FERTILITY PREDICTION BY USE OF CLOMIPHENE CHALLENGE TEST

Abstract: The fertility of females, especially human females can be predicted by use of a clomiphene challenge test. The test is based upon comparison of follicle stimulating hormone and/or luteinizing hormone levels in urine or blood samples at two points in the menstrual cycle. Clomiphene, and most preferably clomiphene in the form of clomiphene citrate, in a dosage of about 100 mg/day, is then administered, preferably on days 5-9. The second test is conducted on days 9-10. The test results are most conveniently compared by colorimetric methods involving monoclonal antibody reactions with follicle stimulating hormone and/or luteinizing hormone.

Description: FERTILITY PREDICTION BY USE OF CLOMIPHENE CHALLENGE TEST FIELD OF THE INVENTION The present invention generally relates to methods f predicting female fertility. More particularly this inventi relates to methods of predicting human female fertility based up chemical tes~s for de~ec~ing the levels of follicle stimulati hormone or luteinizing hormone in zhe female's urine or blood. 3ACKGR0UND OF THE INVENTION The fertility processes which occur in females, especially human females, are highly complex, and continuing efforts are bei made in this area to more fully understand these processes so as facilitate the development: of improved techniques for predicting a creating fertility problems as well as for providing more effecti and reliable bir-h control methods. A number of drugs have be emcioved to treat infertility. Clomiphene citrate is one such drug However, in the fertility problem area, there still is an acute nee for imπroved methods for studying, as opposed to treating, women wh wish to conceive but are unable to do so. For example, a woma see.-cir. r a career outsice tie no e may ccnsiαer ocstnonm childbearing until her thirties, and a prognosticator of fecundit would be of special value to her. It might guide her decision regarding pregnancy attempts and contraception. It also would ai the physician in clinical decisions regarding infertility workup o therapy. Moreover, this need extends to women experiencing othe difficulties such as menstrual cycle variations and other problem associated with the female child bearing organs. However, for th apparently healthy, regularly menstruating woman, age is presentl the only widely accepted parameter of fertility potential Demographic studies indicate that fertility peaks during the earl twenties, decreases noticeably after 30 years of age and markedl after 35 years. However, because of individual variations, age b itself is an inaccurate parameter, and arbitrary limits based on ag alone can influence treatment decisions in a number of patients. In the birth control area, there is also an acute need fo improved birth control methods which are not only effective i preventing conception but also have a reduced incidence of advers physical side effects. There are a wide variety of birth contro methods available, but these are often unacceptable due to the fac that they are unreliable (i.e., prevention of conception is no assured) , or for medical reasons, or on religious grounds. On example of a birth control method which is unreliable is the called "rhythm" method, which is based on the fact that the woman not fertile, i.e., ovulation has not occurred or is not about occur, during a certain period in her menstrual cycle. The maj disadvantage associated with this method is that, while the t period from when ovulation naturally occurs to the next succeed menstrual period is essentially fixed in all women, the time per between the beginning of a menstrual period and the next ovulat can vary considerably depending on the particular woman concern It is during the time period between the beginning of a menstr period and the onset of the next succeeding ovulation that sex intercourse can occur without conception occurring since during t period the female ovum has not yet been produced by the wom However, significant risks do exist with the "rhythm" method sinc even if the woman has not ovulated at the time of sexu intercourse, ovulation occurring one or two days thereafter c result in conception since the life span of male sperm in t vaginal can be as long as one to two days, and sometimes longer. Some methods do exist for determining when a woman is about ovulate but these are inconvenient and difficult to interpret. O such method requires the woman to take her temperature every morni and to plot this on a graph. From the shape of the graph, it possible to see when ovulation has actually occurred, but the maj problems associated with this method is that fluctuations in bo temperature can occur for many reasons other than the ovulati orocess. ;- is also well known to this art that a surge in the level o human luteinizing hormone (hLH or HL) takes place about 34 hour crier to ovulation. It takes