EP0880601A1 - Dosage predictif pour le resultat d'une fiv - Google Patents

Dosage predictif pour le resultat d'une fiv

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Publication number
EP0880601A1
EP0880601A1 EP97903495A EP97903495A EP0880601A1 EP 0880601 A1 EP0880601 A1 EP 0880601A1 EP 97903495 A EP97903495 A EP 97903495A EP 97903495 A EP97903495 A EP 97903495A EP 0880601 A1 EP0880601 A1 EP 0880601A1
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EP
European Patent Office
Prior art keywords
hsd
modulator
activity
ivf
individual
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EP97903495A
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German (de)
English (en)
Inventor
Brian Royal Free Hospital School Medicine COOKE
Anthony Royal Free Hosp. School Medicine MICHAEL
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UCL Business Ltd
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Royal Free Hospital School of Medicine
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Publication of EP0880601A1 publication Critical patent/EP0880601A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Definitions

  • the present invention relates to an assay which can be used to assess the likelihood of pregnancy (or infertility) in a female, and especially is predictive of the outcome of in vitro fertilisation (IVF) in mammals (including humans).
  • IVF in vitro fertilisation
  • modulators such as inhibitors
  • ll ⁇ -hydroxy steroid dehydrogenase 110- HSD
  • IVF is an expensive procedure and can be psychologically traumatic for a patient. Surgical procedures are required to collect eggs from a female for IVF and, following fertilization, further surgery is required to implant the fertilised eggs in the womb. The recipient must then wait for a period of time before it can be determined whether or not pregnancy has been established. In some cases, pregnancy may never be achieved despite repeated attempts, and these cases can represent a considerable expense to the patient and society, both in financial and human terms.
  • the adrenal steroid hormone, cortisol is believed to influence maturation of the female germ cell (the oocyte) and the development of ovarian cells in culture 22 - 23 . Recently, it has been reported that there is an association between the concentration of cortisol in follicular fluid and oocyte maturity. 7
  • the enzyme 11/3-hydroxysteroid dehydrogenase (110-HSD, EC 1.1.1.146) converts cortisol and corticosterone to their inactive forms, cortisone and 11- dehydrocorticosterone, respectively. 3 - 5 16 It is present in rat oocytes 3 and appears to modulate ovarian function. 12 Two isoforms of 11/3-HSD have been characterised; a hepatic form and a renal form. Both isoforms are expressed in ovarian tissue. In this specification, reference to 110-HSD includes both (hepatic and renal) isoforms (unless the context requires otherwise).
  • the present invention thus relates to assay methods and assay kits which can be used to assess the likelihood of pregnancy (or, more accurately, successful implantation), and in particular to predict the outcome of IVF in a female individual.
  • the invention also relates to such methods and kits for use in a method of diagnosis in order to determine the outcome of IVF or the suitability of a female individual for IVF treatment.
  • the invention has been developed from research on human females, it is applicable to any mammalian female and can be used to increase the success of, for example, captive breeding programmes of endangered species or commercial breeding by IVF of livestock such as cattle or horses.
  • a first aspect of the invention comprises a method of predicting the outcome of, or assessing the likelihood of success of, IVF, which method comprises:
  • the (or each) sample may comprise a body fluid or a tissue, such as from the environment of the oocyte. This can comprise granulosa-lutein cells or follicular cells as well as other ovarian cells, recovered for example from the ovarian follicles of women undergoing oocyte recovery for in vitro fertilization and embryo transfer.
  • the (or each) sample may comprise a follicular aspirate, for example obtained on an out-patient basis prior to admission to an IVF programme.
  • the sample may also comprise stored (usually frozen) cells from the environment of an oocyte.
  • Other samples can comprise urine or plasma.
  • a particularly preferred sample comprises follicular fluid.
  • the first aspect may thus additionally comprise, prior to (a), removing one or more biological sample(s) from the female.
  • T e sample may be treated (eg. stored, frozen, washed, cultured, added to a medium, etc) before the determination in (a) is conducted.
  • modulator of 11/3-HSD is meant a substance (whether naturally occurring or not) that affects l l ⁇ -HSD activity.
  • the modulator is endogenous. This may mean increasing 110-HSD activity (an agonist, or cofactor) or decreasing activity (an inhibitor), as well as altering the enzyme's specificity or physiological properties.
