Johnson, 2011; The New York Times, 2010). However, Dr. Steptoe did not share the award because of his death in 1988 and Nobel Prizes are not awarded posthumously (The New York Times, 2010).
Louise Brown (Figure 2) is the first test tube baby born as a result of in vitro fertilization. Louise Brown was born to Lesly Brown and John Brown by a planned caesarean section. She was born in Royal Oldham Hospital, United Kingdom. The birth of her sister, Natalie Brown, also was the result of in vitro fertilization.
Louise Brown has a son, conceived naturally. Her mother, Lesly Brown, died in 2012. Here is a picture of Dr. Robert G. Edwards, Lesly Brown, Louise Brown and her son Cameron (Figure 3)
Good news for sub-fertile women
The in vitro fertilization technique is good news for sub-fertile women as it can help them achieve pregnancy (Kallen, et al., 2012; Goldberg, et al., 2007; Ledger 2002). Originally indicated for women with tubal factor infertility, in vitro fertilization is generally regarded as a “last resort” for the infertility when other conventional therapy fails or is unlikely to be successful for all causes of infertility (Goldberg, et al., 2007; Ledger 2002). Almost all causes of infertility are potentially amenable to in vitro fertilization; other than situations in which gametes cannot be collected, such as after premature menopause or in azoospermia without sperm in testicular biopsy material. In vitro fertilization is seldom the most cost effective primary treatment for infertility, and many couples prefer alternative approaches, even if less successful per cycle, if such treatments are less stressful for them. Increasing attention is being paid to the concept of preparation for in vitro fertilization including pre-in vitro fertilization surgery for endometriosis or hydrosalpinx (a distally blocked fallopian tube filled with serous or clear fluid), collection and cryopreservation of epididymal or testicular sperm or prolonged pituitary downregulation (downregulation is the effect of chronic stimulation of the gonadotrophs releasing hormones receptors which leads to a reduction in the number and activity of this receptor which results in the suppression of secretion of LH and FSH from the pituitary gland) in severe endometriosis (Ledger 2002). Regardless of the cause of infertility, the treatment that leads to the highest pregnancy rate per cycle is in vitro fertilization. Since its inception in 1978, there has been a remarkable increase in the numbers of in vitro fertilization cycles worldwide. Approximately 1 in 50 births in Sweden, 1 in 60 births in Australia, and 1 in 80 to 100 births in the United States now result from in vitro fertilization. In 2003, more than 100,000 in vitro fertilization cycles were reported from 399 clinics in the United States, resulting in the birth of more than 48,000 babies (Voorhis, 2007).
Most of the technological steps needed for the conception of Louise Brown had been developed in veterinary medicine years earlier. The principle of the in vitro fertilization is simple and consists of the following three steps, these are;
- Mature oocytes (eggs) collection from the ovaries,
- Fertilization of the collected oocytes in cell culture in the laboratory,
- Replacement of the embryo into the uterine cavity at such a time in the cycle to ensure a receptive endometrium (Ledger, 2002; Goldberg, et al., 2007; Elder, Dale. 2003).
However, underlying this simple model is a long story of experimentations and improvements leading to the industrial in vitro fertilization of today. One of Dr. Steptoe and Dr. Edwards’ greatest problems was the insufficiency of oocytes. They were able to harvest only one or two oocytes in the natural cycle and, frequently, by the time that laparoscopy had been arranged, general anesthesia induced and surgery undertaken, the follicle had ovulated and the oocyte was lost in the pelvis, therefore success rates were extremely low. In order to improve, the laboratory scientists needed more oocytes and the clinicians needed to be certain that premature ovulation would not occur (Ledger, 2002; Goldberg, et al., 2007). The problem of the insufficiency of the oocytes was then met by super-ovulation for multiple follicle maturation protocols. As a result of these super- ovulation protocols, a large number of ova are released (Goldberg, et al., 2007; Elder, Dale, 2003).
Screening before in vitro fertilization
Before in vitro fertilization, most couples undergo standard infertility tests including; semen analysis, female reproductive track analysis, ovulation detection tests, trans-vaginal ultrasonography. These tests are performed as there are great variations in ovarian fertility and responsiveness at a given chronologic age. A reduced ovarian reserve is manifested by a weaken ovarian response to medications for ovulation stimulation, resulting in fewer eggs recovery, fewer embryos, and a lower pregnancy rate. Many women with unexplained mysterious infertility are found to have a reduced ovarian reserve when they are tested. A reduced ovarian reserve is usually diagnosed on the basis of either an elevated serum follicle-stimulating hormone level on cycle day 3 or trans-vaginal ultrasonographic findings of a low ovarian volume or few antral follicles. The positive predictive value of abnormal test results is lower among women younger than 35 years of age than among older women, and women older than 40 years of age have a reduced chance of achieving pregnancy even with normal test results. All tests are better at predicting ovarian responsiveness to gonadotropins than at predicting pregnancy. Nonetheless, tests of ovarian reserve provide some predictive information, and the test results are sometimes used to select the ovarian-stimulation protocol (Voorhis, 2007).