place according to consideration which can begin by first noting that the normal human menstrua cycle is divided into the follicular phase, ovulation, and th iuteal phase. Normal hormonal control during the follicular phas produces maturation of the primary ovarian follicle. Hum luteinizing hormone and human follicle stimulating hormone (hFSH o FSH) are known to have a sensitizing effect upon the primary ovari follicle. These hormones act in concert to stimulate estrog synthesis. It is also known that estrogen and hFSH achieve the sensitizing effects by inducing expression of gonadotropin recepto in preparation for ovulation. Estrogen in turn acts on t hypothalamus to control pituitary secretion of gonadotropin Estrogen reaches a peak one to two days prior to ovulation. Th peak in turn induces a positive feedback response in the anteri pituitary to hypothalamic gonadotropin releasing hormone. Duri this peak period, estrogen levels decrease while progesterone leve start to increase and thereby stimulate the release αf high leve of hLH. This surge in the hLH level reaches peaks which are usual two to three times the preceding basal concentrations. This h surge in turn induces a rupture of the primary ovarian follicle a the resulting release of a mature oocyte. This phenomenon generally referred to as "ovulation". Thereafter, 1LH promot luteinization and formation of the corpus luteum. This is follow by a decrease in the hLH level to baseline levels within two davs resoor.se to tne sealing progesterone levels wmcn serve to initia the luteal phase whicn lasts about 14 days. In the absence fertilization of the oocyte, a new follicle begins the selecti procedure for maturation in the next menstrual cycle. The methods for detecting the hLH surge have also improved recent times. Before the development of immunoassays, analyses hLH in urine was obtained by bioassay techniques. However, t clinical utility of these methods was limited; they had relative low sensitivity and frequently required that urine extracts tested. In the mid 1960's however, the introduction radioimmunoassay for hLH provided a new tool for quantitating l levels hLH in urine or serum. The later introduction of enzy immunoassays for hLH offered the further advantage of go sensitivity without the use of radioisotopes. Nonetheless, the still exists a need for improved methods for evaluating fertility woman so that fertility problems can be alleviated or so th appropriate birth control methods can be selected. Moreover, t diagnosis of infertility problems is still hampered by the fact th prediction of ovulation does not in and of itself give ' a information as to whether or not the oocyte which is about to released is one that is fecund. Thus even though it is well known that the detection of the h surge can act as an important tool in the detection of ovulati since the onset of the hLH surge precedes ovulation by about hours; and even though it is also known that peak hLH levels occ several hours later in urine than in serum with the onset of t surcre in urine being about 30 hours before ovulation, it has n been heretofore appreciated that differences in hLH and/or hF levels before and after ovulation could be used in a diagnostic te aimed at determining whether or not the female is releasing fecu SUMMARY OF THE INVENTION The disclosed clomiphene challenge test can be utilized prospectively assess future fertility potential in women, especial in women greater than 35 years of age with unexplained infertilit The test has four basic steps. A baseline hormone (most preferab FSH and/or LK) level is established; clomiphene is administered; response hormone level is established; and the response level of t subject hormone is compared to its baseline level. The baseli period hormone level is preferably established on days 2-3. T response period level is preferably established on days 9-11. T human follicle-stimulating hormone (hFSH) and/or, human luteinizi hormone (hLH) levels are measured before and after administration from about 50 mg to about 150 mg/per day of clomiphene between t baseline period and the response period. The clomiphene can be a clomiphene salt in any pharmaceutically acceptable carrie However, in the most preferred embodiment of this inventio clomiphene is administered in the form of clomiphene citra tablets. It can be administered between days 2 and day 9 of t menstrual cycle. However, daily administration on days 5, 5, 7, and 9 is a highly preferred administration procedure and dosaσes about ICO mg / day of clomiphene citrate, in the form cf clomiph ta b lets, administered at the same time daily, is the nest prefer administration procedure. The disclosed clomiphene challenge test can be performed u urine and/or blood samples. However, for reasons of simplici urine is the preferred sample source. Urinary or serum FΞH or each can be measured in a number of known semiquantitative