The process for in vivo fertilization, that is, the natural conception, occurs when the spermatozoid penetrates the ovum inside the oviduct. The fertilized ovum (the zygote) then undergoes cell divisions, and its initial single cell is multiplied into 2, 4, 8 cells, morula and blastocyst. This is the final stage of the embryonic development, when the embryo reaches the uterus and fixes firmly into the endometrium (the internal layer of the uterus).

The development of the fertilized ovum until the stage of blastocyst takes approximately five days and special resources in the laboratory are required for this development. Until a few years ago, the embryo transfer to the uterus was made after two or three days maximum after the ova had been collected. Although the embryos in the blastocyst stage were more capable of being implanted, the methods for the culture of blastocysts were inefficient and delicate.

Then, a new and simpler culture system was created, to develop blastocysts. This resulted in a surprisingly high pregnancy rates. The increase of the pregnancy rates enabled fewer embryos to be transferred to the uterus, and therefore fewer multiple gestations. The culture of embryos to the blastocyst stage brings very important clinic implications.

One of the implications is the better selection of capable embryos, once the embryos that have reached the blastocyst stage and overcome the conditions of the culture in the laboratory have better performance, and thus better chances for implantation. As blastocysts are embryos with greater capacity for implantation, we can decrease the number of embryos transferred to the uterus, without decreasing the pregnancy rates.

The biopsy of the embryo in the blastocyst stage results in that a relatively large quantity of cells are removed. The DNA (the genetic material) of these blastocysts can be analyzed and a number of mutations can be detected by techniques of fluorescent PCR (Polymerase Chain Reaction). The genetic diagnosis in the blastocyst stage has greater accuracy, since more cells are removed and as a consequence pregnancy rates increase.

Regardless of the great progress in the techniques for Assisted Reproduction-mainly after the improvement of the fertilization rates with the Intracytoplasmic Sperm Injection (ICSI)-the implantation rates continued unchanged. One of the possible reasons was that the blastocysts were not able to adhere to the endometrium and experience the hatching process, due to inadequate culture conditions.

The assisted hatching technique consists of the opening of a "hole" in the pellucid zone (the layer that covers the embryo) during the in vitro development period. This is made to facilitate the cells of the blastocyst to pass through and adhere to the endometrium. This technique has been debated and is the origin of controversies in the medical literature. The articles published have shown opposed results and few of them have been prospective and randomized in order to clarify the true usefulness of this technique.

The assisted hatching is performed generally in the third day of the in vitro culture (when the embryo has approximately eight cells), through a number of techniques:

• Partial zone dissection (PZD:): partial dissection of the pellucid zone, using a micro-pipette,
• Zone drilling: chemical opening of the pellucid zone through partial digestion with Tyrode acid,
• Zone thinning: thinning of the pellucid zone with Tyrode acid or laser,
• Point-like opening of the pellucid zone with laser,
• Point-like mechanical opening of the pellucid zone with Piezo's micro manipulator,
• Complete removal of the pellucid zone with acid.

The main cause of embryo loss in the Human Reproduction and in the in vitro fertilization is the high incidence of chromosomal abnormalities originated from chromosomal disjunction in the ovum or in the embryo. These abnormalities are more frequent with increased age. So, how can we determine aneuploidies and other genetic mutations in the human embryos?

The preimplantation genetic diagnosis is the genetic analysis of the embryo before they are transferred to the uterus. This technique consists of the removal of one or more blastomeres (embryo cells) with the micro-pipette, without impairing the subsequent development of the embryo. The chromosomal constitution of these blastomeres can be analyzed by fluorescent in situ hybridization (FISH) or by polymerase chain reaction (PCR). The determination of aneuploidies (genetic abnormalities) can help in the identification of the embryos with best capacity for implantation and normal development.

The preimplantation genetic diagnosis also helps in identifying dominant or recessive mutations that create important genetic diseases. The option of performing the diagnosis before implantation gives the couple, with high genetic risks, the opportunity to avoid a voluntary interruption of the pregnancy, the only choice for the pre-natal genetic diagnosis.

• The Polar Corpuscle: The biopsy of the polar corpuscle (the structure that contains the nuclear material of the embryo) does not affect fertilization and viability of the resulting embryos, as well as their development after implantation. Verlinsky and Cols¹ have analyzed the genetic diagnosis through the polar corpuscle in 700 cycles of assisted fertilization for the preimplantation diagnosis for the cases of cystic fibrosis, a-1-antitrypsin deficiency, pigmented retinitis, A- and B-hemophilia, thalassemia, Alport, the Gaucher Disease, Tay-Sachs disease, falciform anemia, and chromosomal abnormalities. More than 4,000 fertilized ova have been tested. The embryo transference was accomplished in 75% of the cycles, resulting in a pregnancy rate of 23%. More than 90 healthy children were born, confirming the results of the biopsy of the polar corpuscle.

