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IVF Steps

Steps of an IVF Cycle

Micromanipulation Techniques

Superovulation Stimulates Egg Development

The controlled «superovulation» techniques used in IVF are designed to stimulate the ovaries to produce several eggs (oocytes) rather than the usual single egg as in a natural cycle. Multiple eggs increase the potential availability of multiple embryos (fertilized eggs) for transfer and ultimately increase the probability of conception.fertilite_IVFsteps_Superovulation

The medications required to boost egg production may include, but are not limited to the following: Lupron (gonadotropin releasing hormone-agonist), Antagon or Cetrotide (gonadotropin releasing hormone-antagonist), Follistim, Bravelle or Gonal-F (FSH, follicle stimulating hormone), Repronex (combination of FSH and LH, luteinizing hormone), and Pregnyl or Novarel (hCG, human chorionic gonadotropin). Each is administered by injection only. Most medications are given subcutaneously (beneath the skin), though some are intramuscular injections (into the muscle).

Risks associated with injectable fertility medications may include but are not limited to, tenderness, infection, hematoma, and swelling or bruising at the injection site.

Retrieving the Oocytes (egg retrieval)

For IVF, collection of eggs is usually performed under transvaginal ultrasound guidance. To accomplish this, a needle is inserted (under IV sedation) through the vaginal wall into the ovaries using ultrasound to locate each follicle.

The follicular fluid is drawn up into a test tube to obtain the eggs. Although patients are given pain medications intravenously and are carefully monitored by an anesthesiology staff, some women may experience some discomfort during the procedure.

Generally, the oocyte (egg) retrieval takes 20-30 minutes. Patients are usually discharged home within hours after the retrieval. Risks of oocyte (egg) retrieval may include, but are not limited to, the following:

1.– Potential reactions from the drugs and procedures used in the administration of anesthesia.
2.– Risks associated with the passage of the needle through the vagina into the ovaries (including infection, bleeding, inadvertent damage to adjacent structures including, but not limited to, the bowel, bladder, blood vessels, ureter, uterus or ovary(ies), and adhesion formation (internal scarring) following the procedure. Although uncommon, significant bleeding or damage to the bowel may occur, and surgery may be required to repair such damage; this is a very uncommon event. Rarely, infection may become severe enough to require hysterectomy and/or removal of one or both ovaries.

Collecting and Preparing the Sperm

A semen sample will be obtained from the partner by masturbation on the day of the oocyte (egg) retrieval. This is usually obtained while the retrieval is being performed. Abstinence from ejaculation for two to five days prior to providing this semen specimen is recommended. After the specimen is produced, the sperm will be prepared for inseminating the collected eggs in our laboratory.

Because this can be a stressful time period for men, the man/partner may be unable to produce a specimen when needed.

Men who feel that they may have difficulty producing a semen specimen have the opportunity to have their specimens frozen by our laboratory ahead of time for use in this situation. Testicular biopsy can also be performed as a method to extract sperm for IVF.

Insemination of Eggs and Embryo Culture

Following egg retrieval, the follicular fluid is immediately transferred to the adjacent laboratory for identification of eggs, evaluation, and preparation for insemination. In the process of collecting the follicular fluid, it is possible that a large number of eggs may be retrieved. It is strongly recommended that all of these eggs be inseminated to maximize the number of embryos available for subsequent transfer. Any objection(s) to this policy should be stated in writing and attached to the IVF-ET consent form with the understanding that pregnancy success may be reduced. Otherwise, the prepared sperm will be added to each egg and they will be allowed to incubate overnight under controlled laboratory conditions. The next day, each egg is evaluated for evidence of fertilization. However, it is possible that no eggs are fertilized. If this happens, the laboratory staff will re-inseminate the eggs or perform intracytoplasmic sperm injection (ICSI) in hopes of obtaining embryos for transfer. If fertilization still does not occur, the eggs will be discarded and the remainder of the procedure will be cancelled. In the case of severe male factor, the couple may be asked to consider the option of using anonymous donor sperm (obtained through a licensed sperm bank for use as a «backup» or secondary sperm source) if it is not possible to obtain sufficient sperm from the partner at the time of fertilization.

The eggs that have fertilized will be allowed to develop for two or more additional days under controlled laboratory conditions before they are placed inside the woman’s uterus. Depending upon the couple’s wishes, some fertilized eggs/ embryos may be frozen and stored for future use.