fashio The tests can also be based on combined FSH and LH levels. Test FSH alone are, however, a preferred method of conducting the te Again for reasons of simplicity, colorimetric tests are preferred other test procedures which could include other, less preferred t procedures such as, for example, radioimmunoassay or pathologi tests. Most preferably, the colorimetric tests employed are th based upon color reactions of monoclonal antibody-based, enz immunoassay tests. Such colorimetric test results can interpreted by the woman with the aid of color charts which preferably made a part of a test kit. For example, semiquantitative tests of human luteinizing hormo (hLH) in urine can be made by comparing certain color reactio which can be associated with a monoclonal antibody/hLH reactio One such color reaction is described in a publication by Monoclon Antibodies, Inc., entitled, "Ovustick™ Urine hLH Kit" (19S4) a said publication is incorporated by reference into this pate application. Generally, it discloses that human luteinizing hormo is a glycoprotein hormone comprised cf two noncovalently bou polypeptide subunits, designated alpha and beta units, havi carbchvdrate side chains. I e. amino acid sequence of aipha-hLH essen t ially the same as that of human follicle-stimulating hormon has been established that the beta sub πi" of hLH is responsib for the biological and imnunochenical specificity of this particul Hence an hLH test kit normally used to chart ovulation cou also be used as the test method to practice the herein disclos clomiphene challenge test. Preferably it will be in the form of visual determinable, enzyme immunoassay that incorporates t advantages of monoclonal antibody technology. Such antibodies off the great advantage of controlled specificity and affinity. Th will provide consistent assay performance since they enter into specifically determined color reaction whose results can be compar by simple color comparison charts. Hence, tests utilizi monoclonal in semiquantitative, two-site, enzyme link immunospecific assays are most preferred in the practice of the invention. The specificity of these tests follows from the fa that these hormones in a urine specimen can be sandwiched betwe the alpha-subunit-specific antibody, which has been immobilized on plastic dipstick, and a beta specific antibody, such as beta hL which has been linked to the enzyme alkaline phosphatase. Aft unbound enzyme conjugate is removed by washing, the stick can incubated in a substrate solution. The substrate reacts with t enzyme and deposits a blue end product on the reactive end of t stick. By way of example then, a semiquantitative analysis of h in urine can be made by comparing the intensity of the color on ea stick with the color on the sticks incubated in, for examp concurrently run 0 mlU/ml, 20 mlU/ml and 40 lϋ/ml calibrato Color changes corresponding to response levels above one stand deviation, and more preferably those indicating response levels m than two standard deviations, above the base level can be conside as an indication of a low level of fecundity. DESCRIPTION OF PREFERRED EMBODIMENTS Populations and Methods The herein disclosed clomiphene citrate challenge tests w utilized to prospectively assess future fertility potential in wo greater than 35 years of age with unexplained fertility. Basel (day 2-3) and response levels (day 9-11) of follicle stimulat hormone (FSH) , luteinizing hormone (LH) , and 17-beta estradiol ( were measured before and after administration of 100 mg clomiph on days 5-9 of the menstrual cycle. Sixteen women had exaggerated FSH response of over 26 mlU/ml or more (2 stand deviations (SD) above levels found in normal controls) , which considered a diminished ovarian reserve (DOR) . Mean baseline FSH 16 women in the DOR group was 13.5 plus or minus 5 SD mlU/ml wit response level of 38.9 plus or minus 13.8 mlU/ml, while the baseli in 30 women with adequate ovarian reserve (AOR) was 9 plus or mi 4.2 mlU/ml with a response level of 11.5 plus or minus 4.9 mlU/ There was a highly significant difference (p,0.000l) between F response levels of the two groups and between response to baseli levels in the DOR group. LH response was consistently greater th F S H response in the AOR group, while consistently less than FSH the DOR group (?