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(October 2004)
Mark P. Trolice, M.D., FACOG, FACS
Board Certified, Reproductive Endocrinology And Infertility
Director, Fertility C.A.R.E.
Milton McNichol, MD, FACOG, FACS
Board Certified, Reproductive Endocrinology And Infertility
Blastocyst Stage Transfer
Preimplantation Genetic Diagnosis (PGD)
Embryo Cryo-Preservation Techniques
(Part One) In-Vitro Fertilization: Then and Now
Almost three decades have passed since the birth of the first IVF baby, Louise Brown. Since then, the Assisted Reproductive Technologies (ART) has evolved into a highly successful and reliable method to assist couples overcome reproductive obstacles and achieve their goal of parenthood. (Part One) In-Vitro Fertilization: Then and Now reviews the historical developments in the clinical application of ART and their resulting impact on reproductive medicine.
In this article we will review recent breakthroughs in ART resulting from refinements in laboratory culture methods and in micromanipulation techniques. Foremost in these developments are the improvements in blastocyst culture, preimplantation genetic diagnosis (PGD), and the extension of cryopreservation techniques.
Blastocyst Stage Transfer
Under usual conditions, five to seven days after fertilization in the fallopian tube, a single human embryo enters the uterus at the blastocyst stage of development. Approximately 50% - 60% of these blastocysts are able to implant and result in a clinical pregnancy. Because of limitations in laboratory culture techniques during the first two decades of clinical IVF, embryos were routinely transferred to the uterus two or three days after conception, at the four-to-eight cell stage of development. Only 10% - 20% of embryos transferred at this stage are able to implant and result in a viable pregnancy.
To overcome this low "implantation rate," often three or more embryos were routinely transferred to the uterus. As anticipated, this led to an overall improvement in the clinical pregnancy rate but high order multiple gestation was a serious and all too frequent complication. During the late 1990s, further clarification of the nutritional requirements of the embryo facilitated the development of "sequential culture media" laying the foundation for blastocyst stage transfer.
Blastocyst stage embryos are cultured in the laboratory for five to six days prior to being transferred back to the uterus and pregnancy rates of 50% - 60% are routinely achieved. As a result fewer blastocysts, usually one or two, can be transferred to the uterus thus significantly decreasing the risk of high order multiple gestations while maintaining high pregnancy rates. With continued refinements in the classification system and criteria for blastocyst selection, transfer of single blastocysts will soon become a clinical reality.
Preimplantation Genetic Diagnosis (PGD)
Improvements in laboratory science and techniques of blastocyst and embryo culture were closely paralleled by developments in micromanipulation techniques. Application of these techniques extends to the science of PGD and currently holds significant potential to revolutionize the field of antenatal diagnosis.
This "prenatal" diagnosis involves clinical and laboratory evaluation of fetal cells or serum in an attempt to detect fetal biochemical or structural derangements in order to avert the birth of a clinically or genetically affected infant. In current practice this diagnosis cannot be made earlier than the 10th to 12th week of pregnancy but is more routinely delayed until the 16th to 18th week.
PGD was introduced during the early 1990s as an alternative to prenatal diagnosis in order to prevent termination of pregnancy in couples with an affected offspring and was initially offered only to couples with a high risk for sex-linked genetic diseases. Embryos obtained by IVF were tested to ascertain their sex, and only female embryos were transferred. Subsequent developments include techniques for genetic analysis at the single-cell level that involves assessment of blastomeres from cleavage-stage embryos or first and second polar bodies from oocytes.
Additionally, the development of fluorescence in-situ hybridization (FISH) has resulted in a reliable chromosomal analysis and aneuploidy screening. Further applications of molecular and genetic sciences have resulted in the characterization and diagnosis of several of the more common single gene disorders using polymerase chain reaction (PCR).
As currently performed, PGD involves laboratory culture of embryos obtained by IVF for 2 days post oocyte retrieval, to the four cell stage. Micromanipulation techniques are then used to remove a single cell or blastomere from each embryo, for molecular analysis. Under usual circumstances, results are available 48 to 72 hours later thus allowing for blastocyst transfer on day five or six of selected genetically healthy embryos. Widespread application of PGD will ultimately result in improved ART success rates, particularly in older patients by identifying genetically incompetent embryos and precluding these from uterine transfer.
Embryo Cryo-Preservation Techniques
Finally, advances in embryo cryo-preservation techniques and currently ongoing investigations into ovarian and oocyte freezing afford significant optimism regarding future developments in ART.
Embryo cryo-preservation involved controlled "super-cooling" or freezing of supernumerary embryos obtained after IVF. Due to limitations in this technique cryopreservation was feasible only on fertilized oocytes/embryos and sperm cells. Because of their inherent ultra-structural and meiotic apparatus, unfertilized oocytes are not routinely cryopreserved.
However, emerging research has made initial forays into solving this clinical enigma. Ongoing research has also resulted in initial success in ovarian freezing and transplantation. With further development of these techniques, options can soon be extended to the care of reproductive age women undergoing treatment for non-gynecological cancers. These options may soon include oocyte or ovarian cryo-preservation prior to using systemic chemotherapeutic agents, thus preserving a woman's reproductive options. While much remains to be elucidated before these techniques become a routine part of our clinical armamentarium, the history of reproductive developments over the last three decades offer significant hope that these puzzles will be quickly solved and become a routine part in the practice of ART.
(*Of note, at the time this article went to press, the September 2004 issue of The Lancet, Belgian researchers reported the first live birth after laparoscopic orthotopic autotransplantation of ovarian cortical tissue in a patient with Stage IV Hodgkin's lymphoma who had undergone ovarian cryopreservation prior to successful chemotherapy
Read Part One of this article to learn about the history of IVF and its major advances today.
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