  • Suitable 113-HSD inhibitors include glycyrrhetinic acid, glycyrrhetinic acid-like factor (GALF), gossypol and bioflavonoids, as well as glucocorticoid hormones or analogues thereof (which can compete with cortisol for 113-HSD).
  • Other compounds that modulate 110-HSD activity include bile salts, cholesterol and steroid hormones (pregnenolone and progesterone both inhibit). Preliminary studies suggest that oestradiol may, in some cases, act as an inhibitor.
  • Inhibitory activity and site of action can vary. For example, studies have shown that pregnenolone and progesterone both inhibit renal and ovarian 110-HSD activities acutely. Also, it appears that oestradiol inhibits hepatic 113-HSD activity in vivo, but seems to have no acute effect on ovarian 11/3-HSD activity in vitro.
  • the level of the lljS-HSD modulator may be measured directly (eg. by determining the amount or concentration of the modulator) or indirectly (eg. by the level of 113-HSD activity, as that will often be affected by the modulator).
  • Direct med ods can thus include chromatography (TLC or HPLC) while indirect methods may include measuring the effect of any modulator in the sample by, for example, adding 11/3-HSD, a substrate of the enzyme (eg. 3 H-cortisol) and determining the affect, if any, on the enzyme's activity.
  • the sample eg follicular fluid
  • the sample may be contacted with 113-HSD present in another body fluid, or cultured cells (eg human granulosa-lutein cells) or other body-derived substances (eg homogenised animal (eg rat) organs, such as kidneys).
  • a parallel, control, assay may also need to be conducted to allow for any 11/3-HSD already (and naturally) present in the sample.
  • the 113-HSD used in the assay may be from an isolated (or purified) source or can be present in another (human or animal) body or body-derived fluid.
  • the latter includes, for example, (eg human) granulosa-lutein cells and organ (eg kidney) homogenates.
  • the 110-HSD used in the assay may not necessarily be human, it may, for example be from an animal species, such as from a rodent (eg rat). This can allow assays to be performed using relatively cheap and accessible forms of 11 J-HSD (eg rat kidney homogenates).
  • the sample (eg follicular fluid) to be assayed can be removed along with an 110-HSD source (eg granulosa-lutein cells). They are then separated, for example the cells cultured for several days, as a control, in the absence of bodily fluids, and then contacted (i.e. reunited) with the fluid. The effect on 11 / 3-HSD levels can then be assayed.
  • This type of assay uses local fluids/cells and so can be used to test for the presence of a modulator in the sample (follicular fluid).
  • the process may comprise repeating the determination in (a) for: (i) each of a plurality of samples taken from the same female individual; and
  • A. is the amount of modulator (determined directly or indirectly by 110-HSD activity) for sample x, determined in (i), for n samples
  • a m is the modulator level in the mixture (or pool) of (from 2 to n) samples determined in (ii).
  • each sample is preferably taken from a different follicle (or from the environment of a oocyte), and can comprise follicular fluid and/or granulosa cells. It will therefore be apparent that a positive value of ⁇ indicates the presence of an inhibitor (of 11/3-HSD in one of the samples), while conversely a negative value suggests the presence of an agonist.
  • ⁇ will thus give an indication as to the amount of modulator present and indeed is likely to be directly proportional to the concentration of the modulator. Thus a positive value of ⁇ would indicate a greater probability of pregnancy.
  • the invention refers to the level(s) of 11/3-HSD modulator determined (for example in predictions of likelihood or probability of establishing pregnancy) that can include calculating ⁇ and using that value as a basis for any predictions or clinical evaluations.
  • the determination in (a) may be made for two or more biological samples (taken at) different times (eg. in the controlled ovarian hyperstimulation cycle), and any difference noted.
  • the biological samples may be taken from the individual at the same (or similar) time in different cycles. Note that reference is made here to controlled ovarian hyperstimulation cycles because women undergoing preparation for oocyte collection neither menstruate nor ovulate.
  • An increase (or decrease) in ⁇ can thus point to a progressive change in amount (or effect) of the modulator and may give an indication on whether IVF is to be successful. For example, samples may be taken on successive days, and a progressive decline in 110-HSD activity (as opposed to no change, or an increase) may indicate a greater chance of pregnancy.
