The year 1978, when the first IVF baby was born in England, has been the milestone for thousands of couples, which until then were not able to realize the dream of completing their family. Since then, scientific evolution in the IVF field has been tremendous and is expected to be equally impressive in the future; research keeps up lightening unknown features of the miracle of human reproduction while promising more efficient IVF solutions.
Assisted reproduction does not necessarily mean in vitro fertilization. We can describe three assisted reproduction stages of progressive interference: simple ovulation induction with timed sexual intercourse, ovulation induction combined with intrauterine insemination, and application of IVF methods.
- Ovulation induction
- Intrauterine Insemination
- In Vitro Fertilization
- Controlled ovarian hyperstimulation
- Oocyte retrieval (egg collection)
- In-vitro fertilization
- Embryo transfer
- Post embryotransfer period
- Satellite in vitro fertilization techniques
Ovulation induction
Ovulation induction is a relatively simple method of assisted reproduction applied when the cause of infertility is located on the woman's disability to release ova every month (despite the existence of ova in her ovaries). Typical examples of women that may benefit from the method are those suffering from polycystic ovarian syndrome.
Clomiphehe citrate is the most frequently used first-choice drug to induct ovulation; it acts in a quite complicated pattern having as final result a mild ovarian stimulation. This substance is administered orally by form of pills which the woman takes for 5 subsequent days at the beginning of her cycle (usually from the 2nd or 3rd day to the 6th or 7th day of cycle). Clomiphene citrate comes in 50mg tablets. The medication starts with low doses of clomiphene. A few days after the administration of the last pill the woman undergoes serial ultrasonographic scans for the monitoring of follicle(s) growth in her ovaries and for the evaluation of endometrial thickness. When the follicles have achieved a satisfactory size, the day of expected ovulation may either be predicted by detecting an acute rise in blood LH levels that occurs some hours earlier, or be assured through administration of human chorionic gonadotropin which induces final follicle maturation and ovulation. The couple is then advised to have sexual intercourse during the predicted ovulation day. In case that the ovaries do not respond to the initial clomiphene dose, this dose is gradually increased during the subsequent cycles up to a highest 200-250mg daily dose. In the opposite case of an excessive ovarian response after a specific clomiphene dose, sexual intercourse during the fertile days is discouraged to avoid a multiple pregnancy, and the dose is reduced during the next cycle. Clomiphene does not cause any serious side effects; the most commonly reported are temporary disorders of vision, hot flashes, nausea, bloating in the abdomen, and headaches. In the presence of any of the above side effects the woman should contact her attendant physician.
Another way to stimulate ovaries in order to induce ovulation is the administration of fertility drugs called gonadotropins which are the same substances secreted from a woman's own pituitary gland, act on her follicles fostering their growth and maturation, and actually regulate the woman's cycle ensuring the release of her ovum every month. This method of stimulation is applied to women having no ovulation due to absence of own pituitary gonadotropins and to women in whom clomiphene ovulation induction gave no satisfactory results or was related to side effects. Gonadotropins are administered on an everyday basis in form of injection (using small insulin-type syringes or pre-filled pens) usually starting from day 2 of cycle with small doses. According to the selected protocol, the injections are continued for five more days (as in clomiphene induction) or until the ovulation day is determined (by measurements of LH or by chorionic gonadotropin administration). When gonadotropins are used to induce ovulation, the woman's monitoring by serial ultrasonograms and measurements of blood estrogens should be meticulous and performed by experienced physicians to avoid risks arising from excessive ovarian response (such as ovarian hyperstimulation syndrome and multiple pregnancies).
Intrauterine Insemination
Intrauterine insemination is a frequently applied IVF method of moderate interference, during which appropriately prepared sperm is placed into the woman's uterus. This method is adopted as a first-line treatment in couples with mild to moderate sperm disorders or with infertility that is recognized as unexplained. Other indications leading to the adoption of the method are the suspicion of cervical factor infertility (once the sperm is transferred into the uterine cavity the woman's cervix is by-passed), and the cases of azoospermia where sperm retrieval (even by surgical operation to the testicles) is not feasible and thus donor sperm is used (heterologous intrauterine insemination).
