In Vitro Fertilisation (IVF) and Embryo Transfer (ET)
As the acronym denotes, IVF entails the removal of eggs from the ovaries of a female, their fertilisation under precise environmental conditions and the replacement of one or more embryos back into the uterus.
IVF was firstly implemented for the purpose of assisting women with blocked tubes. It was thereafter proven very useful for patients manifesting other fertility conditions inclusive of polycystic ovaries associated with anovulation, endometriosis, poor quality semen and idiopathic infertility. IVF is most often the final resort in a couple’s endeavour to achieve a pregnancy and contemplated when simpler approaches such as ovulation induction and IUI failed to produce a successful outcome. Couples however diagnosed with blocked tubes, severe male factor, and age related infertility IVF represents realistically the only option.
IVF is performed following intensive clinical and laboratory assessment for both partners. The series of pre-treatment testing has already been documented elsewhere in this web page. Rarely, the reason(s) which subject a couple subfertile cannot be determined following utter investigation hence the term unexplained factor infertility. In this respect IVF besides being an effective therapeutic modality can be a very useful diagnostic tool too.The process of conventional IVF is comprised of 5 distinct stages; (1) controlled ovarian stimulation (2) monitoring of ovarian response (3) egg collection (4) egg fertilisation and (5) embryo transfer
1. Controlled Ovarian Stimulation
The aim of this step is to induce the ovaries to respond to hormonal analogs and therefore mature a number of follicles. Each ovarian follicle is composed of roughly spherical aggregations of cells and contains a single egg. Controlled ovarian stimulation regimes endeavour to recruit a number of ovarian follicles at the onset of their administration (days 1-6) and to subsequently promote (day 7 onward) their uniform growth aiming at an optimal egg harvest. The regimen which a physician opts to prescribe depends on a large collection of parameters primarily being the pre-treatment test results, the age of the female partner and the underlying fertility manifestation whilst past medical and surgical history (inclusive of previous IVF attempts) and body mass index are also of importance.
Ovary under controlled ovarian stimulation
Hormonal injections for controlled ovarian stimulation are manufactured either using synthetic (Merional 75/150 IU or Fostimon 75/150 IU or Menopur 75 IU) or recombinant formulations (Gonal-F or Puregon). While several studies have attempted to compare the effectiveness and efficiency between these compounds the results have been contentious. The main hormonal ingredient is FSH while certain analogs may contain low concentrations of the LH hormone too. Based on the parameters described above a high dosage may be initially administered which can either be sustained the same during the course of the ovarian stimulation phase, gradually reduced or indeed subsequently increased pending the initial ovarian response. A high dosage of medication is most often prescribed to candidates with depleted ovarian reserve (so called poor responders) while a low dosage to those who have the propensity toward over responding (i.e. with polycystic or multi-follicular ovaries). The duration of the ovarian stimulation phase may vary between patients but not remarkably. The triggering for final egg maturation is conventionally effected by a single shot of a human chorionic gonadotropin (hCG) injection (Choriomon or Pregnyl or Gonasi) at a dosage of 10000 IU (a recombinant form of hCG namely Ovidrel (0.25 mg) is an equally effective alternative)
The Long Protocol
It works by firstly suppressing reproductive hormone secretion from the organism (by down regulating the pituitary gland) and therefore switching off the action of the ovaries. In this silent state the only stimulatory effect to the ovaries comes from the hormonal injections. The suppression agent known as the agonist can be administered as a single shot injection (i.e. Decapeptyl 3,75 mg or Arvekap 3,75 mg or Lupron 3,75 mg) or in the form of daily injections (Suprefact 1mg/ml or Suprecure 1mg/ml or Decapeptyl 0.1 mg/ml) which can be commenced either from day 2 of a given menstrual cycle in which case successful suppression must be confirmed via an ultra sound scan approximately 12-15 days later (endometrium must be 3mm or less in size) and an estradiol test (a value of 40 pg/ml must be expected) or between day 21 and 23 (in normal menstruating recipients) where a withdrawal bleed signals successful down regulation 10 days post the agonist administration. It must be noted that day 2 suppression may not be effective in approximately 15% of the cases as it may induce the