  • the level of 110-HSD activity can, in turn, also be determined directly or indirectly. That is to say that the level of 11/3-HSD may be measured as an amount (of the protein) or in terms of its activity.
  • Direct methods include enzyme assays to determine the level of 113-HSD activity which involve contacting the sample with a substrate, for example 3 H-cortisol, and measuring the conversion of the substrate (eg. to 3 H-cortisone) by the enzyme. 3 H-cortisol and 3 H-cortisone can be separated by thin layer chromatography and then quantified. This will provide a direct measurement of enzyme activity, and for this reason is preferred.
  • a concentration of about lOOnM of 3 H- cortisol may be used, although a concentration ranging from lOnM to lOOOnM or more can be used.
  • Indirect methods of measuring 113-HSD activity include measuring the levels of cortisol and cortisone in the sample, and determining the ratio of the two as an indirect measure of enzyme (or modulator) activity.
  • the higher the level of cortisone in relation to cortisol the higher the activity of the enzyme (or low level of inhibitor).
  • the levels of cortisol and cortisone can be measured by methods known per se (eg by immunoassay methods having resolved cortisol and cortisone by TLC/HPLC). Kits for the assay of cortisol are commercially available. 34
  • 3-HSD levels can be measured by immunoassay or similar (eg. competitive) ligand-binding techniques. This will provide an indication of the amount of the enzyme, which may be correlated to enzyme activity (and from there to modulator activity) .
  • a ligand (or antibody) capable of binding the enzyme could be used in immunoassay methods such as RIA or ELISA. Methods to determine and obtain ligands which bind with high affinity to a specific analyte are also available in the art.
  • 3-HSD protein, or even its mRNA can be used as a measure of modulator activity since some modulators (eg. oestradiol) exert their effect at the level of mRNA transcription or translation.
  • the expression of the 11/3-HSD enzyme can also be measured by immunocytochemistry using a monoclonal antibody. Such techniques will provide a measurement of the amount of 113-HSD present, which can then be correlated to enzyme activity.
  • the result can be used to predict or assess the likelihood of successful establishment of pregnancy in a female subject undergoing IVF treatment.
  • the level of 113-HSD activity in the sample will be directly affected by the modulator. Therefore, 11/3-HSD activity will be proportional (or inversely proportional) to the level of agonist (or antagonist/inhibitor) and a measurement of the level of the modulator can thus be correlated back to (or provide an indication of) the level of 113-HSD activity.
  • 11/3-HSD activity levels have been measured by the amount of cortisol converted to cortisone per mg protein per 4 hours to obtain a direct measurement of enzyme activity.
  • a level of 110-HSD activity of 10 pmol/mg/4 hr represents the measure that has been used in the past as a suitable limit, and may still be used as a threshold in the practise of the present invention, if a practitioner sees fit.
  • levels of 11/3-HSD activity (or modulator) were to be measured in any of the other ways mentioned above, it would be desirable to conduct, using routine procedures, a control using our method of assay in order to determine the relationship between the results and the results of other methods, in order to make direct comparisons.
  • the level of modulator (or 110-HSD activity) can be used to assess the likelihood of establishing pregnancy by IVF in a patient.
  • the invention can be used in relation to samples from patients who have already had oocytes collected, fertilised in vitro, and implanted. Generally, a number of eggs are collected and fertilised so that in the event of failure to establish pregnancy, more fertilised eggs can be implanted. By conducting the method of the present invention, it is possible to predict, where pregnancy is not established, whether implantation of further stored (fertilised) oocytes is likely to be successful.
  • the methods of the present invention may be performed prior to implantation, prior to fertilization of collected oocytes or even prior to collection of such oocytes. In such cases, the results of such methods may allow the practitioner (or IVF clinic) to decide whether or not to even attempt a first implantation.
  • a second aspect of the present invention also provides a method for predicting the outcome of IVF in a female individual, the method comprising:
  • Suitable biological samples include those mentioned for the first aspect. As previously discussed, more than one sample (not necessarily of the same type, but usually so) may be removed. The determination in (b) may then be performed on each sample, and on a mixmre (or pool) of samples. Each sample is preferably taken from a different follicle present in the same female.
  • This embodiment of the invention can be used to select individuals likely to benefit from an IVF programme. Once an individual has been selected, it will be desirable to confirm their suitability during the IVF procedure by repeating assays for 110-HSD modulators during the initial part of the IVF procedure.