Steps of in vitro fertilization
Following are the steps of in vitro fertilization.
Super-ovulation for multiple follicle maturation
There are several basic super-ovulation protocols available. However, a standard super-ovulation protocol involves three basic steps;
- A gonadotropin-releasing hormone (GnRH) analog, such as leuprolide acetate (Lupron), is given as a daily subcutaneous injection starting around cycle day 21 to induce pituitary suppression. This is done in order to prevent a spontaneous luteinizing hormone (LH) surge, leading to ovulation prior to oocyte collection. Pituitary suppression usually requires 10 to 14 days, after which the injection dose is reduced.
- Recombinant follicle-stimulating hormone (FSH) is given after pituitary suppression. It is given as a daily subcutaneous injection for another 10 to 14 days. Subsequent doses and the frequency of follicular monitoring with trans-vaginal ultrasonography and associated serum estradiol levels are individualized based on the ovarian response.
- Human chorionic gonadotropin is given subcutaneously once the lead follicles have achieved a mature size of 18 to 20 mm. The human chorionic gonadotropin simulates the endogenous LH surge to bring about final oocyte maturation (Goldberg et al., 2007).
Mature oocyte collection
Initially in the first decade of the in vitro fertilization, the mature oocytes were recovered though laproscopy but the laproscopic egg collection was costly and time consuming. After the improvements in the quality of the trans-vaginal ultrasound safe needle puncturing of the ovarian follicles and cysts from the early 1990s and onwards, this could be done by the sedation and local anesthesia. This is more convenient and safer for the in vitro fertilization units. However, the laproscopic egg collection is still done in rare cases like endometriosis, adhesion or prior infections making the ovaries inaccessible by the trans-vaginal route (Goldberg et al., 2007; Ledger, 2002).
The oocyte collection is performed at 34 to 38hours after the delivery of human chorionic gonadotropin. The follicular fluid is obtained and examined immediately by the embryologist for an oocyte with its accompanying cumulus mass of granulosa cells. From one to more than 40 oocytes may be collected, though 10 to 20 are typical. The oocytes are then placed in a culture medium which is based on human fallopian tubal fluid and incubated at 37°C. (Goldberg et al., 2007; Ledger, 2002).
A sperm sample is provided by the male partner on the same day as the oocyte collection, unless previously frozen sperms are to be used (Voorhis, 2007). From 100,000 to 200,000 sperm are then added to the oocytes in a small drop of media, or by direct injection of a single sperm using intra-cytoplasmic injection. By the presence of a male and female pronucleuse after 12 to 24 hours fertilization is documented in about 65% of the oocytes. However, lower fertilization rates suggest intrinsic defects in one or both of the gametes (Goldberg et al., 2007; Ledger, 2002).
Culture is continued for the next 24 to 48 hours. Embryos will divide up to the 4 to 8 cell stage and now can be graded for quality by microscopic assessment of morphology. The best two or three embryos are generally selected for transfer. Other “extra” embryos can be cryo-preserved in liquid nitrogen for later use (Ledger, 2002; Voorhis, 2007), the use of cryo-preserved embryos may be cost effective, since ovarian stimulation is not needed (Voorhis, 2007).
Embryos are usually transferred into the uterus 3 days after retrieval and fertilization via a small flexible transcervical catheter. The implantation of the embryo into the endometrium about 4 days later is the step that limits the in vitro fertilization success rates. A strategy to improve embryo implantation is to transfer the embryos 5 days after oocyte retrieval at the blastocyst stage. In theory, the best embryos are able to continue dividing in extended culture, whereas the poorer quality embryos undergo arrested development (Goldberg, et al., 2007; Voorhis, 2007).
In an effort to reduce high-order multiple pregnancies (triplets or more), the Human Fertilization and Embryology Authority (HFEA) and American Society of Reproductive Medicine guidelines recommend that not more than two embryos should be transferred in women under age 35, three embryos in women 35 to 37, four in women 38 to 40 and five in women above 40, although fewer embryos may be transferred. Ultimately, the goal is to transfer a single embryo and to avoid twin gestations as well. This has already been legislated in several countries (Goldberg et al., 2007; Ledger, 2002).
Embryo transfer is a highly charged moment for the patient and her partner. Successful transfer requires a gentle and unhurried approach to the procedure. Properly trained specialist nurses can perform embryo transfer with equivalent pregnancy rates to medical staff and many patients prefer their familiar nurse to undertake the procedure (Ledger, 2002).
