Corifolitropin –a (single injection)

Corifollitropin-a (Elonva) is a new recombinant gonadotrophin (FSH) designed for ovarian stimulation.

The drugs used in ovarian stimulation are of vital importance for the development of multiple follicles and oocytes, aiming at increasing pregnancy rates. The protocols of ovarian stimulation currently used, include daily gonadotrophin injections, combined with GnRH agonist or antagonist, depending on the protocol. Daily injections cause a physical burden in women undergoing IVF treatment.

Elonva is administrated once as a single injection at the beginning of the follicular phase and can sustain multiple follicle development for up to 7 days. From Day 5 of stimulation we start administration of GnRH antagonist to prevent a premature LH surge.

Two large clinical trials have evaluated positively the effectiveness and safety of corifollitropin-a. These studies showed that following a single injection of corifollitropin-a, ongoing pregnancy rates, the number of oocytes retrieved, and side-effects were similar to traditional protocols (i.e. using daily injections). Therefore, a single injection of corifollitropin-a is more patient-friendly and can substitute the unwanted daily injections for 7 days.

Corifollitropin-a is mainly used in a GnRH antagonist protocol. Therefore, knowledge and experience on the use of this protocol is a prerequisite for the administration of corifollitropin-a.

The scientific team of Eugonia has extensive knowledge and experience, as well as a series of published papers on the use of GnRH antagonist protocols. We now participate in an international study to evaluate corifollitropin-a. The first results are encouraging and a significant amount of experience has been accumulated.

The new generation GnRH antagonists have introduced a new era in ovulation induction. The international medical community tends to recognize GnRH antagonists's protocols as first-line treatment in assisted reproduction, as they offer many advantages such as:The new generation GnRH antagonists have introduced a new era in ovulation induction. The international medical community tends to recognize GnRH antagonists's protocols as first-line treatment in assisted reproduction, as they offer many advantages such as:

• They are friendly to the patient because they have shorter duration, fewer days of injections, lower doses, lower costs and fewer complications.
• They are offered for personalized ivf treatment, according to the ovarian response of each woman.
• They are associated with significantly lower chance of complications (like OHSS) compared to the long protocol, but offer similar pregnancy rates in women with PCOS.Read more in our published study (Lainas et al., 2010 Hum Reprod).
• They offer significantly higher pregnancy rates in poor responders compared to the short protocol.Read more in our published study (Lainas et al., 2008 Hum Reprod)
• They are associated with higher pregnancy rates when the antagonist is initiated earlier than the traditional 6th day of stimulation(Read more in our published study Lainas et al., 2005 Hum Reprod).
• A GnRH agonist can be administered to trigger ovulation, instead of the traditional hCG injection, completely avoiding the incidence of OHSS in high risk women.
• They are useful on Natural Cycles since premature follicular rupture and oocyte loss are avoided.
• They can be combined with the single injection protocol of corifollitropin –α (Elonva)

PICSI is an innovative technique, developed as an improvement of the simpler microinjection ICSI technique. It is recommended to couples with male infertility issues, thus requiring the use of ICSI, but who have in addition low sperm binding to hyaluronic acid (HBA test) or high sperm DNA fragmentation (DFI).

During ICSI, the spermatozoa are selected based on their morphology and motility. However, in this case, sperm selection is more subjective, as the embryologist selects the spermatozoon based on a visual assessment under low resolution, without having any indication on its dynamics and ability to fertilize the oocyte. On the other hand, PICSI, a physiological ICSI technique, provides sperm functionality testing, thus assisting the embryologist to select the best quality spermatozoon.

PICSI is based on the fact that mature sperm head without DNA fragmentation has a specific receptor that provides it with the ability to bind to a protein called hyaluronic acid, the main component of the oocyte's cumulous oophorous, a natural process essential for fertilization.