  • the invention comprises a method for establishing the likelihood of successful IVF treatment in an individual, the method comprising:
  • the invention in a fourth aspect relates to a method which comprises: (a) removing one or more oocyte(s) from a female individual together with a biological sample; (b) determining the level of a modulator of 110-hydroxy steroid dehydrogenase (110- HSD) in the sample; (cl) predicting, from the level of 110-HSD modulator determined, the likelihood or probability of establishing pregnancy in that individual by IVF; and (c2) fertilising an oocyte from those individuals whose 11 -HSD modulator level is above or below a predetermined threshold.
  • these embodiments of the invention further comprise: (d) implanting into the female individual the fertilized oocyte.
  • Preferred biological samples comprise granulosa-lutein cells and/or follicular fluid.
  • both these embodiments are desirably practised on individuals who have already been assayed prior to oocyte collection for suitable levels of 110-HSD, they may also be practised on patients who have not undergone such an initial screen.
  • the invention finds application in large scale screening programs of potential IVF recipients who have been referred to, or present themselves at, IVF clinics.
  • the invention comprises:
  • the type of modulator, and its amount and/or effect can be used as a predictor for the likelihood of pregnancy, for example the presence of an inhibitor in a female with low 110-HSD activity may suggest a greater chance of successful IVF. If the level of modulator(s) is only determined in one cycle then selection (or predictive outcome) may only be possible based on the results for that cycle, although determinations over several cycles may give a more general indication of the outcome of lVF.
  • IVF clinics it will be possible for IVF clinics to allocate resources more efficiently, so that patients with high levels of an 110-HSD inhibitor (or low levels of an agonist) in the environment of a recovered oocyte, who may thus be unlikely to become pregnant by IVF treatment, are not treated.
  • the levels of 110-HSD modulator(s) in a female individual may be monitored over a period of time in order to establish whether or not changes favourable to successful IVF occur.
  • Levels of ovarian 110-HSD in individual patients can vary between consecutive controlled ovarian hyperstimulation cycles (as well as between follicles in the same cycle).
  • Females may thus be monitored in accordance with the invention to obtain an oocyte which is from an environment with favourable (i.e. low) levels of 110-HSD (e.g. high levels of 110-HSD inhibitor or low levels of an agonist).
  • an identification method comprising:
  • Identification may make use of one or more techniques well known in the art, or a combination thereof, such as TLC, HPLC, NMR, IR, mass spectroscopy, etc.
  • the present invention relates to a method of increasing the likelihood of pregnancy, the method comprising:
  • the present invention relates to a method of contraception (or decreasing the likelihood of pregnancy), the method comprising:
  • the inhibitor or agonist administered in (b) can either be the inhibitor or agonist (after isolation and/or purification) identified in (a) or the same substance, except from a different source.
  • the latter is preferable since the inhibitor or agonist may be administered sterile and/or with other substances such as excipients.
  • the inhibitor administered is taken from a commercially available source (e.g. Sigma- Aldrich, Poole, Dorset, UK).
  • a ninth aspect of the present invention thus relates to the use of an 110-HSD inhibitor or a pregnancy enhancing compound for the manufacture of a medicament for increasing the likelihood of pregnancy in IVF. If the inhibitor is progesterone then (since it is administered orally as a contraceptive) it should be given at a dose that will be the same as, or below, physiological level(s) of progesterone.
  • a tenth aspect of the present invention relates to the use of an 110-HSD agonist or contraceptive compound (as defined in the eighth aspect) for the manufacture of a contraceptive medicament.
  • the invention relates to a method of screening potential candidate therapeutic substances.
  • the method may comprise identifying a pregnancy enhancing compound or a contraceptive compound, the method comprising:
  • potential 110-HSD inhibitors include antibodies (or fragments thereof) specific for 11 -HSD (and may thereby reduce or prevent activity by immunoneutralisation).
  • Females may thus be treated to modulate or block 11 -HSD activity in vivo prior to oocyte recovery.
  • antibodies against, or inhibitors of, 110-HSD could be administered to, or introduced into, the individual in order to inhibit enzyme activity.
  • the term "antibody”, unless specified to the contrary includes fragments of whole antibodies which retain their binding activity for a tumour target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies.
  • the antibodies and fragments thereof may be humanised antibodies, eg. as known in the art.
