Schematic representation of fertilization.

Conventional IVF

This represents the main laboratory stage of your attempt. A few hours following the egg collection the embryologist places a specific number of motile spermatozoa in the culture dishes containing the eggs.

In conventional IVF there is no further intervention.

The spermatozoa approach the egg on their own and one of them penetrates it and fertilizes it.

VIDEO: In vitro fertilization (IVF)

In more detail, the spermatozoa digest the layers of granulosa cells, meet the oocyte, attach on the zona pelucida, and only one sperm enters the oocytes.

The oocyte reacts to the penetration of the spermatozoon. First, the zona pelucida and cell membrane change their consistency to prevent the entrance of more sperm. Second, the oocyte resumes the second meitic division and extrudes the second polar body. Simultaneously, the male and female pronuclei form and become visible, marking the fertilization of the oocyte.

According to the recently revised definitions and terminology of the International Society for Mild Approaches in Assisted Reproduction (ISMAAR), mild stimulation protocols include:

a) natural cycle,
b) modified natural cycle and
c) mild stimulation protocols.

For further details click on Recent Developments in IVF – Mild Stimulation Protocols

The transfer of frozen thawed embryos may be scheduled in a pharmaceutically controlled cycle or in a natural cycle by monitoring ovulation.

Pharmaceutically controlled cycle

In this protocol, only the endometrium is prepared with drugs for the embryo transfer. Initially, the pituitary function is suppressed in order to prevent a premature LH surge and therefore ovulation, which would result in the release of progesterone and its effect on the endometrium at the wrong time. A cycle with hormonal replacement therapy would be the best solution.

Suppression starts on the 21st or the 2nd day of the cycle, as with the long protocol, the same steps of controlling the suppression are followed. Stimulation of ovarian function is not necessary. During the 2nd phase of the treatment, the endometrium is prepared with pills or patches of 17-β oestradiol (white pills of Estopause or Cyclacur and Dermestril Estraderm patches. Monitoring is performed by vaginal ultrasound, during which the thickness and the structure of the endometrium are checked. At the end, progesterone pills are also administered and the day of embryo transfer is scheduled.

Natural cycle

The transfer of frozen thawed embryos can be performed in a natural cycle. In this case, the development of the follicle is monitored along with the endometrium with a series of transvaginal ultrasounds and hormone measurements. Ovulation is predicted or induced and the day of embryo transfer is scheduled.

These protocols are rarely used and only in special cases. In these protocols, which are usually used in women with particularly poor response to ovarian stimulation, the GnRH agonists are not used and there is a risk of a premature LH surge. In such instances, a GnRH antagonist may also be used. The start date and the dosage of the drugs administered are set by the doctors of our Unit.

Ultra short protocol

The difference of this protocol when compared to the usual short protocol is in the very short duration of the GnRH analogue administration (about three days). In this way, only the stimulatory action of the analogue on the pituitary (which results in an increase in endogenous FSH) is used and not its suppression effect.

Clomiphen-gonadotropin protocol

In the clomiphene-gonadotropin protocols, clomiphene citrate is usually administered from the 2nd to the 6th day of the cycle, and then the gonadotropin administration follows

ΜΑP

In the MAP protocol a contraceptive pill is used in the previous menstrual cycle. The GnRH agonist administration starts on the 2nd day of the cycle in microdoses. The administration of gonadotropins starts on the 3rd day of the cycle.

See more: Mild stimulation protocols

In the short protocol the GnRH agonist is administered almost concurrently with the gonadotropins and it is also known as the Flare-up GnRH agonist protocol. The short protocol is usually selected for women with poor response to ovarian stimulation. In the short protocol:

• The administration of GnRH agonist usually starts on the 1st-2nd day of the cycle and the gonadotrophins on the 2nd-3rd day.
• It is not divided into a downregulation and stimulation phase,
• its duration is about 10-14 days in total.