Although intrauterine insemination can be applied during the woman's natural cycle (through simple monitoring of her ovarian follicle), it is usually combined with mild ovarian stimulation and ovulation induction to achieve better results. Ovulation induction is performed in the ways described in the 'ovulation induction' paragraph. During the predicted or scheduled ovulation day the couple arrives to the IVF Unit, and the husband gives sperm which is appropriately prepared in the laboratory. A small quantity of the elaborated and improved sperm is injected through thin catheters that pass through the cervix up to the uterine cavity. This technique is almost painless for the woman and does not require any kind of anesthesia. The woman needs to stay at bed for a few minutes and then is able to leave and resume everyday activities.
The possibilities of acheiving a pregnancy after intrauterine insemination depend on how serious the infertility problem is and on the final quality of sperm (after its preparation); it should be noted however that these possibilities do not exceed an 18-20% per effort for the best of cases. Moreover, most studies agree on the point that if there is no pregnancy after 3 or 4 inseminations then we should proceed to the application of IVF.
In Vitro Fertilization
In Vitro Fertilization (IVF) is the most advanced method of assisted reproduction during which the fertilization and first steps of the fertilized ovum's development take place outside the woman's reproductive system, in the laboratory. The candidate couples to this method are those with: obstructed or destructed fallopian tubes in the woman, sperm that presents serious disorders, repeated failures after the application of simpler assisted reproduction methods, advanced age of the woman posing the application of an assisted reproduction technique with more chances of success, or when the aim is a genetic diagnosis before implantation or a pregnancy into the uterus of a surrogate mother. The IVF umbrella covers a series of techniques aiming at optimizing the results.
The main stages of routine IVF programs are as follows:
- Controlled ovarian hyperstimulation:
- Gonadotropins: they stimulate the ovaries; are administered by injection (usually subcutaneous through the abdomen or thigh using a small insulin-type syringe or an easy-to-use pen); must be administered the same time on an everyday basis; their dosage (number of units to be administered) is adapted according to the woman's features and re-adapted according to the ovarian response. Several gonadotropin preparations exist according to the prevalent hormone (FSH, LH, or their combination) and to their origin (such as recombinant hormones or purified human hormones)
- Hypothalamic Gonadotropin releasing hormone's (GnRH) analogues: The administration of such substances suppresses the production of endogenous gonadotropins (i.e. those produced by the woman's pituitary gland). This suppression is essential for our stimulation protocols as it allows us to gain full control of the situation (once we are able to regulate ovarian stimulation by exogenous gonadotropin injections) and it also ensures us that no acute increase in endogenous LH production will occur as the latter would unavoidably result in untimely ovulation leading to failure of the whole attempt. There are two basic analogue types (agonists and antagonists) that differ in the time span needed to achieve pituitary suppression. The analogues are administered the same time on a daily basis by form of subcutaneous injection or more rarely via nose inhalation.
- Chorionic gonadotropin (human or recombinant): it is administered by a single dose injection at the appropriate time (depending on the follicles' growth and the scheduled time for egg collection); it induces final follicle maturation and ovulation.
- Oocyte retrieval (egg collection)
Oocyte retrieval, that is the collection of oocytes (eggs) contained in follicles grown in the ovaries after drug stimulation, is performed 32-36 hours after injecting chorionic gonadotropin. The woman comes to the IVF Unit without having taken any food or drink for the previous 8 hours at least (as she will be administered a mild sedation against pain). Oocyte retrieval requires a thin pipe fitted on the transvaginal ultrasound probe through which a needle that is connected to a suction system is inserted. Under direct transvaginal ultrasound guidance the needle advances towards the ovaries through the vaginal walls to puncture the follicles and draw up the follicular fluid. The embryologist examines the fluid under the microscope to retrieve the oocytes contained. After having punctured all the follicles, the egg collection ends; the woman awakens and is ready to leave the Unit 1 or 2 hours later having received all necessary advice. In the meanwhile and after having confirmed the success of egg collection, the husband is asked to give semen sample to be used for the in vitro fertilization. From the day of oocyte retrieval and on, the woman must commence the appropriate supportive treatment (basically comprising administration of progesterone in form of vaginal gel or suppository, low-dose aspirin and cortisone, antibiotics and sometimes estrogen tablets) aiming at preparing the endometrium to accept the fertilized egg for the achievement of a pregnancy.