  • Antibodies against 11 -HSD for use in the present invention may be monoclonal or polyclonal antibodies.
  • Monoclonal antibodies may be prepared by conventional hybridoma technology using the proteins or peptide fragments thereof, as an immunogen.
  • Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a peptide of the invention and recovering immune serum.
  • 11 -HSD it may also be possible to inhibit the activity of 11 -HSD using other glucocorticoid hormones or analogues thereof which compete with cortisol for 110-HSD. Suitable inhibitors of 11 -HSD have already been mentioned (see page 4). Such hormones or analogues thereof can be identified by a screening process where the candidate hormones or analogues thereof are assayed to determine whether they can compete with 3 H-cortisol for 110-HSD, and thus inhibit the activity of the enzyme. Candidate hormones or analogues thereof may then be screened (for in vivo efficacy) by administering an effective amount of a hormone or analogue thereof to female subjects
  • inhibitor eg.hormone or analogue thereof
  • the amount of inhibitor (eg.hormone or analogue thereof) to be administered will need to be determined by the physician, taking into account its activity and the condition of the patient. This can be achieved without difficulty for hormones since they are often used in clinical practice in fertility clinics. Indeed, it is likely that the inhibitor administered will be naturally occurring, and may already be present in the recipient female.
  • the inhibitor(s) thereof may be administered by any suitable route, e.g. orally or by injection.
  • the inhibitor may be formulated with a pharmaceutically acceptable carrier or diluent.
  • 11 -HSD inhibitors may also permit in vitro treatment of collected oocytes to reduce enzyme activity prior to fertilization of oocytes. This may be achieved by bringing into contact an effective amount of such an inhibitor, for example a hormone or analogue thereof as previously mentioned, with a sample comprising an oocyte and surrounding tissue such as the granulosa lutein cells, in order to inhibit the activity of 110-HSD in the sample.
  • the sample and inhibitor may be brought into contact under sterile conditions such as those typically used for IVF.
  • 11 -HSD inhibitors may also permit in vitro treatment of collected oocytes to reduce enzyme activity prior to fertilization of oocytes. This may be achieved by bringing into contact an effective amount of such an inhibitor, for example a hormone or analogue thereof as previously mentioned, with a sample comprising an oocyte and surrounding tissue such as the granulosa lutein cells, in order to inhibit the activity of 110-HSD in the sample.
  • the sample and inhibitor may be brought into contact under sterile conditions such as those typically used for IVF.
  • a method of increasing the likelihood or probability of pregnancy comprising:
  • the method additionally comprises:
  • kits for use in performing the assay of the invention include at least one reagent useful for the detection of a modulator of 110-HSD activity.
  • Suitable reagents include antibodies, or other suitable ligand-binding reagents, against the 110-HSD modulator optionally linked to a label.
  • Typical labels are those commonly used in immunoassay procedures, for example horse radish peroxidase.
  • the kit may contain antibodies, or other suitable ligand-binding reagents, against cortisol and/or cortisone.
  • kits may be suitable for indirect assays (for measuring the level of 11 -HSD activity) such as the determination of cortisol: cortisone ratio or radiometric conversion of pH]-cortisol to [ 3 H]-cortisone.
  • the kit may also contain standards, for examples predetermined amounts of cortisone, cortisol and/or 110-HSD, any or all of which may be labelled with a detectable label.
  • the kit may also contain enzyme cofactors, for example, NAD or NADP which are converted to NADH or NADPH respectively.
  • the kit may comprise agents such as oxidised tetrazolium salts to serve as a colorimetric substrate for the re-oxidation of the reduced NAD(P)H.
  • agents such as oxidised tetrazolium salts to serve as a colorimetric substrate for the re-oxidation of the reduced NAD(P)H.
  • the change in optical density of the indicator salt at the appropriate wavelength (for its reduced form) may be directly proportional to the rate of reduction of the NAD(P) + cofactor, which may in turn be directly proportional to 110-HSD activity and hence inversely proportional to the concentration of, for example, 110-HSD inhibitor in the sample.
  • the kit may contain an 110-HSD modulator (such as an inhibitor, eg. glycyrrhetinic acid) as a standard for comparison.
  • an 110-HSD modulator such as an inhibitor, eg. glycyrrhetinic acid
  • This may be present in a known concentration (or amount) or at several concentrations (or amounts) so that a calibration curve may be derived for comparison.