In the short protocol, with the simultaneous administration of GnRH agonists and gonadotropins we are taking advantage of three stimulation signals of the gonadotropins on the ovaries in order to recruit multiple follicles and to collect more oocytes. These three "triggers" are applied by the increased endogenous FSH that normally increases in the beginning of the cycle, by the increase of endogenous FSH due to the initial stimulating effect of the GnRH agonist in the pituitary and by the exogenously administered gonadotropins via injections. Towards the last days of the short protocol, the suppressive effect of the GnRH agonist in the pituitary also takes place. Variations of the short protocol include the ultra-short and the Microflare (Microdose flare) protocols.

The modified natural cycle is recommended in cases of extremely poor response to ovarian stimulation and also as an alternative in women with multiple failed attempts and in women that do not wish to take any drugs for stimulation. It may also be an alternative solution before oocyte donation is suggested in selected cases.

The scientific team of Eugonia has many years of clinical experience and the know-how in modified natural cycles and has to present an encouraging number of cases with successful pregnancies.

It is recommended as an alternative to patients with particularly poor response to ovarian stimulation, such as patients with several previous failed attempts and embryos of bad quality. The development of the follicle and the endometrium is monitored with a series of ultrasounds and hormone measurements. The disadvantage of the natural cycle is that we place our hopes in one follicle from which we have to retrieve one egg that needs to be mature and fertilize normally, divide on time and create an embryo of a satisfactory quality to proceed with an embryo transfer. Of course, in the natural cycle, there is also a chance of a premature LH surge and follicle rupture that would lead to failed oocyte retrieval.

Corifollitropin is a new drug developed for controlled ovarian stimulation, mainly used in a GnRH antagonist protocol. One single subcutaneous injection on the 2nd day of your menstrual cycle can replace a whole week (7 days) of daily FSH injections. On the 5th day of your menstrual cycle your doctor will prescribe you an antagonist. You may need to continue treatment with extra FSH injections until enough follicles of the adequate size are present.For further details click here

The advantages of the antagonist protocol

It has been quite some time that the focus of the pharmaceutical companies that specialize in the production of fertility treatment drugs has shifted towards the production of drugs that could provide immediate suppression of LH and quick reversibility of that effect. GnRH antagonists met these specifications, although their safety and efficacy had to be proven in everyday clinical practice.

Nowadays, following numerous published studies and metanalyses that followed the first five multicentre phase III studies (2000-2001), we can say that antagonist protocols are patient friendly because:

• They have fewer injections
• smaller treatment duration (at least 12-15 days less when compared to the long protocol)
• smaller gonadotropin dosage in total
• less side effects (hot flashes, night sweats, nervousness, insomnia etc.)
• smaller chance of OHSS (statistically significant)
• Are suitable for modified natural cycles (M.N.C.)
• Are suitable for the application of mild stimulation protocols

Antagonist protocols demand a long learning curve and keeping up to date with all the latest studies, which could explain their initial smaller acceptance by the international scientific community.

The scientific team of Eugonia has proven its everyday knowledge and experience in the antagonist protocols, with original studies published in internationally accepted scientific journals. The effort of the scientific team of Eugonia is internationally recognised as a significant contribution to the assisted reproduction field.

Application of the antagonist protocol

In this protocol, gonadotrophic stimulation begins on the 2nd or 3rd day of the cycle, while the downregulation using the antagonist GnRH analogue follows. The duration of the antagonist protocol is approximately 12-15 days.

In the gonadotropin and antagonist protocol, the ovary receives two consecutive stimulation signals: initially from the increased endogenous FSH in the beginning of the cycle, and then from the exogenous injected gonadotropins. This means that follicle recruitment does not cease from the normally increased endogenous gonadotropins at the beginning of the cycle and that the gonadotropins administered from the beginning of the cycle reinforce follicle recruitment.

