Ovulation detection methods for urinary hormones: precision, daily and intermittent sampling and a combined hierarchical method

BACKGROUND: We evaluate the performance of ovulation detection methods and present new approaches, including evaluation of methods for precision, combining multiple markers into a hierarchical system and using ovulation markers in intermittent sampling designs. METHODS: With serum LH peak day as the 'gold standard' of ovulation, we estimated accuracy and precision of ovulation day algorithms using 30 ovulatory menstrual cycles with daily urinary and serum hormones and transvaginal ultrasound. Sensitivity and specificity for estimating the presence of ovulation were tested using visually assessed ovulatory (30) and anovulatory (22) cycles. RESULTS: Sensitivity and specificity ranged from 70 to 100% for estimating presence of ovulation with twice-per-cycle, weekly, twice weekly, every-other-day and daily specimens. A combined hierarchical method estimated ovulation day using daily specimens within +/-2 days of the gold standard in 93% of cases. Accuracy of estimating ovulation day within +/-2 days using intermittent sampling ranged from 40% (weekly sampling) to 97% (every-other-day). CONCLUSIONS: A combined hierarchical algorithm using precise and accurate markers allows maximal use of available data for efficient and objective identification of ovulation using daily specimens. In intermittent sampling designs, the presence and the timing of ovulation can be estimated with good sensitivity, specificity and accuracy.     

Validating signals of ovulation: Do women who think they know,really know?

This study was carried out to test whether women who think they know when they ovulate, really know. Fifty-three women of age 18.7 to 46.1 (mean age 28.4 years) participated in initial interviews about ovulation. Criteria for recruitment included perceived ovulation, regular menstrual cycles, and not using hormonal contraception. Women collected and refrigerated urine samples from day 5 until they thought they ovulated. Samples collected within 48 h of the perceived signal were then tested for a pre-ovulatory LH surge. Of the 53 original participants, 36 women provided urine samples for 1-6 cycles, so that 87 cycles were tested. Subjective signals of ovulation varied between women and between cycles but included abdominal pain and changes in cervical discharge, libido, and mood. Of the 87 cycles tested, during which women identified one or multiple signals of ovulation, 37 of the 87 urine specimens tested positive for an LH surge for a concordance rate of 42.5%. Using the first tested cycle from the 36 women who provided urine specimens, 13 of those specimens demonstrated an LH surge, for a concordance rate of 36.1%. That rate dropped to 28% (7/25) when women who used basal body temperature as an ovulatory signal were excluded. Finally, the mean level of accuracy among the 15 women who contributed 3-6 urine specimens for testing was 48.9%. The results of this study demonstrate a low degree of concordance between LH surge and perceived ovulation among women who think they know when they ovulate. The most motivated study participants were right about half of the time. Although there is variation among women in their ability to know when they ovulate, this study suggests that, for most women, ovulation is concealed.     

Food intake and the menstrual cycle in rhesus monkeys

Fourteen adult female rhesus monkeys were observed for 1 complete menstrual cycle, five of them for 2 cycles. Changes in LH, FSH, estrogens and progesterone were monitored daily. Mean hormonal concentrations followed patterns previously demonstrated in primates, with a typical late follicular phase estrogen peak preceeding the ovulatory LH and FSH surges. Maximal sexual skin color intensity paralleled the midcycle increase in estrogens. The results indicate that food intake fluctuated with changes in hormonal secretion. A significant decrease in the amount of food consumed correlated well with the midcycle estrogen and gonadotropin surges. The amount of food consumed during the luteal phase was greater than that of the early follicular phase.     

Endogenous overnight creatinine clearance compared with 51Cr-EDTA clearance during the menstrual cycle

Fourteen healthy women aged 21-41 years, who did not take oral contraceptives and who all had ovulatory cycles, were examined once in the follicular phase and once in the luteal phase of a single menstrual cycle. The glomerular filtration rate (51Cr-EDTA clearance) increased from the follicular to the luteal phase by a median of 7.0% (95% confidence interval: 0.7-10.3%). Endogenous overnight (22.00-08.00 hours) creatinine clearance increased by a median of 7.3% (95% confidence interval: 1.0-14.6%). The urinary creatinine excretion rate also increased with a median of 7.3% (95% confidence interval: 1.5-11.9%) whereas the serum concentrations of creatinine and beta 2-microglobulin, urine flow and urinary excretion rate of urea did not change. The results confirm previous observations of an increase in creatinine clearance in the luteal phase of the menstrual cycle and indicate that the increase in overnight creatinine clearance reflects a true change in glomerular filtration rate.     