- In-vitro fertilization
After the egg and sperm collection, the fertilization takes place at the same day into the laboratory. Ova and spermatozoa are placed in a special culture medium; if there are no problems in the spermatozoa, the latter fertilize the ova. The next day the success of fertilization is evaluated (i.e. the number of embryos that have occurred), the couple is informed about the results, and the embryo transfer day is scheduled. Until then, the embryos remain in the appropriate culture media and grow by continuous cell divisions.
Oocyte in metaphase II 2-pronuclei Stage 2-cell Embryo 
4-cell Embryo 8-cell Embryo Morula - Embryo transfer
The embryo transfer, i.e. the placement of in vitro fertilized ova into the woman's uterus, is usually performed at the 2nd or the 3rd day after the egg collection. In some instances, embryo transfer may be performed at the 5th or 6th day (at the embryonic stage of blastocysts). The woman comes to the Unit at the time agreed, with no need of not having eaten (once she won't be administered any anesthesia); and the embryo transfer is performed under a simple process (not more painful than a Pap smear test): the embryo(s) graded as the 'best' (considering specific morphological criteria) is/are chosen among the available embryos; it/they are then placed in a special syringe containing a small quantity of culture fluid, and through an extra thin catheter inserted by the physician via the cervix is/are transferred into the uterine cavity. The woman stays in bed for a while and then is able to leave the Unit. The number of embryos transferred each time is determined by factors like the woman's age and the quality of available embryos, and is further restricted to avoid multiple pregnancies (as those are the main cause of premature labors with all subsequent negative effects). According to the official instructions given by the Greek Human Fertilization and Embryology Authority, this number should not exceed 3 embryos for women aged up to 40 years, and 4 embryos for women aged over 40. - Post embryotransfer period
The post embryo transfer period is undoubtedly the most nerve stressful of all. The woman should follow strict drug directions and not take any initiatives to modify the treatment scheme without prior communication with the Unit. Staying in bed all this time is unnecessary and not recommended (only a two- or three-day stay in bed after the embryotransfer might be of some benefit). The woman may resume all usual activities as well as her job, provided that this is not extremely tiring). The establishment of pregnancy achievement is done by measuring blood b-chorionic gonadotropin levels (and not by urine pregnancy test) during the 14th day after the embryotransfer. Even in the case that the woman sees vaginal bleeding around the scheduled day of blood test, she must do the test and not interrupt the drug scheme before getting the test results. In the case of a positive result, the woman must contact the Unit to obtain all necessary instructions to proceed with the supportive treatment.
To increase efficiency of IVF methods, we aim at obtaining more than one woman's ova (wherever this is feasible or not contra-indicated) and this is because: one or more of the ova may not be fertilized in vitro, or some of the fertilized ova may not continue their development; we wish to transfer embryos of best quality and therefore we want to have selection choices at the stage of embryotransfer; we may wish to obtain spare embryos which will be cryopreserved for future use in case of failure or of desire for a second child without the woman undergoing a new ovarian stimulation with fertility drugs. The controlled ovarian hyperstimulation protocol should be tailored individually for each couple to achieve best results and avoid complications. For this reason, the treatment of women undergoing ovarian stimulation must be exclusively performed by experienced physicians who are specialized in IVF. Women who follow an ovarian hyperstimulation program may resume all everyday activities while devoting some of their time to a close monitoring whenever this is indicated by the attendant physician. Ovarian stimulation is achieved by applying a specific stimulation protocol which is individually selected for each case. The drugs used in ovarian stimulation protocols are the following:
There are several controlled ovarian hyperstimulation protocols that differ in when the analogue-induced suppression begins. According to the long protocol, we begin with a daily analogue injection at the cycle preceding the one of stimulation (about seven days before the expected first day of menstruation); after the beginning of menstruation and after the success of suppression is proven by measurement of blood estradiol levels we begin stimulating the ovaries through gonadotropin administration. According to the short protocol, we begin the administration of analogue injections at the 2nd day of menstruation, and at the 3rd day we begin the administration of gonadotropin injections. Finally, according to the antagonist protocol we start with gonadotropin injections at the 2nd day of menstruation, adding the antagonist injections when the growing follicles reach an average diameter of 14-15 mm which happens at the 6th to 7th day of stimulation.