PICSI is particularly useful in cases of men who have:

• Low HBA indicator
• High DFI (DNA fragmentation index)
• Previous failed attempts with ICSI
• Embryo development failure in previous attempts
• Bad spermatozoa morphology
• Reduced sperm motility
• Sperm collected through the techniques of TESA or MESA

Next generation sequencing (NGS) constitutes one of the major advances in molecular biology of the last 20 years and has revolutionized genomic research. As a method, it has been applied in multiple fields, such as the food industry, botany, microbiology, forensic genetics and of course, the health sector and particularly in preimplantation genetic diagnosis (PGD).

NGS is a method for sequencing millions of DNA bases in parallel (the DNA's building blocks). More simply, it allows us to "read" almost all "letters" of the "book" that is our DNA. It constitutes one of the outcomes of the Whole Genome Sequencing Program, a fundamental program carried out by international research groups between 1990 and 2003. Since then, the cost and time required for DNA sequencing have dramatically dropped and NGS is now considered an accessible and affordable method for the identification of genetic anomalies.

Regarding preimplantation diagnosis, NGS allows us to simultaneously diagnose a high number of genetic disorders, from the same biological material. Compared to previous diagnostic methods, NGS has a higher sensitivity and accuracy, while it can also diagnose mosaicism in the embryo (differences in the genetic makeup between the cells, leading to false diagnoses). It is capable of identifying changes not only on a chromosomal level, but also in a single gene, by essentially combining the older PGD and PGS methods, and thus providing us with multiple information regarding the embryo’s health. Hence, the healthy embryo that will be selected, has the best chances for a successful pregnancy.

The process itself is similar to the older methods of PGD. The embryos generated in the laboratory following microinjection (ICSI), are cultured until the blastocyst stage on the 5^th day of development. The embryologists are then performing a biopsy, removing a small number of cells from the blastocyst, which are then sent to a genetics laboratory for NGS analysis. The analysis may concentrate on the whole genome, or just a part of it.

Vitrification- Oocyte cryopreservation

Until recently, oocyte cryopreservation remained a challenge in human IVF, as the low survival and fertilization rates of cryopreserved oocytes led to low pregnancy rates. Recently however, the method of vitrification has revolutionized oocyte freezing and it is associated with high rates of oocyte survival, fertilization, embryo quality and pregnancy rates. The method can be applied on fertilized oocytes, cleavage stage embryos and blastocysts.

Vitrification is a method of ultra-rapid freezing. The oocytes are placed in special cryoprotectant solutions and are then plunged directly in liquid nitrogen, where they are stored until future use. Through this technique, the interior of the oocytes reverts to a form of glass, avoiding the formation of ice crystals that are detrimental to oocyte viability.

Numerous studies have been conducted on the success rates and safety of this technique and all report encouraging results regarding the health of children born following the method.

Applications of vitrification

Oocyte cryopreservation is performed during an IVF cycle following ovarian stimulation. It is a method for storing genetic material for women who wish to postpone having a child until later in life, as well as in cases of premature ovarian failure, lack of spermatozoa in the male partner’s semen sample (azoospermia) or following an unsuccessful testicular biopsy on the day of oocyte retrieval.

The experience of Eugonia

In Eugonia, we use vitrification for oocyte and embryo cryopreservation, following the international scientific developments, informing the patients about the method's advantages and disadvantages and we have several successful pregnancies and live births following the use of this technique.

The development of array Comparative genomic hybridization (a-CGH) for preimplantation genetic diagnosis (PGD) provides useful information on the frequency and type of anomalies in gametes and embryos. The technique allows screening of all the chromosomes, contrary to the technique of FISH, which evaluated only a limited number of chromosomes (usually X, Y, 13, 16, 18, 21, 2) and can be applied at the stage of the mature oocyte (biopsy of 1st polar body), zygote (biopsy of 1st and 2nd polar bodies), day-3 embryo (biopsy of one blastomere) or blastocyst (trophectoderm biopsy). a-CGH showed that 20-30% of oocytes are aneuploid in women aged 20-30, while aneuploid oocytes from women over 40 are 50-80%.