Use of a technic of bioluminescence for determining estrogens and LH during static and dynamic study of the ovary

Estrogens and LH are necessary among other assays for ovulation diagnostic and ovarian monitoring during stimulations. They can be both measured with a method based on a bioluminescent reaction. This method measures, with a standard luminometer, the reduced NAD produced and accumulated by the reaction of two dehydrogenase enzymes: the estradiol dehydrogenase for direct assay of estrogens (estrone + estradiol), the glucose-6-phosphate dehydrogenase for gonadotrophin determination. These techniques can be applied to all biological fluids. They are simple and straightforward, do not need extraction and can be automated. Urinary assays are very useful in clinical practice to appreciate ovarian function, either during a spontaneous cycle or under stimulation, and are mandatory to decide the timing of hCG injection. The reported studies can be listed as follows: determination of preovulatory LH rise (LH greater than 10 Ul/g of creatinine) prior to embryo transfer after cryopreservation and thawing, detection of LH surges during ovarian stimulation, either premature surges causing premature luteinization, either normal surges when follicular maturation is adequate (296 cycles), confirmation of pituitary desensitization when using GnRH agonists (43 cycles), study of the initial stimulatory effect of GnRH agonists (13 patients). This effect can be responsible for the inadequate results obtained with the so-called "short protocol" in this experience when compared with the "long protocol" in the author's experience with compared with the "long protocol" (8 p. cent pregnancy rate per stimulation cycle versus 20 p. cent respectively, intrafollicular LH in 129 follicular fluids (86 with GnRH agonists and 43 without) has no correlation with the fecundability of the ovum. These results lead to extend bioluminescent techniques to the study of other parameters, and in particular FSH.     

Infertility: Corpus luteum function and pregnancy rates with clomiphene citrate therapy: comparison of human chorionic gonadotrophin-induced versus spontaneous ovulation

A total of 508 clomiphene citrate cycles with intra-uterineinsemination (IUI) performed in 233 consecutive patients werestudied. In 247 cycles insemination was performed 36–38h after human chorionic gonadotrophin (HCG)-triggered ovulation;in the remaining 261 cycles IUI was performed 18–20 hafter urinary luteinizing hormone (LH) kit detection of a spontaneousLH surge. Corpus luteum function, as determined by luteal phaselength and midluteal progesterone concentrations, together withpregnancy rates were analysed. There was no difference in lutealphase parameters between spontaneous and HCG-triggered cycleswhen adjusting for patient age. Furthermore, the pregnancy ratesdid not differ between the HCG and LH kit groups, even afteradjusting for patient age and number of motile spermatozoa inseminated.Additionally, the large numbers of cycles analysed providedsufficient power to detect increases in clinical pregnancy ratesin spontaneous ovulatory cycles and HCG-induced ovulation of10.1 and 2.4% respectively, using the customary significancelevel (alpha-type error) of 0.05. These findings indicate thatpregnancy rates and corpus luteum function in carefully monitoredclomiphene citrate/IUI cycles do not differ between HCG-triggeredand spontaneous ovulatory cycles.     

Reliability of home urinary LH tests for timing of insemination: a consumer's study.

In this study, intrauterine insemination (IUI) was timed either after the detection of a urinary luteinizing hormone (LH) surge at home by the patient (group A), or following a positive LH test as interpreted by the gynaecologist (group B). Afterwards, samples tested by the patient were retested by the gynaecologist and vice versa. The gynaecologist also rechecked his own findings and the results were correlated with ultrasound data and charts of basal body temperature. Forty-seven cycles were evaluated. The patient's and the gynaecologist's readings agreed (+/- 12 h) in 42 of the cases (89%), and in five cycles (11%) a difference greater than 24 h was found. The intra-observer variation in the gynaecologist's results was +/- 12 h in four cycles (8.5%). These findings suggest that the LH test can be used as a reliable home device for the prediction of pending ovulation and timing of IUI.     