  • the invention also provides a kit for the identification of, or measurement of a level of, a modulator of 110-HSD (eg. in a sample) for use in a method of diagnosis, prognosis, and or IVF treatment of a female individual.
  • a modulator of 110-HSD eg. in a sample
  • the invention also comprises the use of the above mentioned antibodies, fragments and variants thereof, and other suitable ligand-binding reagents, which may optionally be labelled with a detectable label for the manufacture of a diagnostic kit for use in the treatment or diagnosis of suitability for IVF.
  • Levels of 11 -HSD activity may also be assayed via analysis of the levels of 11 -HSD mRNA present in samples obtained.
  • 11 -HSD cDNA 30 or fragments thereof may be used as a probe to determine levels of 110-HSD in the environment of the oocyte.
  • Such probes may also be formulated into kits in a manner analogous to those described for antibodies, and may contain control nucleic acids.
  • Probes for the 110-HSD gene may be designed for use as probes, for example for use in a nucleic acid amplification assay.
  • Figure 1 is a graph of 110-HSD activity against patient number showing the variation of ll ⁇ -HSD activities in individual follicles from 12 different patients.
  • the asterisk (*) indicates significant variation between follicles ( ⁇ 0.05 by ANOVA) for a given patient.
  • the dotted line at lOpmol cortisone formed/mg protein.4h indicates the non ⁇ specific assay detection limit);
  • Figure 2 is a graph of 110-HSD activity against oocyte maturity showing the relationship between ovarian ll ⁇ -HSD activities and oocyte maturity on an individual follicle basis. (The data relate to 34 follicles from 9 different patients; the dotted line at lOpmol cortisone formed/mg protein.4h indicates the non-specific assay detection limit); and
  • Figure 3 is a bar graph of 110-HSD activity showing the relationship of the 116HSD activity of a multi-follicular pool of cells (shaded vertical bar) to those activities of the constituent individual follicles (open circles) and the arithmetic mean of the latter individual values (horizontal line) for three different patients.
  • the dotted line at lOpmol cortisone formed/mg protein.4h indicates the non-specific assay detection limit).
  • one objective was to evaluate the variation in ll ⁇ -HSD activities between granulosa-lutein cells from different individual follicles in a given patient, and to appraise the relationship between follicular 1 l ⁇ -HSD activities and oocyte maturity scores.
  • Also determined was whether the l l ⁇ -HSD activity of a pool of granulosa-lutein cells combined from several patients differed significantly from the mean of the activities in the multi-follicular pools of cells from each individual patient.
  • Granulosa cells were obtained from patients undergoing assisted conception by IVF-ET following controlled ovarian hyperstimulation as described previously 8 . Follicular aspirates were stored (for up to 3 days) and transported at 4°C before the preparation of cells. Granulosa cells were isolated from follicular aspirates on 60% (v/v) Percoll (Sigma Chemical Co., Poole, Dorset, UK) and washed repeatedly in Dulbecco's modified phosphate-buffered saline 33 (Life Technologies Ltd., Paisley, Scotland, UK). Cells were counted by haemocytometer and viability was assessed by the exclusion of 0.4% (v/v) trypan blue dye.
  • Example 1 In the second series of experiments, the procedure of Example 1 was followed except that all follicular cells aspirated from a given patient (i.e. from several different follicles) were combined prior to the isolation of granulosa cells on a single Percoll preparation. Having counted the total number of granulosa cells obtained from that patient, 150,000 viable cells were allocated for the triplicate assay of ll ⁇ -HSD activity in that multi-follicular pool. Any rernaining cells were then combined with cells from the multi-follicular pools of different patients to form a single multi-patient cell pool (where that multi-patient pool contained equal numbers of cells from each patient to a total in excess of 150,000 viable cells at a density of 50,000 viable cells/ml).
  • 1 l ⁇ - HSD activities were calculated as the rate of conversion of [ 3 H]-cortisol to [ 3 H]- cortisone (quantified by liquid scintillation counting), corrected for the specific activity of the substrate, the amount of cellular protein per well and the non-specific rate of generation of [ 3 H] -cortisone.
  • This assay was found to have a finite detection limit of lOpmol cortisone formed/mg protein per 4h which equates to the rate of oxidation of cortisol in the presence of lOO ⁇ g bovine serum albumin (BSA) (Sigma, UK).