Exogenous progesterone is given after embryo transfer to optimize endometrial receptivity for embryo implantation. It may be given by intramuscular injections or various vaginal formulations during the luteal phase and continued until gestational week 8 or 10 (Goldberg et al., 2007; Ledger, 2002).
However, luteal support remains an under-researched area of in vitro fertilization practice, which is prone to superstition and unquestioning belief in favored course of therapies. “Implantation failure” remains the commonest result of IVF treatment and progress will come from basic research on endometrial function, receptivity and implantation in “in vitro” models of embryo – endometrial interaction (Ledger, 2002).
Risks of in vitro fertilization
The risks of in vitro fertilization are;
In general, the transfer of more than one embryo results in a higher rate of pregnancy than single embryo transfer, but it is also associated with a high risk of multiple gestations. Multiple births are the most frequent complication of IVF. Although children from triplet and higher-order multiple gestations are at greatest risk, IVF twins are more likely than IVF singletons to be admitted to a neonatal intensive care unit, to require surgical intervention, and to have special needs and poor speech development. The mothers of these IVF twins gave lower ratings for their children’s general healths than did the mothers of the IVF singletons, and twin births were associated with more marital stress. As compared with women who carry one fetus, women who carry multiple fetuses have a greater need for bed rest and higher risks of premature labor, hypertension, postpartum hemorrhage, cesarean delivery, and, although rare, death. The American Society of Reproductive Medicine has issued voluntary guidelines that have resulted in the transfer of fewer embryos in the United States. This reduction, in turn, has led to a lower rate of triplet and higher-order gestations; however, the rate of twin gestations remains high. One obvious way to reduce multiple gestations is to transfer a single embryo. Although commonly used in Europe, single embryo transfer has not been widely adopted in the United States because patients and clinicians think that the chances of conception are lower with a single embryo than with multiple embryos. In addition, many infertile couples do not recognize the risks of multiple gestations and actually prefer twins as a way of attaining their ideal family size more quickly (Voorhis, 2007; Halliday, 2007).
Adverse Perinatal Outcomes
Even singleton in vitro fertilization pregnancies are associated with a significantly higher risk of adverse outcomes than natural singleton pregnancies, after adjustment for maternal age and other confounding variables. The risk of such results, which include perinatal (period around child birth) death, preterm delivery, low or very low birth weight, and delivery of small-for-gestational-age infants, is approximately twice that associated with naturally conceived singletons. Additional risks include gestational diabetes, placenta previa (the complication in which the placenta is inserted partially or wholly in lower uterine segment), preeclampsia, and stillbirth. The causes of these adverse perinatal outcomes remain poorly understood (Voorhis, 2007; Halliday, 2007).
The majority of in vitro fertilization-conceived infants do not have birth defects. However, some studies have suggested that assisted reproductive technology is associated with an increased risk of birth defects. In the largest U.S. study, which used data from a statewide registry of birth defects, 6.2% of in vitro fertilization conceived children had major defects, as compared with 4.4% of naturally conceived children matched for maternal age and other factors (Halliday, 2007; Voorhis, 2007).
Maternal health risks
The ovarian hyper-stimulation syndrome is a short term result of gonadotropin stimulation and early pregnancy. This syndrome, which occurs in less than 5% of in vitro fertilization cycles, consists of ovarian swelling, pelvic pain, and hemodynamic fluid shifts, often accompanied by ascites (the accumulation of fluid in the peritoneal cavity, causing abdominal swelling). The disorder almost always resolves after several weeks, although in rare cases, death due to thromboembolism (the blocking of a blood vessel by a particle that has broken away from a blood clot at its site of formation) has been reported (Voorhis, 2007; Halliday, 2007).
- Elder K., Dale B., 2000. In vitro fertilization, second edition. Cambridge university press. ISBN: 0 521 77863 8.
- Goldberg J. M., Falcone T., Attaran M., 2007. In vitro fertilization update. Cleveland clinic journal of medicine, Volume 74(5), pp. 329-338.
- Grady D., 2012. Lesley Brown, Mother of World’s First ‘Test-Tube Baby,’ Dies at 64. New York Times. Retrieved 23 June 2012.
- Halliday J., 2007. Outcomes of IVF conceptions: are they different? Best Practice & Research Clinical Obstetrics and Gynaecology, Volume 21(1), pp. 67-81. doi: 10.1016.
- Ledger W. L., 2002. In vitro fertilization. Current Obstetrics & Gynaecology, Volume 12, pp. 269-275. Doi: 10.1054.
- Voorhis B. J. V., 2007. In vitro fertilization. The New England journal of medicine, pp. 356:379-386.
- Wade N., 2010. Pioneer of in Vitro Fertilisation Wins Nobel Prize. New York Times. Retrieved 5 October 2010.
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