The technique’s major advantage is that allows the identification of aneuploidies, deletions, duplications and/or amplifications of any locus represented on an array. It has also proven to be a powerful tool for the detection of microscopic chromosomal anomalies in individuals with idiopathic mental retardation and various genetic abnormalities. Several large-scale studies have demonstrated that a-CGH has a 10%–20% detection rate of chromosomal abnormalities in children with mental retardation/developmental delay with or without congenital anomalies; only 3%–5% of these abnormalities would be detectable by other means. It may also provide accurate information regarding the extent and possible implications of the identified genetic aberrations.

Therefore, PGS using a-CGH is an important development of molecular genetics. The technique can lead to increased pregnancy rates because it can detect chromosomal abnormalities with high accuracy, resulting in the transfer of healthy embryos; efforts are being made to improve the technique even further, so that we can gain information on the chromosomal complement of all embryos, more quickly and with great accuracy. In addition, it is a promising method in cancer research, as well as the diagnosis, classification and prognosis of various malignancies.

Metabolomics is a new method of embryo assessment by studying embryo metabolism. Using this method, we can analyze in a rapid and accurate way the metabolism of embryos, and in combination with their morphological characteristics we can have useful information on the viability of each embryo from a patient's cohort. The method is non-invasive and does not harm the embryos, as it entails the analysis of the culture medium that the embryo has developed in.

Metabolomics can increase pregnancy rates and also reduce the rate of multiple pregnancies, as we can reduce the number of transferred embryos.

The principles of the method are pioneering, but the reproducibility of the measurements needs to be improved, following international preliminary studies, in which Eugonia actively participated. For this reason, the method has been temporarily suspended and an upgraded version is awaited.

The research interest of several scientific teams has been recently focused on the ability of certain embryonic cells to multiply and differentiate in culture leading to the formation of organ tissues. The embryonic stem cells are derived from the inner cell mass of the blastocyst.

Experimental data have demonstrated the ability to direct the differentiation of these cells, under specific culture conditions, into haemopoetic cells, neurons, hepatocytes and myocardium cells.

Further development in this area is an exciting scientific challenge as it would form the base for new therapeutic prospects for many diseases.

At present, pluripotent stem cells from the umbilical cord of newborns can be isolated and frozen for a possible future transplantation and treatment of blood diseases.

The method intends to preserve a woman's reproductive ability prior to aggressive treatments for malignant conditions, aiming to restore normal ovarian function in the future.

A small part of the ovary which contains several immature follicles is removed by laparoscopic surgery or laparotomy and is frozen in Cryopreservation banks.

This confers the following future prospects:

• In vitro maturation of primordial follicles and immature oocytes until they reach the stage of full maturity and are able to be fertilized by IVF.
• Autografting, i.e. transplantation of the tissue in the same woman in order to restore her ovarian function.
• Heterografting, i.e. transplantation of the tissue in another woman who has lost her reproductive ability.

Freezing of ovarian tissue is internationally accepted, but restoration of ovarian function following transplantation is still under investigation. Progress is continuous giving hope to young cancer patients, who may wish to have a child later on in life.

Transplantation of ovarian tissue

A piece of frozen ovarian tissue can be transplanted in the body of the woman from whom it was taken (autografting) after total treatment from a malignant condition.

When the location of transplantation is the anatomic location of the ovary the transplantation is termed orthotopic. If the tissue is replaced in a different part of the body the transplantation is termed heterotopic. In heterotopc transplantation the ovarian tissue can be grafted in a more convenient anatomic position (e.g. under the skin) which facilitates oocyte recovery from follicles developing under drug stimulation

Experimental data have shown restoration of ovarian function for a limited period. Researchers investigate the factors that are responsible for the re-establishment of the function of the transplanted ovarian tissue and the restoration of the woman?s reproductive ability. Autologous and heterologous transplantation is only recommended in special cases after the approval of specialized scientists.