<0.001). One of 16 patients in the DOR gro conceived; 14 of 30 (47%) conceived in the AOR group (p,0.005) These tests indicate that despite apparently "normal" ovulato cycles, the DOR group has compromised follicular apparatu Disparity between normal Ξ 2 secretory capacity of the granulosa a diminished capacity to secrete inhibin could explain t inappropriately high FSH levels in response to the clomiphe citrate challenge test. Hence it would appear that this clomiphe challenge test can reliably predict fecundity irrespective of ag It can provide a prospective test of the reserves cf the gonad hypothalamic axis as an indicator of female fecundity. The basic study to establish this invention was conducted up fifty-one patients seeking counsel for infertility between Jun 1983, and January, 1986. Criteria for inclusion were age of years or above, regular menstrual periods (23-35 days) , norm seminal fluid analysis of the partner, and no history suggestive mechanical causes of infertility. All patients were followed unt conception or up to 3.5 years. Within one year, if no pregnan ensued, infertility workup was completed or re-evaluated. Fo patients were found to suffer from multiple pelvic adhesions, a one husband had repeatedly subfertile semen analyses. These fi were excluded from the statistical analysis ^of pregnancy. The ovarian challenge test was -based on the administration clomiphene citrate (CC) , 100 mg/day for five consecutive day starting on day 5 of the menstrual cycle. Peripheral venous blo was drawn during the eaπy (days 2-3 } ana late (days 9-1 follicular phase. Hormone levels were measured by standa radioim unoassay (RIA) for follicle stimulating hormone (FSH luteinizing hormone (LH) (Amersham Radiochemical Center, Amersha England, according to 2nd IRP human enopausal gonadotropin (HM reference standards) , 17-3 estradiol ( 2 ) , progesterone a dehydroepianirosterone sulphate (DKΞA-S) all by commercial available RIA kits. Hormone levels on cycle day 2 or 3 are referr to as the baseline hormonal profile, while days 9 to 11 are t preferred times to establish the response values. An FSH value higher than 26 mlU/ml, measured on days 9-11, w defined as an abnormal response and was designated as the diminish ovarian reserve (DOR) group. Patients whose response was within t normal range were defined as the adequate ovarian reserve (AO group. 26 mlU/ml of FSH is more than two standard deviations abo the mean value on days 9-11 as observed in 8 healthy volunteers ag 22-28 years, undergoing the same clomiphene challenge tests. T results of the clomiphene challenge tests were separately file Fertility promoting treatment was conducted irrespective of t clomiphene challenge test results or interpretation. Nearly all t women (90%) eventually has ovulation stimulation with gonadotropin The one DOR patient who conceived did so during an IVF cyc subsequent to failures of human menopausal gonadotropin (hM treatment for 12 previous cycles. All values are expressed as me plus or minus standard deviation (SD) . Statistical analysis w performed employing student's T test or the Fischer exact test appropriate. 12 Results Of the 51 women, 18 had an abnormal response to the clomiphe challenge tests; 23 had FSH response levels within 2 SD of me normal value as defined above. Table 1 details the age, duration infertility, duration of follow-up, gonadotropin treatment, a outcome in the 51 patients. The two groups of patients did n differ in age, duration of infertility, treatment, or mean follo up, (see Table 1) . Sixteen of eighteen women in the DOR group (89 and 31 of 33 in the AOR group were treated with hMG and hum chorionic gonadotropin (hCG) for ovulation induction, perform according to individually adjusted schedules. During treatment a follow-up, only 1 of 18 women in the DOR group conceived (6.3 while 14 of 33 (46.7%) conceived in the AOR group. The differen in conception rates is statistically significant (p<0.005). T results of these tests are summarized in Table I. It should be noted that the baseline hormonal profiles differ only in respect to FSH levels. Although well within the norm range (13.5 plus or minus 5 mlU/ml) , the mean FSH level w significantly higher (p<0.002) in the DOR group than in the A group. No other baseline values differed between the two groups. highly significant increase in serum FSH levels was observed aft clomiphene citrate administration in the DOR group (p<0.0001), whi only moderate increase was detected in the AOR population (p=0.05 The difference between the two groups in response levels of FSH w highly significant (p<0.0001). In both groups a signific elevation in LH levels from baseline was documented, although levels on days 9-11 were significantly higher in the DOR group t in the AOR group (p<0.02). In 15 of 13 (33%) in the DOR group, FSH levels more t doubled, while in the AOR group only 3 of 33 (9%) had FSH lev twice as high after clomiphene citrate administration. T difference is statistically significant (p<0.0001). In the group a reversal of the ratio of the FSH/LH response was also no in the same proportion of patients; in 15 of 13, FSH levels w higher than LH levels on days 9-11, while only 3 of 33 in the group had higher FSH levels that LH levels (p<0.0001). There was obvious rise in E after clomiphene citrate was administered in bo groups, and higher levels were attained in the normal respo group. However, none of the values differed significantly, probab because of the wide range of E