development of a single follicle. Similarly if a day 21-23 agonist administration is advised, this must be preceded by a baseline ultra sound scan to confirm that ovulation had occurred. If ovulation did not happen and suppression is initiated then the risk exists that the non-ovulating follicle will become cystic incorporating delays in the normal progress of the treatment. The long protocol is the commonest regimen of choice. It has been proven to be consistent with a more uniform ovarian response, superior egg harvest and quality. Furthermore it allows for better programming since a patient can sustain the down regulation phase for a prolonged period before controlled ovarian stimulation is commenced without any compromise to the treatment outcome. The Short Protocol
It is most commonly prescribed to candidates who have poor ovarian reserve. Its principle of action is to induce a stimulatory effect early in the cycle by contrast to the long protocol which aims at suppressing. Normally the agonist is administered as from day 2 of a given cycle with contemporaneous injections of stimulation hormones. When injected at an early stage the agonist exerts a stimulatory effect (so called flare up effect) onto the hypophysis which releases for the first 2-3 days the reproductive hormones FSH and LH. Continuation of the agonist thereafter suppresses the pituitary gland therefore inhibiting premature ovulation. The main aim is to increase the stimulation response of the ovaries by combining the volume of hormone released by the organism with that being injected. A shortfall of this protocol may be a discrepancy noted in the size of follicles which gain growth by contrast to the down regulation long protocol counterpart. A more uniform development of ovarian follicles during controlled ovarian stimulation is consistent with a superior number of mature eggs at retrieval. The Short Antagonist Protocol with HCG or Agonist Triggering
This protocol is commenced early in the cycle using similar stimulation drugs to the ones already described. Premature ovulation is prevented by the use of injection compounds namely antagonists (e.g. Cetrotide or Orgalutran). By contrast to agonists which have short lived stimulatory effects antagonists work by desensitizing the pituitary gland immediately.
The administration of the antagonist is commenced once at least one follicle had reached a mean size of 14-15 mm and it is sustained until the day prior to the triggering injection or at least 12 hours before that. This approach to ovarian stimulation most often results in a number of follicles developing faster than others and therefore the number of eggs at retrieval is usually smaller than with the agonist down regulation protocol. Moreover it has been shown to produce inferior pregnancy rates when used in unselected patients. The antagonist protocol is advantageous to poor responders since it avoids suppressing the ovary at the time of follicle recruitment. Patients who have been diagnosed with polycystic or multifollcular ovaries and therefore have propensity to over response are the best candidates for this protocol but a prerequisite is that the triggering for final egg maturation is managed with the use of an agonist rather than the conventional hCG injection. This reduces the possibility of ovarian Hyperstimulation to literally zero since the latter condition is caused by the hCG injection and the associated estrogen release from the ovaries. The agonist injection triggers the release of LH and FSH (the reproductive hormones) at different patterns (4 hours and 12 hours respectively). It has been shown that these hormones remain elevated in the blood stream for 24 and 36 hours respectively. While the amplitude of the LH surge following the agonist triggering is similar to that occurring during the natural menstrual cycle the pattern of release differs between the two (two phases for the agonist and three during the natural cycle). For this reason the overall amount of LH released to induce ovulation with the agonist triggering is much lower than that during the natural cycle. The lower volume accounts for the impaired efficiency of the ovulated follicle (corpus luteum) to support effectively the uterus for embryo implantation. Fresh embryo replacements following controlled ovarian stimulation using antagonists with agonist triggering are consistent with lower pregnancy rates. However, this approach is extremely beneficial for women who consent to the cryopreservation of their eggs following fertilisation for subsequent utilization since the larger number which can safely be harvested will allow a luxury of embryo choice at the time of replacement. For the same reason it can be employed during egg donation cycles or for those who wish to freeze eggs in the context of fertility preservation treatments.