  • BSA bovine serum albumin
  • this radiometric conversion assay provides a measure of the net conversion of [ 3 H]-cortisol to [ 3 H]-cortisone by intact granulosa-lutein cells cultured in the presence of a concentration of cortisol (lOOnM) known to approximate to that concentration typically measured in follicular fluid (i.e.200nM). 6,7
  • the assay was not designed to discriminate between the dehydrogenase activities of isoforms of the ll ⁇ -HSD enzyme, nor does it measure the gross rate of cortisol oxidation to cortisone since, as wim any biochemical reaction, the true rate of the enzyme catalysed reaction will be decreased by the opposing reaction: i.e. the reduction of cortisone to cortisol, catalysed by the 11-ketosteroid reductase (1 IKSR) activities attributable to one or more 11BHSD isoforms (see conclusions). Assessment of Oocyte Maturity.
  • Oocyte maturity was assessed by the observation of oocytes at the time of collection under a dissecting stereo-microscope as described previously. 8 Oocytes were scored for maturity as follows:
  • 1.0 immature oocytes (i.e. dense/compact cumulus mass with no evidence of germinal vesicle breakdown (GVBD));
  • 3.5 to 4.0 post-mature/luteinized oocytes (i.e. diffuse/dispersed cumulus cells and evidence of oocyte degeneration).
  • the ll ⁇ -HSD activities of different follicles were subjected to one-way analysis of variance (ANOVA) and comparison of these values to the corresponding scores of oocyte maturity was made by Spearman's rank correlation analysis.
  • ANOVA analysis of variance
  • the 1 l ⁇ -HSD activities of the multi-follicular pools of cells were compared to the arithmetic means of the activities of the corresponding constituent individual follicles by unpaired f-tests.
  • the ll ⁇ -HSD activities of the two multi-patient pools of cells were compared to the respective arithmetic means of the activities of the constituent multi-follicular pools for each patient by unpaired t- tests.
  • the decision to compare the multi-follicular pool and multi-patient pool measurements to the ari ⁇ metic means for the appropriate individual follicle/patient values respectively was justified on the basis that equal numbers of cells from each follicle/patient were used to derive the pooled cells in each case.
  • the expected llflHSD activity for the multi-follicular/multi-patient pooled cells was calculated to be the simple arithmetic mean of the appropriate individual values, independent of the frequency distribution of the latter data.
  • the coefficient of variation (CV.) for triplicate determinations did not differ significantly between the pooled cell assays and the individual follicle/patient measurements
  • the ll ⁇ -HSD activities of different individual follicles from a given patient were found to vary significantly (P ⁇ 0.05, ANOVA) for each of the 12 patients studied ( Figure 1).
  • Table 2 shows the relationship of the ll ⁇ -HSD activity (pmol cortisone/mg protein.4h) of a multi-patient pool of cells to those activities of the constituent multi-follicular pool of cells from each patient in two independent experiments.
  • the main parameter for the selection of oocytes for IVF has been their maturity, as assessed by a scoring system based on the morphological appearance of the cumulus- oocyte complex (COC). It was recently proposed that measurements of ovarian ll ⁇ - HSD activities may provide a more objective parameter for assessing the probable outcome of IVF-ET in a given patient. 13 15 In these Examples the 1 l ⁇ -HSD activity of granulosa-lutein cells varied dramatically (from undetectable to in excess of 500pmol/mg protein per 4h) in different follicles from a given patient, and ll ⁇ -HSD activities did not relate to the maturity of the oocyte contained within each follicle.
  • ovarian ll ⁇ -HSD activity has been measured for each patient in a pool of cells derived from all of the granulosa-lutein cells aspirated from several different follicles. Whereas none of me 101 cycles with detectable ovarian ll ⁇ -HSD activity were associated with a clinical pregnancy, the clinical pregnancy rate for the 71 cycles with "ll ⁇ -HSD negative" cells was 63 %.
  • a pool of human granulosa-lutein cells will only manifest high ll ⁇ -HSD activity if all of the constituent follicles are "l l ⁇ -HSD positive" .
  • the activity of the multi-follicular pool of cells will be low/undetectable, irrespective of the enzyme activities in the other constituent follicles.