A close surveillance of the woman undergoing ovarian hyperstimulation is imperative, and must include serial ultrasound scans and blood estrogen measurements. When short protocols are applied, an ultrasonographic and/or hormonal evaluation should be done before starting with the administration of injections, while in the case of long protocols it would be useful if the suppression success was established by hormonal evaluation and then the stimulation injections would follow. At the 5th-7th day of stimulation the first ultrasonographic evaluation of the treatment is done, and thereafter this evaluation is repeated every 24-48 hours.
Whatever the applied protocol is, when the follicles have gained a satisfactory growth and thus produce adequate estradiol levels the woman will be asked to do the chorionic gonadotropin injection at some time late in the evening (32-36 hours before the scheduled oocyte retrieval). A non-injection day follows, and one day later the oocyte retrieval is scheduled.
Satellite in vitro fertilization techniques
Beyond classic IVF techniques, there is a wide variety of satellite in vitro fertilization techniques applicable to indicated cases
- Intracytoplasmic Sperm Injection (ICSI)
- Assisted Hatching
During the first days of its growth, an embryo is surrounded by a shell called zona pellucida from which the embryo has to escape in order to be implanted. If the zona pellucida is too hard or thick (as it may happen in women of advanced age) then the embryo may have difficulty in exiting the zona which would inhibit implantation. With the Assisted Hatching technique, a hole is opened in the zona pellucida just before the embryotransfer hoping that this will facilitate implantation.
- Blastocyst culture
In human reproduction, the fertilized ovum is implanted into the endometrium when it has reached the stage of blastocyst that is at the 5th or 6th day after fertilization and not at the 2nd or 3rd day. The question was whether the transfer of day-2 or day-3 embryos is practically an obstacle to the synchronization between embryo and receptive endometrium which is essential for a successful implantation. Moreover, a theory was expressed that only embryos of good dynamics will manage to develop until the stage of blastocyst, having therefore more possibilities of being implanted. In vitro blastocyst culture has been made possible after the development of new appropriate culture media; however the results from the application of blastocyst culture and transfer did not fully meet the expectations. There is not a common agreement among international IVF results published in medical literature whether a routine adoption of blastocyst culture and transfer gives better results than the traditional 2 or 3 day embryo transfer. Besides that, a large number of embryos do not manage to reach at the blastocyst stage in the laboratory without being sure that those embryos would not develop if they were transferred into the uterus at an earlier stage; this results in a reduced number of spare embryos for cryopreservation or even in IVF cycle cancellations due to lack of available embryos for transfer. However, nowadays it is believed that the option of blastocyst culture and transfer should be offered for couples with numerous (more than 3) failed implantations, provided that there is a large number of available embryos.
- Embryo cryopreservation - Thawed embryo transfer
- Egg cryopreservation
- Egg donation
- GIFT, ZIFT: gamete or zygote intrafallopian transfer
- Microsurgical sperm retrieval
- Preimplantation Genetic Diagnosis (PGD)
The application of this technique is essential for couples facing serious sperm disorders. This is because in such cases the spermatozoa may not be able to fertilize the ova during in vitro culture.Thus with the help of special micro-instruments a small cut is done on the oocyte's surrounding zona and a spermatozoon is inserted into it, offering great chances of fertilization. The ICSI technique should be performed only when this is necessary as this involves a larger degree of intervention.Watch the related video
Spare embryos that remain available after an embryo transfer during ovarian stimulation/IVF cycles may be cryopreserved at extremely low temperatures (-196°) and remain intact for a long period of time. The cryopreservation procedure may be applied in 1 day-old embryos (at the pronucleate stage) or later in 2 or 3 day-old embryos. Whenever the couple decides so, an embryo thawing program may be arranged with an 80% of cryostored embryos usually surviving, and subsequently a thawed embryo transfer with good chances to achieve pregnancy can be scheduled. Thaw cycles are organized in a way to synchronize the development stage of thawed embryos with the endometrial receptivity. A thaw cycle can be totally 'natural', with a simple monitoring of the developing follicle during a woman's natural cycle and the estimation of the time when her endometrium becomes receptive to the embryo in order to proceed to embryo transfer; or it can be 'artificial', where the woman's natural cycle is inhibited and the necessary estrogens and progesterone are administered by us in order to enable the endometrial receptivity and perform the embryo transfer. It should be noted that the use of frozen-thawed embryos has not been associated to any risks of disorders in children born from those embryos.