The antagonist administration can start either blindly on the 6th or the 1st day of stimulation with gonadotrophins (fixed antagonist protocol), or based on ultrasound and hormone criteria (flexible antagonist protocol).

The contribution of Eugonia to the antagonist protocol

The scientific team of Eugonia has published numerous studies on women undergoing assisted reproduction using the antagonist protocol in internationally renowned scientific journals. These are:

Women with polycystic ovarian syndrome

Comparison of long versus flexible antagonist protocol

Our study is the largest one internationally. It is a randomized prospective study that compares long with flexible GnRH antagonist protocol.

The results show that pregnancy rates are similar in both protocols and there is also a significant reduction (by 20%) of severe ovarian hyperstimulation syndrome when the antagonist protocol is used.

The antagonist protocol is suggested as a protocol of choice for women with polycystic ovaries. The conclusions of this study are adopted by independent editors of international organizations (Faculty of 1000 Medicine) and are honorary selected in "Editor's choice" by Professor Andre Van Steirteghem, who is the editor of scientific journal Human Reproduction.

The flattering comment that the suggested protocol can change the routine clinical practice refers to the finding that the flexible antagonist protocol is the safest protocol in women with polycystic ovaries without reducing their pregnancy rates.

See 1000 Medicine
See Editors choice
See our publication

Comparison of long versus antagonist protocol (D1)

In this study, the long protocol is compared with an alternative antagonist protocol. In this antagonist protocol, the first day of antagonist administration is on the 1st day of stimulation instead of the standard day 6 of stimulation./p>

In this original randomized prospective study the hormone levels are examined, along with the follicle development and the differences between the two protocols are highlighted. The study shows the differences in the hormonal environment during the stimulation and that the fastest follicle development happens with the antagonist protocol. This study has a great theoretical interest in the effort of optimizing the antagonist protocols/p>

The results show similar pregnancy rates for both protocols and reduced length of stimulation duration and reduced appearance of OHSS with the antagonist protocol.

See our publication

Poor responders

In our study on poor responders we achieved significantly higher ongoing pregnancy rates using the flexible antagonist protocol, as compared to the short agonist protocol. This is by far the largest randomized controlled trial in the literature on poor responder patients undergoing IVF treatment, and has been included in several meta-analyses.

See our publication

Antagonist administration according to individually selected criteria increases pregnancy rates

When antagonist administration is applied according to individually selected criteria, this correlates with statistically increased pregnancy rates. A relevant study of the Eugonia scientific team was recently published in scientific journal of the European Society of Human Reproduction and Embryology (ESHRE) titled «In a flexible antagonist protocol earlier, criteria-based initiation of GnRH antagonist is associated with increased pregnancy rates in ΙVF» (Lainas et al., Hum Reprod 2005;20(9):2426-2433).

See our publication

The administration of antagonist in the luteal phase can manage already established severe OHSS

See our publications
Live births after management of severe OHSS by GnRH antagonist administration in the luteal phase.

Management of severe OHSS using GnRH antagonist and blastocyst cryopreservation in PCOS patients treated with long protocol.

Management of severe early ovarian hyperstimulation syndrome by re-initiation of GnRH antagonist.

• Long protocol
• Onset of downregulation in the long protocol
• Downregulation check during the long protocol
• Long protocol starting on the 2nd day
• Long protocol starting on the 21st day
• Long protocol under contraceptive

Long protocol

The long protocol has been in use for quite some time. The first publication was in Lancet scientific journal by Porter and colleagues in 1984 and it is also known as the GnRH agonist down regulation protocol. The basis of this protocol is the down regulation of the pituitary and therefore the prevention of a premature LH surge. It is well known, that a premature LH surge would result in follicle rupture prior to the egg collection and thus in loss of the oocytes.

Pituitary down regulation is achieved with the continuous administration of GnRH agonist analogues. Having established a continuous suppression of the pituitary, the stimulation of the ovaries whose main goal is the recruitment and development of multiple follicles then follows. This means that the communication between the pituitary and the ovaries is cut off and that we are the ones to take over.