values. P levels were less than 1 mg/ml in all but one patient sampl on days 2-3 and all but two patients sampled on days 9-11. Me mid-luteal P levels are suggestive of ovulation in both groups: 11 plus or minus 3.4 vs 18.3 plus or minus 3.4 mg/ml in the DOR and A groups respectively. However, peak luteal P levels we significantly higher in the AOR group (p<0.05), see Table 1. Despite a definite trend of elevation in delta 4 A, T, and DHEA levels after clomiphene citrate challenge, no statistical significant differences were found between baseline and respon levels, and no discrimination could be demonstrated on the basis ovarian reserve. Prolactin levels were somewhat lower after t clomiphene citrate challenge test, but the differences were no statistically significant. Discussion These results indicate that a diminished fertility potential c be detected through the utilization of a clomiphene citra challenge test. This longitudinal study demonstrates that fecundi can be predicted on the basis of gonadotropin response to clomiphe citrate stimulation. The challenge test has a 93.5% sensitivity recognizing the population with diminished fertility potential and 47% specificity in identifying the fertile population. T specificity is apparently low; however, the 47% conception rate remarkable in a relatively old group of apparently normal patien undergoing ovulation induction. It may be that older patients w have unexplained fertility have subtle ovulatory abnormalities. T favorable results of hMG/hCG augmentation of ovulation suggest th a hypersti ulated cycle may overcome those undefined abnormaliti and can achieve pregnancy when the natural cycle have failed. T fact that all patients had normal baseline FSH and LH values a none had entered menopause during the follow-up period suggests th an inadequate ovarian reserve may be detected a considerable ti before actual cessation of menses. During the early stages perimenopause, there is a gradual rise in serum gonadotropins due decreasing members of oocytes and follicles. The rise in FSH leve is earlier and more marked than LH levels. In contradistinction the perimenopause, with clo ipnene citrate administration during reproductive span of life there is an early rise in pituit gonadotropin levels which is more marked and sustained for LH t for FSH. In DOR patients, gonadotropin response to the clomiph citrate challenge is reversed, being much more pronounced for than LH. This peculiar response pattern of gonadotropins is simi to premenopausal FSH/LH ratio and may be attributed to a decrease the ovarian elaboration of inhibin. Inhibin can specifica suppress plasma FSH levels, probably by reducing the release of from adenohypophysis. Like steroidal hormones, inhibin is secre by granulosa cells to secrete inhibin may also be related to ooc quality. A reduced capacity to secrete inhibin could explain specific overshoot in FSH levels after the clomiphene citr challenge. If that capacity is also related to oocyte quality, may explain the severely compromised chances to conceive. Thus, clomiphene citrate challenge may unmask incipient failure in apparently normal follicular apparatus. The increase in T and delta- 4 A following clomiphene citr treatment is an established phenomenon. This increase steroidogenesis is partly accounted for by the increase in LH lev which accelerate ovarian steroidogenesis. A direct action clomiphene citrate on ovarian 3B-ol dehydrogenasedelta-5 isomera is an additional route through which generation of delta- 4 A increase. The elevated androgen levels during clomiphene citra levels on days 9-11 were similar for the AOR and DOR groups; b this could not account for the significant difference in concepti rates between tne groups. 1 ° Hence this study shows that the herein disclosed clomiphe citrate challenge can be used as a stress test for the reserve the ovarian-follicular apparatus. Therefore, it will prove helpf in clinical decisions regarding contraception, type and timing profertility treatment, prognostication, and-above-all- knowing wh to abandon further efforts in an already over treated inferti woman whose failure to conceive remains unexplained. From the above discussion, those skilled in this art wi appreciate that although the application of clomiphene citrate f treatment of infertility and for testing the integrity of t hypothalamic-pituitary-ovarian axis is well established, it has n been described in the context of evaluating fecundity in patien seeking treatment for infertility. Those skilled in the art wi also appreciate that the herein disclosed clomiphene challenge te also can be modified without departing from the limits and spirit this patent disclosure. At the very least, the form of t clomiphene (e.g., administration via various salt forms), the da of testing and the adminstration dosage can be varied somewhat the practice of the hereinafter claimed invention.