This is an option of great value for young women who are going to receive chemotherapy that may damage their ovaries. In addition, many women who are not planning to have family in the near future for social or personal reasons discuss the option of egg cryopreservation. The development of advanced freezing protocols allows for the cryopreservation of oocytes with very good survival rates on future thawing. However, it has to be noted that according to the most recent ASRM guidelines, women who adopt this option should be informed that the chances to deliver a baby from every single frozen embryo are not more that 2%; for this reason a quite large number of eggs must be available for freezing.
The performance of IVF by use of donated eggs is an option that may help certain categories of couples. In couples where the woman suffers from premature failure of ovarian function, early menopause, or has her ovaries been removed, as well as in couples having experienced numerous IVF failures or multiple unexplained miscarriages or recurrent production of very bad quality embryos, the possibility of using donor eggs that will be fertilized by the husband's semen should be discussed. According to the Greek legislation in force, the egg donor must be and remain anonymous, which means that the donor should be one of those women undergoing in vitro fertilization for their own reasons; the donor's identity should not in any case be revealed to the recipient woman and vice versa; the donor should not donate her eggs for any amount of money; the donor must be under 35 years of age; finally the donor together with her husband must sign a consent for donation in written form. A similar consent for egg acceptance must be signed by the couple that will use the eggs.
At the day that the donor eggs are found, they will be fertilized by the semen of the husband of the recipient couple; if the female recipient is synchronized to her current cycle the embryotransfer is organized; if not, the occurring embryos are cryopreserved and transferred during the next properly prepared cycle. It is worth saying that the application of IVF by use of donated eggs gives a boost to pregnancy possibilities for couples having the indication to. Finally, the use of spare embryos from a donor couple is possible in special occasions, and is of course regulated by the relevant medical and legal rules.
These techniques are variations of the classic IVF technique; however as years go by they are less and less applied because they do not give better results than the classic IVF method. The woman follows a typical controlled ovarian stimulation program, and the egg collection is performed; in the GIFT technique the woman undergoes a laparoscopy where ova and spermatozoa are inserted through special catheters into the woman's fallopian tube to achieve fertilization inside the lumen, while in the ZIFT technique in vitro fertilization is performed and the woman undergoes laparoscopy one day later to have 1 day-old embryos transferred into the fallopian tube. These techniques were formerly applied in women with endometriosis, however today the only indications suggesting their application are the intention to perform for some reason a diagnostic laparoscopy at the same time of performing IVF, or in the case of couples having religious restrictions against in vitro fertilization (during GIFT the fertilization occurs inside woman's body).
In selected cases of men with azoospermia there is the option of sperm retrieval using microsurgery techniques followed by fertilization of eggs retrieved from the woman by use of the ICSI technique. The most frequently used techniques are: microsurgical epididymidal sperm aspiration (MESA), testicular sperm extraction (TESE), and testicular sperm aspiration (TESA). These techniques have given impressive results and much of success in cases of men with azoospermia who in the past would have to seek use of donor sperm.
Preimplantation Genetic Diagnosis is a recent medical development that is always combined with application of IVF. Couples with indication to PGD are those with a partner suffering from a genetic disease or with both partners bearing a genetic disease trait, and thus having strong possibility of transferring the disease to their child. In the past, such couples having achieved pregnancy were tested with amniocentesis at the 2nd trimester of pregnancy; if the embryo was found to be suffering from the genetic disease then the couple would proceed to termination of the (already advanced) pregnancy which is a traumatic and quite dangerous experience. Today, these couples have the opportunity to follow a typical IVF program that includes the delicate removal of 1-2 cells from each of the occurring embryos usually at the 3rd day of their growth. The examination of these cells using advanced techniques may help to detect whether the specific embryo from which the correspondent cells were obtained suffers from the disease or not. Thus in a short period of time (1 or 2 days) we are able to know which of the embryos are healthy and proceed to transfer of these embryos only. Common indications for PGD in Greece are couples with both parents bearing b-thalassemia trait or cystic fibrosis trait who have a 25% chance to give birth to a child suffering from the disease. Finally, during the recent years a long international debate exists on whether PGD should be also suggested to couples with history of numerous IVF failures or numerous unexplained miscarriages arguing that the cause may be located to the creation (by those couples) of embryos bearing chromosome disorders that are unable to be implanted or that are aborted. This knowledge is of great value for those couples as they can receive now reliable genetic advice on their reproductive future.