After the embryos are transferred to the womb, the woman will continue progesterone supplementation that begins on the evening of your egg retrieval procedure. Progesterone can be taken as a combination of oral troches and rectal/vaginal suppositories or by injections. Insemination of Eggs and Embryo CultureAdministration of these medications after egg collection has been shown to create a more favorable uterine environment for the embryos, which therefore increases pregnancy rates. Side effects of progesterone may include, but are not limited to the following:

1.- Vaginal dryness;
2.- Bloating, breast tenderness;
3.- Depression, mood swings;
4.- Delay of menses.

Synthetic progesterone-like medications have been associated with certain birth defects. By using only natural progesterone, the risk of drug-induced birth defects is significantly reduced. It is important to note, however, that birth defects occur in approximately 3% of spontaneously-conceived pregnancies in the USA. Therefore, use of natural progesterone does not guarantee a child without a birth defect.

Transferring Embryos to the Uterus

Embryos are transferred on either day three or day five of development. The embryologists at FERTILITE are highly-skilled in identifying «healthy» embryos and in some cases will recommend that a patient extend embryo development to day five, known as the blastocyst stage. Blastocyst transfer has become quite common in IVF cycles as it can increase chances for success while decreasing the likelihood of multiples. Your physician will work closely with the embryologists to determine if a day three or day five transfer would be ideal for your cycle.

Embryos are transferred to the uterus through a small tube (catheter). This procedure is much like a pap smear and does not require any anesthesia and is usually painless. The embryos are placed in a small amount of fluid inside the catheter, which is passed through the cervix at the time of a speculum examination. The embryos are placed in a manner so they reach the top part of the uterus. The number of embryos transferred depends on individual circumstances of the couple, and this decision will be made collectively by you, your physicians and the embryologist. Typically, two to four embryos are be transferred in one treatment cycle.

Embryo transfer can cause mild cramping. Although unlikely, during the embryo transfer the embryo(s) may be displaced through the cervix (causing loss of embryos) or into the fallopian tubes (causing possible tubal pregnancy). There is a small risk of bleeding or infection as a result of the transfer procedure.

After transfer, the woman may get dressed and leave after a brief recovery period. A pregnancy test will be done twelve to fourteen days after the transfer, regardless of the occurrence of any uterine bleeding.

The transfer of several embryos increases the probability of success. A multiple embryo transfer also increases the risk of a multiple pregnancy.Any multiple pregnancies carries an increased risk of miscarriage(s), premature labor and premature birth as well as an increased financial and emotional cost. Pregnancy-induced high blood pressure and diabetes are more common in women pregnant with more than one fetus. Prolonged hospitalization may be necessary for these pregnant women and for the mother and babies after delivery. Tubal (ectopic) pregnancy is also possible, and a combination of normal pregnancy and ectopic pregnancy may occur. A tubal pregnancy is a condition that may require laparoscopy or major surgery for treatment. Like spontaneous (natural) conceptions, pregnancies that arise through IVF may result in miscarriage. In the event of a miscarriage, a dilatation and curettage (D&C) may be necessary.

Couples going through therapy must choose and formalize their choice in the appropriate FERTILITE consent form by indicating one of the following options for handling of any remaining embryos:

1.– Freezing (cryopreservation) of remaining embryos for use by the couple in future treatment cycles

2.– Anonymously donating the embryos for use by another infertile couple(s), if the donating couple and the donated embryos meet the screening criteria (You will not receive any money for this donation, nor will FERTILITE «sell» them.

FERTILITE reserves the right to cryopreserve (freeze) any donated embryos as well as the right to discard any donated embryos if a suitable woman cannot be identified to receive the embryos)

3.– Allowing the embryos to develop in the laboratory until they perish, at which time they would be disposed of in a manner consistent with professional ethical standards and applicable legal requirements (This usually occurs within six to eight days after egg collection).

4.– Alternatives to IVF-ET:
Depending upon the individual and unique cause(s) of infertility for each couple, the chance of conception through alternative means, including intrauterine insemination (IUI) and medicinal therapy, other than IVF-ET may or may not exist. Possible success rates of these alternatives may vary depending upon the type and severity of the cause of the infertility. For some couples, it may even be possible to conceive spontaneously without a physician’s help. You should discuss these alternative treatment methods with your physician before you proceed with IVF-ET therapy.