Claims: 1. A method for predicting female fertility comprising: establishing a baseline level of follicle stimulat hormone on days 2-3 of the menstrual cycle; administering clomiphene on days 2-9 of the menstr cycle; establishing a response level cf follicle stimulat hormone on days 9-11 of the menstrual cycle; and comparing the follicle stimulating hormone of the basel level to the follicle stimulating hormone of the response level. 2. A method for predicting female fertility comprising: establishing a baseline level of follicle stimulat hormone, on days 2-3 of the menstrual cycle; administering clomiphene citrate on days 5-9 in a dosage about 100 mg/day; establishing a response level of follicle stimulat hormone on days 9-11 of the menstrual cycle; and comparing, on a basis of color differences produ reactions of monoclonal antibodies to the follicle-stimulat hormone, the follicle stimulating hormone of the baseline level the follicle stimulating hormone of the response level. 3. A method for predicting female fertility comprising: establishing a baseline level of luteinizing hormone, days 2-3 of the menstrual cycle; administering clomiphene on days 2-9 of the menstr cycle; establishing a response level of luteinizing hormone days 9-11 of the menstrual cycle; and comparing the follicle stimulating hormone of the basel level to the follicle stimulating hormone of the response level. 4. A method for predicting female fertility comprising: establishing a baseline level of luteinizing hormone days 2-3 of the menstrual cycle; administering clomiphene citrate on days 5-9 of about 1 mg/day; establishing a response level of luteinizing hormone days 9-11 of the menstrual cycles; and comparing, on a basis of color differences produced reactions of monoclonal antibodies to the luteinizing hormone, t luteinizing hormone of the baseline level to the luteinizing hormo of the response level. 5. A method for predicting female fertility comprising: establishing a baseline level of follicle stimulati hormone and luteinizing hormone on days 2-3 of the menstrual cycle administering clomiphene on days 2-9 of the menstru cycle; establishing a response level of follicle stimulati hormone and luteinizing hormone on days 9-11 of the menstrual cycl and comparing the follicle stimulating hormone and luteinizi hormone of the baseline level to the follicle stimulating hormo and luteinizing hormone of the response level. 6. A method for predicting female fertility comprising: establishing a baseline level of follicle stimulati hormone and luteinizing hormone on days 2-3 cf the menstrual cycle administering clomiphene citrate on days 5-9 in a dosage about 100 mg/day; establishing a response level of follicle stimulati hormone, luteinizing hormone on days 9-11 of the menstrual cycl and comparing on the basis of color differences produced reactions of monoclonal antibodies to follicle-stimulating hormo and luteinizing hormone, to the follicle stimulating hormone a luteinizing hormone of the baseline level to the follic stimulating hormone and luteinizing hormone of the response level.

Inventors: NAVOT DANIEL, NAVOT, DANIEL
Applicants: NAVOT DANIEL, NAVOT, DANIEL
Application year: 1988
Main classification: A61B10
IPC codes: A61B10, A61K49, G01N33