  • bile salts 4,25 cholesterol, 4 lanosterol 4 and a number of steroid hormones 10,24 have been shown to regulate ll ⁇ -HSD activities in both the liver (low affinity, NADP + -dependent, type 1 ll ⁇ -HSD activity), distal nephron and placenta (high affinity, NAD + -dependent, type 2 ll ⁇ -HSD activity).
  • sex steroids e.g.
  • progesterone and oestradiol by human granulosa-lutein cells in vitro, and that this relationship might form the basis for the paracrine suppression of ovarian ll ⁇ - HSD activity indicated by the observations reported here. Indeed, it has been demonstrated that in cultured human granulosa-lutein cells, pregnenolone and progesterone (but not oestrone nor oestradiol) can inhibit ll ⁇ -HSD activity 31 .
  • This isoform designated 11B-HSD2
  • ll ⁇ -HSD2 is capable of metabolizing the synthetic glucocorticoid, dexamethasone 2,29 and is susceptible to inhibition not only by derivatives of glycyrrhetinic acid, but also by the end-products of ll ⁇ -dehydrogenase action 2,28 (i.e. cortisone and 11- dehydrocorticosterone).
  • ll ⁇ -HSD2 has recently been cloned and sequenced 1,2 and has been shown to be expressed in tissues other than the kidney, including the human ovary.
  • low "ll ⁇ -HSD activities" in a given follicle or pool of granulosa cells could reflect the presence of cells in which the 11KSR activity of one or more ll ⁇ HSD isoforms predominates to such an extent tiiat any cortisone generated by the ll ⁇ -dehydrogenase activities is immediately reduced back to cortisol.
  • Follicular fluid aspirated from die ovarian follicles of women undergoing oocyte retrieval for IVF, has been found to contain at least one compound tiiat, when added to cultured human granulosa-lutein cells in vitro, is capable of inhibiting 110HSD activities (Table 3). After removal of me sample from me female the follicular fluid was separated from the granulosa-lutein cells, and the latter were cultured separately (for 72 hours) before being brought back into contact wim die follicular fluid, in order to test whe ier there was an 110-HSD inhibitor present in me fluid.
  • kidney 110-HSD Effect of 10% follicular fluid on kidney 110-HSD.
  • Lax ER, Ghraf R & Schreifers H The hormonal regulation of hepatic microsomal 1 l ⁇ -hydroxy steroid dehydrogenase activity in die rat. Acta Endocrinologica (Copenhagen) 89: 352-358 (1978).

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Abstract

L'invention concerne des dosages (et des trousses) qui permettent de prédire les chances de réussite d'une grossesse par fécondation in vitro (FIV) en déterminant le taux d'une modulateur de la 11β-hydroxystéroïde déshydrogénase (11β-HSD) dans un échantillon biologique prélevé chez une patiente, par exemple dans l'environnement d'un ovocyte, liquide folliculaire ou cellules de la granulosa en particulier. La quantité de modulateur (par exemple d'inhibiteur) peut être déterminée par référence à son influence sur l'activité de la 11β-HSD, et l'administration d'un modulateur (par exemple d'un inhibiteur de la 11β-HSD) peut accroître la probabilité d'une grossesse, tandis que les agonistes de la 11β-HSD sont des agents contraceptifs potentiels.
EP97903495A 1996-02-16 1997-02-17 Dosage predictif pour le resultat d'une fiv Withdrawn EP0880601A1 (fr)

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US5994148A (en) * 1997-06-23 1999-11-30 The Regents Of University Of California Method of predicting and enhancing success of IVF/ET pregnancy
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CN1161615C (zh) * 1998-06-22 2004-08-11 梅迪-卡尔特公司 预测取出用于体外受精的卵细胞的时间的方法和应用
WO2007130673A2 (fr) * 2006-05-05 2007-11-15 Beth Israel Deaconess Medical Center Procédé de diagnostic et de traitement de la stérilité féminine au moyen de marqueurs moléculaires
US9176145B2 (en) 2006-07-21 2015-11-03 Femalon S.P.R.L. Kit for predicting implantation success in assisted fertilization
MY150687A (en) * 2006-07-21 2014-02-28 Femalon S P R L Assay and kit for predicting implantation success in assisted fertilisation
KR101112748B1 (ko) * 2009-10-22 2012-03-13 중앙대학교 산학협력단 소의 체외 수태능력 예측 방법

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