Since the birth of the first baby achieved through conception outside of the human body in 1978, the principles of «in vitro» (literally «in glass») fertilization and culture have remained the same – careful establishment and maintenance of a well-controlled, sterile environment in which the normal physiology of fertilization and early development can be played out relatively undisturbed to provide healthy embryos for transfer back into the body. During the ensuing two decades, much has been learned, however, about the tolerances of such a system and how this technique can be exploited to treat a widening range of infertility cases. There have been great strides made in development of more appropriate culture media that has enabled embryos to be grown for extended periods of time in culture. Surplus embryos and possibly eggs may now routinely be cryopreserved in liquid nitrogen for use in subsequent attempts at pregnancy. Fertilization itself is no longer a hit-and-miss affair with the advent of assisted fertilization through micromanipulation. Embryos can be micro-manipulated for cell biopsy to determine their genetic status as well as aid in their ability to implant through drilling into their outer shell (assisted hatching).


Through the controlled application of ovarian hyperstimulation, it is current practice to time the retrieval of mature oocytes (eggs) from a woman’s ovary. The yield may vary anywhere from one to 30 or more eggs that may be retrieved depending on the responsiveness of the ovaries to the gonadotropins used to stimulate them. These eggs are gathered by the embryologist into an appropriately balanced salt solution and maintained at body temperature (37°C) until such time as they are ready to be inseminated. Meanwhile, a sample of semen containing the sperm destined to be used for each specific set of eggs is collected and processed by cell separation techniques to provide as clean and active a sample of sperm as possible. A major emphasis of the IVF laboratory is directed toward guaranteeing that the correct sperm go with the right eggs through good labeling and check systems. Ultimately, following several hours in culture, eggs and sperm can be mixed and allowed to bind and fertilize in a relatively natural fashion. Depending on the quality and maturity of both eggs and sperm, it is common for fertilization rates to vary considerably relative to the original number of eggs collected. Twenty eggs retrieved in no way guarantees 20 embryos. Likewise, 20 fertilized eggs in no way guarantees that there will be 20 embryos of sufficient quality for both cryopreservation and fresh transfer to the woman’s body.

Central to the question of how many embryos are actually utilized in any IVF treatment cycle is the period during which the embryos are cultured in vitro. This can be as little as one day, or up to seven in the case of blastocyst growth and transfer. Assuming that culture conditions are relatively optimal, there is less and less reason not to culture embryos throughout their pre-implantation stages to allow the embryos to «select» themselves for transfer or cryopreservation. The blastocyst is the term given to the very last stage of an embryo prior to it implanting into the endometrial lining of the uterus. The poorer the rates of blastocyst growth are, the more restricted the choice of embryo is at this stage of development. In any event, growth of any embryos to the blastocyst stage improves the level of discrimination of embryo viability available to the embryologist, and is a key to reducing the numbers of embryos used for uterine transfer. The more confidence a clinic has in the viability of the embryos it transfers, the less need there is for multiple transfers of three or more embryos. Thus with the transfer of three or less embryos, the risk of multiple pregnancies is significantly reduced, in turn minimizing risks of pregnancy loss or fetal abnormalities common in multi-fetal pregnancies.

Micromanipulation Techniques


Micromanipulation is the technique whereby sperm, eggs and embryos can be handled on an inverted microscope stage, performing minute procedures at the microscopic level via joysticks that hydraulically operate glass micro tools.


Micromanipulation first saw clinical use in IVF for purposes of assisted fertilization in the treatment of male factor infertility, where fertilization potential was low in cases of poor sperm quality. The ultimate evolution of this approach has been the development of the single sperm injection procedure referred to as Intracytoplasmic Sperm Injection, or ICSI. Sperm of virtually any quality and from any level of the male reproductive tract may be used with the only criterion for use being that the sperm is alive even if it is not moving (motile). Dead sperm may be able to achieve fertilization; however, the DNA or genetic material from such sperm is too degenerate to form a viable embryo. Immature sperm from the testicle or the epididymis can be retrieved for use with ICSI for men who possess no sperm in their ejaculated semen (azoospermia). This azoospermia is either due to an obstruction in the tract (obstructive), or to extremely low production of sperm in the testicle itself (non-obstructive).


With the almost unlimited potential to achieve some level of fertilization with ICSI regardless of sperm quality, it would seem that male factor infertility would no longer be of concern. It must be noted, however, that sub-fertility in men can be related to certain numerical and structural defects of the chromosomes and, therefore, there is a strong recommendation for all couples that achieve pregnancies from ICSI to undergo prenatal screening. In certain cases of obstructive azoospermia, there is a higher incidence of cystic fibrosis in the male. Hence, before embarking upon treatment of the more extreme forms of male factor infertility, it is advisable to have some cytogenetic screening performed. Incidentally, very subtle compromise in sperm quality may well be responsible for a marginally lower embryonic viability rate and a slightly higher early miscarriage rate even if such embryos implant. Such observations have led to the suggestion that the technique ICSI itself is at fault; but this misses the point that ICSI per se is not causing the problem, merely facilitating the use of sperm, which under other circumstances would never have even achieved fertilization.