• Blastomeres from embryos with at least 4 cells: After the in vitro fertilization, the embryos can undergo a biopsy during several stages of the cleavage (division), and the removed blastomeres are prepared to the genetic analysis. On the early 90s, the preimplantation genetic diagnosis was used when the couple showed risk of having babies with diseases related to the chromosome X. Today, the fluorescent in situ hybridization with probes combined for both autosome and sex chromosomes is used for sex diagnosis. The polymerase chain reaction (PCR) is generally used in cases of specific genetic imperfections. The worldwide statistics of all cases of preimplantation genetic diagnosis shows pregnancy rates of approximately 26% per embryo transference².

• Blastocyst biopsy:
The biopsy of blastocysts is becoming much more affordable due to the progresses in culture systems that enable the stage of blastocyst to be reached more easily³.
The benefits are:
a) Highly viable due the natural selection of the embryos,
b) The biopsy is performed on the trophoderm, with minimal injuries to the mass of cells,
c) A larger number of cells is available for the genetic analysis.
The cons are:
a) It is technically difficult to remove cells that adhere one another,
b) The embryos must be kept in culture for 2 or 3 days more. The experience with biopsy in blastocysts is still very recent and the clinical data available are not enough to draw consistent conclusions.

1. Verlinsky, Y.; Cieslak, J.; Ivakhnenko, V.; Lifchez, A.; Strom, C.; Kuliev, A. Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body FISH analysis. Fertil. Steril. 1996;66:126-9.

2. Verlkinsky, Y.; Handyside, A.; Grifo, J.; Munne, S.; Cohen, J. et al. Preimplantation diagnosis of genetic and chromosomal disorders. J. Assist. Reprod. Gen. 1994;11(5): 236-43.

3. Carson, S.A.; Gentry, W.L.; Smith, A.L.; Buster, J.E. Trophectoderm microbiopsy in murine blastocysts: comparison of four methods. Assist. Genet. 1993;10:427-33.

Advances in Reproductive Medicine have occurred in parallel with advances in Human Genetics. The presence of deletions (modifications) of genes in chromosome Y and mutations on the gene of the androgenic receptor (the receptor for substances that develop the masculine characteristics) have been related to the masculine infertility. The increased knowledge of the human genome and of the DNA variations in the genes controlling the maturation of the gamete, fertilization, implantation and fetal development, will become key factors in the fertility of the couples.

The chromosome Y mapping identified regions with deletions, like the region AZFc, which is the most common molecular cause identified for failure in spermatogenesis (the process for the production of spermatozoa).*

*Reijo, R.; Alagappan, R.K.; Patrizio, P.; Page, D.C. Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet 1996;347:1290-3.

Oocytes recovered from non-stimulated follicles in the ovary can be maturated in vitro, fertilized and a few can develop and be transferred to patients.

The in vitro maturation of oocytes has many benefits. The most important one is the cost reduction due to the simpler, non-invasive treatment.

The systems for the maturation of the oocytes can be specially useful, for example, to patients with polycystic ovaries. These patients often have a large number of oocytes that can be obtained from small follicles. There is no need to treat with gonadotrophin (hormones) to stimulate the follicular growth.

The successful use of this technique in those patients can expand its application to other patients that might want to avoid the undesired effects of the high doses of drugs for ovarian hyperstimulation.

Until this moment, there have been very few pregnancies reported that do not allow us to draw conclusions about how safe this technique is.

Another aspect that we need to highlight is the possibility of combining this technique with the cryopreservation of oocytes. This enables a more efficient use of the oocytes, and collaborates to the establishment of a donation program and the creation of a oocytes bank. *

*Trounson, A.O.; Wood, C.; Kausche, A. In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil. Steril. 1994;62:353-62.

The nuclear transference can be used to correct mitochondrial genetic diseases.* The nuclei of embryo cells in the early stages of development can be isolated from the mitochondrial elements of the cytoplasm and inserted in a anucleate cytoplasm of the ovum of a donor, with normal mitochondrial function.

Another alternative is the transference of a small portion of the cytoplasm of the ovum from a donor to the ovum of the receptor to correct genetic anomalies.

*Cohen, J.; Scott, R.; Schimmel, T.; Levron, J.; Willadsen, S. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 1997;350:186-7.