The effects of clomiphene citrate upon ovulation and endocrinology when administered to patients with unexplained infertility

Twenty-four couples with unexplained infertility were studied in a spontaneous cycle followed by a clomiphene citrate (CC) cycle (150 mg, days 5-9). All spontaneous cycles were ovulatory, as defined by follicular collapse determined by transvaginal sonography. In CC cycles, 6/24 (25%) cycles demonstrated luteinized unruptured follicles (LUF). In 2/6 LUF cycles there was no apparent luteinizing hormone (LH) surge. LUF cycles had significantly elevated LH levels in the follicular phase compared to ovulatory CC cycles. There was no apparent difference in serum oestradiol. In CC cycles multifollicular development occurred in 87.5% of cycles, with significantly elevated serum oestradiol. Luteinizing hormone and follicle-stimulating hormone were elevated in the follicular phase compared to spontaneous cycles. This study suggests a high incidence of LUF when CC is administered to ovulatory patients, and its use in patients with ovulatory infertility is questioned.     

Volumetric self-sampling of cervicovaginal fluid to determine potential fertility: a multicentre pre-effectiveness study of the Rovumeter

The aim of this study was to assess how effectively the Rovumeter, designed for the volumetric self-sampling of cervicovaginal fluid (CVF), can be used to locate the minimum period of potential fertility (PPF) during ovulatory cycles. A multicentre, prospective study was undertaken of volunteers (attending natural family planning clinics) over three consecutive, apparently normal, menstrual cycles. All women collected daily samples of early morning urine and CVF and recorded the volumes (to the nearest 1.0 and 0.1 ml respectively). The concentrations of oestrone glucuronide (EG), luteinizing hormone (LH) and pregnanediol glucuronide (PG) were measured in all samples of early morning urine by immunoassay. A preliminary data set was used to optimize an algorithm to detect the start and end of potential fertility from the volumes of CVF. The end-points used were the normality of each menstrual cycle from its length, the length of luteal phase, and concentrations of EG, LH and PG, the start and end days of potential fertility from CVF volumes, and the minimum PPF, which was defined as the day of the LH peak minus 3 to day plus 2 inclusive. Overall, 72 women (median age 30 years, range 24-38) were recruited from three centres (23 from Birmingham, 24 from Milan, 25 from Santiago) and contributed data from 235 menstrual cycles (median length 28 days, range 23-44). The urinary LH peak was identified in 228 cycles (97%; median time, day 15 from day 1 of last menses, with range day 10 to day 35). The use of the Rovumeter gave start and end signals of potential fertility during 138 cycles (59%). The median length of the derived PPF was 8 days (range 4-18). The signals covered the defined, minimum PPF in 113 cycles [i.e. 50% of those with an LH peak; range 28% (Milan) to 62% (Birmingham)]. Overall 16/72 women (22%) had successful tests over three consecutive menstrual cycles [range 2/24 (8%; Milan) to 8/23 (35%; Birmingham)]. We conclude that signals from daily changes in the volume of CVF as determined by the use of the Rovumeter consistently locate the minimum period of potential fertility in only a small proportion of women.      

Comparison of a rapid, quantitative and automated assay for urinary luteinizing hormone (LH), with an LH detection test, for the prediction of ovulation