The use of ICSI is now routinely applied to a range of clinical situations wherever there is a possibility that conventional in vitro fertilization may be suppressed or not occur. Such situations include the following: idiopathic or unexplained fertility; hyper-responsive ovarian stimulation cases where egg quality may be reduced; post-thaw sperm samples that survive poorly; post-thaw egg insemination; generation of embryos for pre-implantation genetic screening where embryos «clean» from any extraneous contaminating sperm is needed; or, indeed, any case where there is an extreme need to maximize normal fertilization, for example, when a woman has only a few eggs retrieved. It is possible to «rescue» cases following complete failed conventional fertilization with ICSI. The viability potential of these «late-fertilized» embryos is approximately half of timely fertilized embryos; nevertheless, they do generate successful live births. ICSI has become such a common feature of IVF therapy that it is fast becoming the insemination technique of choice.


It has been proposed that a certain number of otherwise viable embryos do not implant simply because they are unable to break free from the surrounding «jelly coat» (zona pellucida) when they reach the blastocyst stage of development. Around an unfertilized egg there exists a transparent glyco-protein coat that acts to protect the egg and regulate normal fertilization by any penetrating sperm. This jelly-like coat continues to protect the early preimplantation embryo until, as a blastocyst, the embryo fills itself up with fluid like a water-filled balloon, pumping itself larger and larger until it ruptures and «hatches» from the zona pellucida. The embryo is now ready to make contact in its naked form with the endometrium and implant. Inappropriate ovarian environment due to advanced maternal age or other factors that may compromise the follicular environment may in certain cases render the zona pellucida thicker or tougher. Such IVF cases may benefit from the application of a form of micromanipulation referred to as «assisted hatching» In this process, the embryo has a hole made in the surrounding zona pellucida prior to transfer to enable it to «hatch» free from the zona pellucida more easily when it expands as a blastocyst in the uterus. This technique has never been unconditionally proven to be effective in any well-defined group of IVF patients, and as such remains essentially an experimental procedure. Holes in the zona pellucida may be made mechanically, chemically, or by laser. With the advent of more routine transfer of blastocyst stage embryos, the future of this technique, usually carried out on day three of development, may seem in question. Indeed, at the blastocyst stage in vitro, it may be most appropriate to dissolve off the entire zona pellucida prior to transferring naked embryos into the uterus. This could be considered the ultimate form of assisted hatching without the need for micromanipulation. Currently, however, assisted hatching can be easily performed using an infrared laser to create a hole in the zona pellucida that allows the embryo an easy means of escape when it is time to try and implant into the uterine wall.


fertilite_IVFsteps_EMBRYO BIOPSYBriefly, it is of relevance in any discussion of micromanipulation techniques to mention the potential to biopsy both eggs and embryos. This approach is known as preimplantation genetic diagnosis (PGD) and enables the screening of both the unfertilized egg by removal of the first polar body, or the fertilized multi-cellular embryo by removal of one or more cells either at the 6-12 cell stage or from the trophectoderm of the blastocyst. This material can be probed for either genetic mutations or gross chromosomal errors. This technology remains in its infancy and can be of profound importance clinically, but at this time only for cases with very clear medically-defined needs. The biopsy procedure requires very exacting skills of the IVF laboratory, and the egg or embryo is not entirely free of risk during the procedure. Hence, couples whose offspring have a high chance of inheriting a genetic disorder may have their embryos screened. Women who are at risk of generating eggs with a high risk of chromosomal anomalies can benefit from having their eggs or embryos screened for chromosomal normality. While embryos can have their sex determined through this procedure, the FERTILITE team considers it inappropriate to do so except in cases of sex chromosome-linked disorders.


It is a privilege for any scientist or clinician to have access to the earliest stages of human development through culturing gametes and embryos in vitro. And, as such, it requires a high degree of ethical responsibility to provide as safe and optimal an environment as possible for these microscopic changes. While much has been done to maximize IVF pregnancy rates over the last two decades, it nevertheless remains to improve individual embryo selection to the point where we can routinely transfer only one embryo at a time, while being able to successfully and consistently freeze all surplus embryos of sufficient quality for later use in attempting pregnancy.

For futher information of Human Embryo Cryopreservation visit our section

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