2.Monitoring of Ovarian Response
The response of the ovaries to hormonal stimulation is monitored via ultra sound scan examinations and estradiol blood tests. The estradiol hormone is secreted from specialised cells present within the follicles namely granulosa cells and its value along with the size of each developing follicle is predictive of the egg maturation level. Assessment can commence as early as from day 4 from the onset of controlled stimulation and repeated usually every other day thereafter.
Consistency should exist between the results of the blood tests and the number and size of the follicles developing. During the natural cycle ovulation occurs when the ovarian follicle has developed to approximately 24 mm in mean diameter and when the estradiol level had reached 250-300pg/ml. With controlled ovarian stimulation, the final injection is administered when at least two follicles had reached a mean size of 18 mm with at least one or two at 16 mm. The duration of the stimulation phase can vary but not dramatically. Unpublished institutional data suggest that triggering for egg collection when the stimulation phase endured 9 days or less or 16 days or more is not consistent with good prognosis. The stimulation phase is expected to last for approximately 12 days with a contingency of maximum 2 days prior to triggering for egg collection
3.The Egg Collection Procedure
Egg collection is normally performed between 34-36 hours following triggering for final egg maturation. The patient is asked to fast overnight and arrive at the clinic approximately 15 minutes prior to the designated time of the operation. An interview with the anaesthetist will precede the preparation of the patient for the procedure. The anaesthetist will take records mainly associated with the health status of the patient, her past medical and surgical history and known inherited conditions. The patient will then be asked to change into appropriate gowns and empty her bladder prior to being accompanied by qualified staff into the egg collection theater. The anaesthitist will administer anaesthetic drugs and immediately the gynaecologist surgeon will start the retrieval of the eggs. This procedure is undertaken via ultra sound guidance where a very fine needle targets each of the ovarian follicles so their contents (follicular fluid containing the eggs and other types of cells) are aspirated and emptied into sterile containers. The whole process is performed under a sterile positively pressurized environment in the dark avoiding any temperature fluctuations.
The sterile containers with the follicular contents are passed through to the embryologist who recovers the eggs and stores them in specialty culture solutions under physiological conditions (37 °C, 6% CO2 and 99% humidity) in IVF incubators. The culture media are mainly composed of ultra purified embryo grade water containing salts solutions, amino acids, antibiotics, energy source compounds and human serum albumin as the protein supplement. They provide all the necessary support for the development of pre-implantation embryos. While such materials can be produced ‘in house’, clinics tend to purchase them readymade from commercially available sources mainly for quality control reasons and time effectiveness.
The duration of the egg collection procedure depends on the number of follicles the surgeon has to retrieve but in general it takes no more than 15 minutes. Following completion of the procedure the patient is moved under the guidance of the anaesthetist and a staff nurse to her recovery room where she rests for approximately 20-30 minutes. During the course of the egg collection the male partner is requested to dispose a fresh semen specimen. This is processed similarly to the IUI procedure discussed previously and incubated under physiological conditions until the time of egg fertilisation. A meeting with a specialist takes place following complete recovery of the patient so an update on the results of the egg collection and details on the progress of the treatment are discussed.
The post egg collection medication and mode of administration are also prescribed and explained. Normally the patient is prescribed progesterone and estrogen supplements for uterine embryo implantation support (Cyclogest Suppositories 400 mg/12hours, Progynova 2mg/12 hours respectively), baby aspirin for improved blood circulation (NuSeals 75 mg/24 hours), low dose cortisone (Medrol 16mg/24 hours) for suppressing mildly the immune system and reducing inflammation and broad spectrum antibiotic (Doxat 100 mg/12 hours) as a precautionary measure. The patient should not experience any major discomfort or bleeding which may be a consequence of the egg collection procedure. Certain advice pertaining to the lifestyle and diet which the patient should pursue is given at the time.
