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The IVF Cycle
The therapeutic efficiency of IVF treatment is linked to the number of zygotes (fertilized oocytes) obtained, which in itself depends on the number of retrieved oocytes. Therefore, an ovarian stimulation leading to a “super ovulation” is necessary to induce women to produce a larger number of oocytes (which is 1 under a natural cycle).
Various stimulation protocols and various drugs have been used so far: clomifene citrate, human menopausal gonadotropins (hMG), purified follicle stimulating hormone (FsH), agonists or antagonists of gonadotropin releasing hormone (GnRH). All these substances tend to increase the number of oocytes produced.
In IVF cycles, a so-called “long protocol” is most often used. It includes a complete pituitary suppression of FSH and LH secretions by an analogue of GnRH and a stimulation of follicular recruitment and growth by gonadotropins FsH (Gonal-F, Puregon, Fostimon) or hMG (Merional or Menopur).
Other protocols are considered for particular cases such as high- or low- responders. The doctor always presents the specificities of the chosen treatment before any protocol is started. In general, short use of anti contraceptive pill is applicable (Microgynon 30). This approach synchronizes all available follicles in available at that moment. It results in a smoother stimulation and probably optimises the chances of success. In addition, this approach allows a better control of the precise timing (sequence number of the week) for IVF punction. The brief use of the pill (1 – 30 days), sometimes called “double suppression” is common use by the majority of IVF programs throughout the USA.
Below is a brief description of an IVF cycle.
To stimulate follicular recruitment and growth, two drugs are used:
1.An analogue of GnRH (Decapeptyl®) inhibits the synthesis of pituitary hormones (FSH, LH) and therefore induces a blockade of the natural cycle. The daily subcutaneous administration starts usually at day 4 of the cycle before ending the intake of the anti contraceptive pill. It is continued up to the ovulation induction (during the following cycle (around 20-25 days in total).
2.A follicle-stimulating hormone (FsH; Gonal-F®, Puregon®, Fostimon®, HMG Merional®, Menopor®), administered subcutaneous daily, induces the recruitment and growth of the follicles (ovarian stimulation). The growth of the follicles is monitored with ultrasound imaging (see picture) and by hormonal dosages in blood samples.
Scan of an ovary showing the presence of several follicles (round dark areas). The largest ones measure around 2 centimetres
Step 2: The ovulation induction
A spermatozoon can fertilize the oocyte only when it has reached its maturity (meiotic division has taken place and a polar body has been expelled).
Example of a mature oocyte. We can observe the polar body positioned at 15:00 hours, which indicates that the meiosis has started.
During a natural cycle, the oocyte maturation and the ovulation are induced by a rise in LH, the luteinizing hormone. In a stimulated cycle, the administration of hCG (the pregnancy hormone) mimes the action of LH. This is the ovulation induction. Different hormones, natural or synthesized such as urinary hCG, recombinant hCG or recombinant LH, can be used to perform this ovulation induction.
The ovulation induction is usually carried out 35 hours before the moment of the oocyte pick-up.
Step 3: The oocyte pick-up (OPU)
Most of the OPU are performed under light general anaesthesia in the operating theatre. Exceptionally, a local anaesthesia can be offered. In this case, it is discussed with the doctor and the anaesthetist before the operation.
The follicles are punctured very accurately via the vagina using a needle, which is introduced in each follicle and is attached to a vaginal ultrasound probe. The follicular fluids pass into plastic tubes labelled with the name of the patient. The tubes are then placed in a “hot block” and conveyed to the laboratory. There the oocyte/cumulus complexes (OCC) are searched for (see picture) under a heated dissecting microscope and are transferred immediately to an equilibrated cultured medium and placed in an incubator.
Three cumulus-oocyte complexes. Each COC has a 2-3 mm diameter. Culture dish with 4 wells were the COC are placed in culture.
Step 4: Sperm collection and preparation
The sperm collection is performed in a special room in the laboratory between 09:00 and 10:00 am on the day of the OPU. Intercourse abstinence of 2-3 days is recommended in order to obtain a maximum of motile sperm. When an ejaculation by masturbation is not possible, the collection of sperm can take place during an intercourse using a special condom distributed on request. However, this possibility has to be discussed first with the doctor and the laboratory.
In very rare cases, when the patient produces a sample with a low number of motile sperm, he is asked to produce a second ejaculate within 2 hours of the first. This second sample often contains a higher proportion of motile sperm, although the sperm concentration might be reduced.
Sperm observed after preparation and coloration with Papanicolaou
The head measures 4-5 µm long and 1.50-1.75 µm large; the length of the flagella is about 45 µm (this might vary between individuals).
Preliminary cryoconservation of sperm
In order to avoid functional “breakdown” problems on the day of the punction, with recommend to the husbands to envisage cryoconservation of a “spare” sperm sample which could be used in this particular case. Once informed, it is up to the husband to decide whether this precaution will be applied. If the husband decides to do so, he should get in touch with the laboratory for an appointment. The same abstinence norm (2 – 3 days) applies.
In classic IVF, a few hours after the OPU, 50'000 spermatozoids are joined with the oocyte/cumulus complex. This insemination is performed in four-well dishes (see picture) containing the culture medium. Each dish is identified with the name of the patient and is left inside an incubator at 37°C and in atmosphere of 5% CO2, 5% O2 et 90% N2 in air.
Incubator with controlled atmosphere (CO2 et de l'O2)
Oocyte/cumulus complexes (2 mm large). The oocyte (0.2-0.3 mm in diameter) appears within varying quantities of cumulus cells. Culture dish in which the insemination takes place.
When a low sperm count makes a classic IVF impossible, an l'ICSI is performed.
Step 6: The scoring of fertilization, the culture of embryos and the cryopreservation of surplus zygotes (pronucleate embryos)
Once a spermatozoid has penetrated inside the oocyte, the genetic material from both parents appears as two structures called pronuclei. 16-20 hours after the insemination, zygotes (fertilized oocytes) are identified by the presence of two pronuclei (female and male) and two polar bodies.
Zygotes containing two pronuclei positioned side by side in the centre. The two polar bodies are observable at 12:00 hours.
A maximum of 3 zygotes are left in culture for a transfer of fresh embryos though in most cases the actual transfer takes 2. The surplus zygotes are cryopreserved immediately after the scoring of fertilization. In conformity with the LPMA), cryopreservation has to be performed before the 20th hour after insemination, time which corresponds to the moment when pronuclei disappear and mix their contents before the first division can take place.
The transfer of embryos in the uterus is usually performed 48-72 hours following the OPU. The transfer is done by inserting a fine catheter through the vagina and the cervix into the uterus and is painless.The transfert is donue under US with half full bladder.
Catheter used for the embryo transfer.
It is sometimes necessary to seize the cervix with forceps in order to straightened the uterus and make easier the passage of the catheter through the cervical canal
Once the embryos have been replaced inside the uterus, the patient rests lying down for 20-30 minutes.
Progesterone is administered per vagina is prescribed from the same evening of the day when the punction took place. Our team will explain the doses and modalities of this treatment (support).
Thawed embryo transfers are usually performed in natural cycles substituted with the intake of oestrogen (Progynova® or patches of Estradot®), and progesterone (Utrogeston®, Crinone®) administration.
Mostlyf two embryos are replaced in the patient’s uterus.
Embryo at 4 blastomeres (or cells) stage (45 hours following insemination) and at the eight-blastomeres stage (72 hours after the insemination)
Embryo at the morula stage (96 hours after the insemination) and at the blastocyste stage (120 hours after the insemination)
Embryo at the full-blastocyste stage (144 hours following the insemination)
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