Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-04-17
2002-12-03
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S019000, C435S025000, C435S026000, C435S975000
Reexamination Certificate
active
06489135
ABSTRACT:
FIELD OF INVENTION
The present invention enables the determination of various biological characteristics of in vitro fertilized embryos, including overall embryo health, implantability, viability, and likelihood of successful development to term. More specifically, the present invention concerns analyzing media specimens of in vitro fertilization cultures for levels of bioactive lipids in order to determine the optional characteristics for media formulation to increase embryo viability and reduce the frequency of multiple births.
BACKGROUND
As many as 30% of couples attempting to conceive suffer from infertility. Due in part to the increasing age of pregnancy, infertility is on the rise. For men, infertility is usually caused by low sperm count or poor motility. For women, common causes include oviduct occlusion, abnormal ovulation cycles, and inhospitable vaginal mucus. Treatment options are available, depending on the source of the problem. However, even with treatment, many couples are not able to conceive in the traditional fashion.
In vitro fertilization (IVF) has been used increasingly to assist infertile couples in becoming pregnant. IVF generally includes the steps of: recovery of mature eggs from the female patient or a donor; incubation of the eggs in culture media; collection of sperm from the male patient or a donor; culture and preparation of the sperm for addition to the egg culture media (including capacitation procedures); fertilization of the eggs by the sperm; monitoring the embryos during early embryogenesis; and, finally, transfer of fertilized oocyte into the uterine cavity.
To harvest the oocytes, gonadotropin is administered to the ovaries of the donor to stimulate follicle growth and to increase the number of oocytes that mature in a single cycle. Just prior to ovulation, oocytes in the late stages of first meiotic division are aspirated from mature ovarian follicles. Generally, harvested oocytes are placed in a culture medium and fertilized by adding semen or cultured sperm. In some cases, sperm are micro-injected directly into the oocyte, bypassing the normal sperm/oocyte fusion process. After fertilization, the zygotes are microscopically monitored to the eight-cell stage, and further into blastocysts. Healthy embryos with normal phenotypic development and morphology are chosen for transfer by visual inspection. The embryos are then transferred directly into the uterine cavity through the cervical opening using a thin catheter.
Only about 20% of the couples utilizing IVF, however, carry an infant successfully to term. To increase the chances for a successful pregnancy, three or four embryos are usually transferred at the same time. Although a single pregnancy is desired, multiple pregnancies often result. Multiple pregnancies pose health risks for the mother during pregnancy, and can cause emotional and economic problems in family planning. The alternative of terminating one of the pregnancies poses a difficult ethical and emotional dilemma for many couples, and successful delivery of the remaining fetus cannot be ensured. Therefore, there is a need for the ability to select single embryos which are likely to develop successfully upon transfer into the uterus. Similarly, there is a need for the ability to de-select embryos that are unlikely to survive to term. As an embryo's probability of survival generally decreases with culture time, a need also exists for the ability to rapidly assess embryo health as early as possible after fertilization.
In recent years, platelet activating factor (PAF) has been discussed as a possible indicator of embryo viability. PAF (1-alkyl-2-acetoyl-sn-glycero-phosphocholine) seems to be involved in an autocrine hormonal signaling pathway in early embryogenesis. PAF also seems to be involved in paracrine pathway between the mother and the child, and has been detected in the culture medium of embryos that develop successfully to term. However, hormonal signaling in the early stages of development is complex, and a multitude of pathways and signaling mechanisms are involved in embryogenesis. PAF alone has not proven a sufficient indicator of embryo viability, and the role of small hormone molecules both in indicating and maintaining embryo health is not well understood. There is no current adequate molecular marker, or combination of markers, which indicates proper embryonic development at the very early stages of life. The structural formula for PAF is shown below (although the alkyl chain may be of varying lengths):
SUMMARY OF INVENTION
Thus, in a primary aspect, the present invention provides the ability to determine the implantability, overall viability, and likelihood of successful development of an embryo fertilized in vitro. The present invention comprises analyzing the presence or concentration of one or more bioactive lipids in culture media specimens obtained from IVF cultures containing the embryo. Differences in levels of specific types of bioactive lipids in an IVF culture sample specimen where the bioactive lipid concentrations deviate significantly from the levels of bioactive lipids found in culture specimens from embryos which successfully developed to term, those deviations reveal differences in the biochemical environment in which the embryo develops and are indicative of a difference in probability that the embryo will implant or develop normally to term. The present invention contemplates analyzing bioactive lipids from either single or multiple embryo cultures in media, as well as co-cultures of single or multiple embryos grown on a bed of feeder cells and using this analysis to make informed choices about the likelihood of successful fertilization and full-term development.
Specifically, the invention contemplates measuring the presence or concentration of bioactive lipids, comparing these concentrations to a normal, pre-existing, or established values for the specific embryo or media, and correlating these differences to the underlying biochemical status of the embryo, including the probability that the embryo will accede through viability to normal development, or will result in a multiple-birth pregnancy. Also, The ability to analyze the underlying biochemical status of the embryo provides the ability to analyze the chemical composition of the media and the ability to alter the chemical composition of the media through supplementation with chemical or biochemical agents to affect the viability of the embryo or the probability of a multiple-birth pregnancy. By analyzing one or more lipids, a multi-component lipid profile can be constructed for the culture media such that the lipid profile of the culture media can be correlated to viability, or, where appropriate, the composition of the culture media may be changed according to the concentration of one lipid or the multi-lipid profile. Moreover, an individual embryo, or group of embryos, can be monitored over time to determine how the lipid profile and the culture media is altered through natural development or through chemical or biochemical intervention to titrate the composition of the culture media to reach a desired level indicative of viability or the avoidance of multiple pregnancies. Therefore, the invention enables both the selection of healthy embryos and the deselection of unhealthy embryos to increase the probability of success of in vitro fertilization procedures. The ability to transfer a single healthy embryo with an increased probability of success, instead of several embryos with unknown probabilities of success, will result in fewer undesired multiple birth events.
Suitable bioactive lipids for analysis in the present invention include lysophospholipids, phospholipids, sphingolipids, and combinations and subspecies thereof. In addition to analyses of bioactive lipid species, precursors and metabolites of these bioactive lipids, such as glycerophosphatidyl compounds, may also be analyzed in the method as indicators of embryo health and viability. Various analytical methods of measuring the levels of bioactive lipids in the emb
AtairginTechnologies, Inc.
Leary Louise N.
Orrick Herrington & Sutcliffe LLP
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