4.The Egg Fertilisation Stage
Approximately 4-6 hours post completion of the egg collection procedure the embryologist will manage the fertilisation of the eggs retrieved. This is undertaken via one of two different laboratory procedures.
(I) Conventional IVF
This procedure is in essence the mixing of each egg with approximately 100000 motile spermatozoa in a culture dish well containing 1 ml culture medium. The spermatozoa will actively swim towards the egg which is covered with a mesh of cells namely cumulus oophorus. The sperm must push through this layer of cells to reach the outer egg membrane called the zona pellucida to which they attach through specialised receptors present on its surface. This triggers the release of digestive enzymes from the acrosome of the sperm (a cap-like structure over the anterior half of the sperm head) facilitating their penetration into the perivitelline space which is a narrow fluid filled area between the zona pellucida and the egg cell membrane. The first spermatozoon to achieve contact will fertilize the egg. Within a very short time the membranes of the cells fuse and the contents of the sperm head are released inside the egg. This event causes changes on the cell membrane which together with chemicals which the egg releases form a barrier for further sperm to penetrate. At this stage the zona pellucida hardens preventing any spermatozoa to attach to it. Meanwhile the tight genetic material of the sperm spreads out and a new membrane forms around it creating the male pronucleus. Inside, the genetic material reforms into the 23 chromosomes. The egg which is activated by the fusion of the cell membranes (sperm and egg) completes division resulting in the female pronucleus which also contains 23 chromosomes. The two pronuclei which signal successful fertilisation appear under microscopic view as to round circles
Occasionally two spermatozoa adhere simultaneously on the egg cell membrane incorporating their genetic material to the egg (polyspermy). Such fertilized ovums will arrest early in development due to deranged chromosomal constitution. The chances of an egg fertilizing with IVF is approximately 75-80% when the quality of both gametes is good.
Following fertilisation the eggs are cultured under physiological conditions and monitored for their quality and developmental potential every 24 hours. The implantation potential of an embryo using mere microscopic inspection is based on its capacity to progress consistently with time in culture, the constituent cells (blastomeres) being void of fragmentation and overall uniform appearance of the cells with respect to relative size, shape and distribution within the zona pellucida. Embryos progress by dividing in multiples of 2 every 24 hours from the time of fertilisation. A zygote should develop to 4 cells on day 2 of development, 8 cells on day 3, 16 cells on day 4 etc.
(II) Intra Cytoplasmic Sperm Injection
This technique which was accidentally invented towards the end of 1993 is used to achieve egg fertilisation mechanically with cases of impaired sperm motility, count and morphology in normally ejaculated spermatozoa and during microsurgical sperm aspiration or testicular sperm extraction. Other indications include repeated fertilisation failures using conventional egg insemination and for couples where a fertility presentation could not be elucidated at the time (idiopathic infertility).
Most IVF clinics worldwide use ICSI as the preferred method of choice for managing egg fertilisation regardless the indication. In essence the technique which necessitates the use of delicate micromanipulation systems entails the selection and activation of a single morphologically normal motile spermatozoon and its subsequent release into the inner part of the egg (cytoplasm) by direct injection. Fertilisation rates after ICSI vary between 70%-90% and these are dependent on the skills of the embryologist performing the procedure and the quality of the gametes themselves.
Since the cascade of mechanisms involved during the normal fertilisation procedure is circumvented with ICSI, the egg does not recognize the presence of the sperm in its cytoplasm unless this is immobilized prior to its injection. Immobilization causes a disruption to the sperm tail triggering the gradual disruption of other parts of the sperm membrane facilitating the entry of the sperm nucleus decondensing factor of the egg to induce the swelling of the head. It is because of this swelling that the sperm head plasma membrane breaks releasing sperm associated oocyte activating factors into the cytoplasm of the egg necessary for its activation.