The long protocol involves two phases:

• 1st phase: Downregulation using GnRH antagonists (Arvekap, Daronda, Suprefact, Gonapeptyl). Duration approx. 10-14 days.
• 2nd phase: Ovarian stimulation using gonadotrophins (Puregon, Gonal-F, Altermon, Pergoveris, Merional, Menopur, Bravelle) during continuous downregulation of the pituitary. Duration approx. 10-14 days.

The total duration of the long protocol is about 1 month.

Onset of downregulation in the long protocol
Downregulation of pituitary and ovarian function is checked at the end of the 1st phase. You may be informed of the exact date of your downregulation check you may contact the midwives of our unit either on the day of analogue administration or on the first day of your period, so they can schedule your next appointment. The downregulation check usually involves a transvaginal ultrasound and measurement of certain hormones. If downregulation is sufficient you will be given instructions to start the gonadotrophins.

Onset of stimulation in the long protocol
We will contact you in the late afternoon of the downregulation day to confirm the completion of your downregulation and the start of your stimulation. You will be given instructions on the dose, start date and times to take your gonadotrophins. Meanwhile, you will continue to take the GnRH analogue until the end of the treatment at the dose suggested by us.

Onset of downregulation in the long protocol

The administration of the GnRH agonist (Arvekap, Daronda, Suprefact, Gonapeptyl) can start:

1. on the 21st day of the cycle, in a normal 28 day menstrual cycle,
2. on the 2nd day of the cycle,,
3. three days prior the end of the treatment with contraception pills.

Right ovary during downregulation. The ovary appears without any cysts during a transvaginal ultrasound (EUGONIA archive).

Downregulation check. The endometrium appears thin during a transvaginal ultrasound(EUGONIA archive).

Downregulation check during the long protocol

Downregulation of pituitary and ovarian function is checked at the end of the 1st phase. You may be informed of the exact date of your downregulation check you may contact the midwives of our unit either on the day of analogue administration or on the first day of your period, so they can schedule your next appointment.

The downregulation check usually involves a transvaginal ultrasound and measurement of certain hormones. If downregulation is sufficient you will be given instructions to start the gonadotrophins.

Onset of stimulation in the long protocol

Long protocol starting on the 2nd day

In this protocol, the administration of the GnRH agonist starts on the 2nd day of the cycle. In this case, there is an initial is stimulation, followed by suppression of the pituitary function, due the GnRH agonist mechanism of action. As a result of this initial stimulation of the ovary, there is a 15-20% chance of a functional ovarian cyst appearing.

Functional cycst of the right ovary (transvaginal ultrasound) (EUGONIA archive).

Transvaginal aspiration of a functional ovarian cyst by ultrasound guidance (EUGONIA archive).

During the check of downregulation, increased levels of oestradiol are also observed. If an ovarian cyst appears, it is aspirated transvaginally by ultrasound guidance and it usually subsides after few days, as the GnRH agonist administration continues. Downregulation check is then repeated after one week and the treatment continues after confirming downregulation (by ultrasound and blood eostradiol level measurement).

Long protocol starting on the 21st day

In this protocol, the administration of the GnRH agonist starts in the middle of the luteal phase of the cycle. If the cycle is regular (28 days), the start date coincides with the 21st day of the cycle. In the case of an irregular cycle, the ovulation needs to be determined and the start of the GnRH agonist administration is scheduled seven days after that, once a progesterone measurement has been performed in order to confirm that we are indeed in the middle of the luteal phase of the cycle.

Long protocol under contraceptive

In the long protocol under contraceptive, the GnRH agonist administration starts three days prior to stopping the contraceptives (3 pills before the end). This protocol is usually suggested in cases of irregular menstrual cycles, polycystic ovarian syndrome, or in cases of ovarian cysts etc.