The cryopreservation (freezing) of oocytes can offer solutions to a series of ethic, religious and legal problems related to the freezing of embryos. With this technique, it is possible to keep the fertility of women who suffer from pathologies that can impair the ovarian function, like precocious ovarian failure, endometriosis, cysts and pelvic infections. The cryopreservation of oocytes can also make the logistics easier for the programs of ovule donation.

However, the protocols for cryopreservation of oocytes show technical problems that result in low survival rates after unfreezing and low number of gestations. One of the reasons raised would be the possible induction of aneuploidies after the exposure to cryoprotective agents and to the freezing itself.

More recent researches have shown, however, normal chromosomes and karyotypes after unfreezing. By using the technique of intracytoplasmic sperm injection (ICSI), better fertilization rates have been achieved.

Another important improvement in Reproductive Medicine is the possibility of cryopreservation of ovarian tissue for women who have the objective of auto-transplantation, culture of primordial follicles, or the use of xeno-transplant techniques to recover ovules with development ability. This technique is particularly recommended to young patients with cancer, and who wish to preserve their germinating cells in order to avoid the damages caused by radio- or chemotherapy.*

Experimental studies in animals demonstrate that after freezing, unfreezing and auto-transplantation of fragments of the ovarian cortex, ovulation cycles could be established as well as fertility. Progresses in follicle maturation of frozen and unfrozen tissues for in vitro fertilization, however, still evolve slowly.

*Newton, H.; Fisher, J.; Arnold, J.R.; Faddy, M.; Gosden, R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum. Reprod. 1998;13:376-80.

The birth of the "Dolly" female sheep was one of the most debated events in the last few years. For the first time it was demonstrated what we can call "nuclear equivalence" in adult cells of any species, the proof that the nuclei of somatic cells contain all the genetic material necessary to create a living animal through nuclear transference. It also demonstrated that different phenotypes (at nuclear level) can be reversed.*

The technology that was developed during these studies made it possible not only the accurate genetic modification of several species of animals, but also enabled the cellular differentiation process to be disclosed, which can lead to a new range of therapeutic possibilities for human diseases.

The production of animals from one single cell makes it possible that the cells be genetically modified in culture and selected before the animal is produced. The production of animals with multiple genetic modifications requires the addition, removal or modification of the sequence of genes.

The genetic modification of cells in culture can provide not only the development of the transgenic technology, but it can also facilitate genetic modifications that otherwise would be unlikely. Transgenic animals can have an important role in a series of therapies for human diseases:

• Production of human proteins in transgenic animals. Human proteins can be produced in several tissues and body fluids like blood, urine and milk. The greatest benefit of the bio-pharmaceutical production in transgenic animals resides in the fact that high volumes can be produced with low cost;
• Modification of the animal milk to increase its nutritional value or to remove allergenic substances;
• Use of animal organs and other tissues in transplants to humans. Organs of the pig, for example, are very similar to those of the man, and they are considered appropriate for transplantation. The largest problem, however, is the rejection. Considering the fact that not all rejection mechanisms are completely understood, we know that the most important antigen is the a-1,3 galactose, which is found in the pig but not in humans and therefore they trigger the immunological response. The nuclear transference of cells in culture can make it easier to break the genetic code of the pig for the a-1,3 galactosyl transferase, thus eliminating the risks of rejection. Potential organs for human transplantation can be the heart, lungs, kidneys, liver and pancreas,
• The technology of nuclear transference can also be useful to produce models of diseases in species that are physiologically similar to the man, in order to study their evolution and possible therapies.

The development of the techniques for Assisted Reproduction represents a huge progress in the treatment of infertile couples. This technology, however, gets into important and fundamental aspects of the life of these couples, and of the society. Like any other area of the human knowledge where the scientific progress go faster than the debate in the society and the creation of new laws, the Assisted Reproduction involves legal and ethical aspects whose debated is essential.

Some of the aspects for thought:

• Who can decide about the right of performing the treatments of the assisted fertilization?
• Who can decide which couples should be treated?
• Who can decide about the cryopreservation, the number of embryos frozen, the embryonal reduction, or about the donation of gametes and embryos?
• With the improvement of the techniques for preimplantation genetic diagnosis a key question arises: How far can we go?
• Regardless of the clear and diversified benefits of animal cloning, what can we say about cloning of humans?

As a conclusion, we can say that the rapid development of new techniques and resources in Human Reproduction have been able to help countless of couples. It is necessary and essential that these techniques be used skillfully, based in well-proved clinical conclusions and based in serious scientific research and ethics. The debate in the society and in the appropriate agencies must be stimulated in order to guide the scientific community with a clear and ethical line of direction, in the future development of this technology.