A few hours following these molecular mechanisms, the sperm and egg chromosomes become indistinguishable from each other and further decondensation causes the development of the male and female pronuclei. During the injection procedure aspiration of the egg cytoplasm is necessary to ensure that the oocyte’s membrane is broken therefore the incorporation of the sperm is successfully implemented. The proper orientation of the egg at the time of injection and the needle position are essential parameters towards successful fertilisation minimizing thereby the risks of damage to the egg
5.The Embryo Transfer
Embryo transfers are optimally undertaken on day 5 of embryonic development (blastocyst) for at least two important reasons. Firstly, only embryos which have reached the blastocyst stage have implantation potential therefore embryo transfers which are convicted to a negative outcome are avoided. Besides enabling the correct choice (quality wise) to be made by the embryologist, one or maximum two embryos are selected for replacement in order to minimize the risks of high order pregnancies resulting.
On the morning of the embryo transfer procedure the patient will be advised to arrive at the clinic with a full bladder at least 15 minutes before the designated replacement of her embryos. This minimal time is necessary for discussing with a specialist the results of the laboratory culture pertinent to the number and quality of her blastocysts. Advice is also given with regards to the optimal number which should be replaced.
The patient will have to wear special gowns prior to entering the embryo transfer suite’s sterile environment. All embryos which developed will be demonstrated on a monitor at the time and live images will be taken. These are kept in the clinic’s records for future reference. A copy can be given to the patient if requested. Surplus good quality embryos are cryopreserved for the patient’s subsequent utilization following informed consent.
The embryo transfer at Genesis is performed by initially accessing and cleaning aseptically the orifice of the cervix with sterile water followed by the insertion of the embryo transfer introducer which serves as a guide to the actual catheter the embryos are to be loaded. The exact position of the guide is confirmed amid abdominal ultrasound guidance. Once in position the embryologist will load the embryo(s) onto a very thin and flexible embryo transfer catheter and within seconds this is threaded through the guide into the uterus. The surgeon will inject the embryos with minimal volume of culture medium at a specific area of the endometrium (1 cm from the fundal end) while the patient can witness the procedure.
The successful replacement of the embryo(s) into the uterus is confirmed immediately thereafter by the embryologist who will flush the catheter several times into a sterile dish in the lab. Although extremely rare an embryo may get stuck within the catheter during the procedure. If this occurs it will be reloaded using a fresh catheter so it is replaced successfully. The patient is advised to rest for approximately 10 minutes following the embryo transfer before she is accompanied back into her recovery room. A final consultation then takes place with a specialist so the couple can address any queries which may have at the time while medication administration is revised and a pregnancy test date is assigned.
Following an embryo transfer the recipient is asked to resume normal life and certainly not restrict herself in bed between that time and the day of her pregnancy test. Literally there is nothing one can do to influence the outcome of the IVF procedure other than taking the prescribed medication.
An initial positive pregnancy blood test will be assessed approximately 10-12 days post the embryo transfer procedure and a preliminary indication of how valid the pregnancy is can be determined at the time. A second blood test will be advised so to confirm on an ongoing pregnancy. Traces of the hormone hCG secreted by the embryo confirms on the occurrence of a pregnancy. The units of this hormone should double every 48 hours for an ongoing pregnancy to be confirmed. With the onset of a pregnancy the clinic advises the administration of folic acid at a dosage of 5 mg although this compound can be taken before and during IVF. A date will be given to the couple for the first ultra sound scan which aims at assessing the order and validity of the pregnancy. Normally this is arranged at 6.5 weeks gestation and at the time a fetal heart pulse should be evident on the monitor. When unfortunately the pregnancy test proves negative the patient is advised to discontinue medication so a withdrawal bleed is allowed to occur. A new appointment is scheduled for the couple to meet with a specialist very soon thereafter.