The prediction of ovulation is necessary for oocyte aspiration in a spontaneous cycle and can be reliably achieved only by measuring luteinizing hormone (LH). Since radioimmunoassays of LH take too long for repeated measurements on the same day, we evaluated the possibility of adapting a rapid and fully automated assay of serum LH for use with urine samples. The study group comprised spontaneously ovulating women (38 cycles) who requested artificial insemination. Their serum oestradiol (E2) levels, ultrasound profile (US) and thrice daily urinary LH levels were determined from day 10 of their menstrual cycle. These patients were followed until US signs of follicular rupture were recorded. In all patients, a well-defined LH peak was measured in the urine. This peak lasted 12-15 h and was followed in 35 cycles (no US available for 3) by follicular rupture 9-51 h later. The data were grouped according to the time of the LH peak on day 0. Patients experiencing an LH peak between 0300 h and 0700 h on day 0 had significantly lower levels of E2 on day 0 compared to those with an LH peak between 2200 h and midnight. This is due to the fact that in the patients with an LH peak between 0300 h and 0700 h, E2 levels were already decreasing (from day 1 to day 0), whereas in those with the LH peak between 2200 h and midnight E2 levels were still increasing on the morning of day 0. We conclude that the 30-min IMX LH assay is a reliable, rapid and readily acceptable method for measuring urinary LH and for the prediction of ovulation.     

Adjustment of urinary mercury in health risk assessment of mercury.

The determination of adjustment method of urinary mercury in spot urine is one of the important issues in assessing the health risks of mercury workers. But there have been debates about whether creatinine or other forms of correction for urinary concentration are better in reducing the variation of urinary mercury. We evaluated four adjustment methods-specific gravity, creatinine, log creatinine and excretion rate-by correlation between values adjusted by the four methods and individual exposure levels which were the geometric mean of daily air mercury level for 2 or 5 days, and mercury concentrations in 24 hour urine were also investigated to compare the results of spot urine. The correlation between values of spot urine and mercury exposure level was over 0.8 in all adjustment methods for workers who worked over 1 year. All four adjustment methods for urinary mercury were found to be similar in assessing the exposure, log creatinine and excretion rate method however were not practical to use due to lack of reference values, and variable standard values of specific gravity. And the creatinine adjusted values were more sensitive in low mercury exposure level. We therefore recommend the creatinine adjustment method for adjustment of urinary mercury.     

Prediction of the potentially fertile period by urinary hormone measurements using a new home-use monitor: comparison with laboratory hormone analyses

BACKGROUND: The study compared a new urinary hormone monitoring system, Clearview Primera Fertility Monitor (CPFM), with laboratory hormone analyses in the prediction of the potentially fertile period. METHODS: Thirty healthy female volunteers provided blood and early morning urine samples for one cycle. Serum oestradiol, progesterone and luteinizing hormone (LH), and urinary LH and oestrone-3-glucuronide (E3G) were measured. The fertility status of volunteers; Low, High or Peak, was collected from monitors and compared with the hormone measurements. RESULTS: There was agreement between the first day of peak fertility and the urinary LH peak day in 65.6% of cycles and detection 1 or 2 days before the urinary LH peak day in 24.1 and 6.9% of cycles respectively. In 58.6% of cycles the system detected up to 5 days of increased fertility prior to the urinary LH peak day. Warning days of the urinary LH peak were similarly determined using defined thresholds of E3G and oestradiol providing up to 5 days warning in 82.8 and 96.6% of cycles respectively. CONCLUSIONS: The system can provide couples attempting to conceive with information about the potentially fertile days in the cycle in order that they may time intercourse. It also has potential for use in evaluation and treatment of infertile couples.     

Elimination of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol when normalized to urinary creatinine

Gas chromatography mass/spectrometry quantitative analysis of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH), the major metabolite of delta-9-tetrahydrocannabinol (THC) found in urine following marijuana use, was performed on serial urine specimens collected from an inpatient adolescent population of marijuana users. Creatinine normalization of THCCOOH was used to compensate for dilute or concentrated urine specimens. The urinary terminal elimination rate constant and terminal half-life was calculated for each subject. The mean urinary elimination rate constant for THCCOOH normalized to creatinine was 0.08433 days(-1) (range 0.05408-0.16544) reflecting a 8.22 day terminal half-life. A half-life of 1.15 days was observed for the initial decline phase of THCCOOH corrected by creatinine suggesting that reuse of marijuana can be detected after this phase ends. The creatinine normalized THCCOOH level was a better indicator for predicting reuse of marijuana than urinary concentrations of THCCOOH. The Mean Residence Time (MRT) of THCCOOH/Cr (5.7 days) correlated well with the length of time a subject will have detectable urinary THCCOOH concentrations (20.8 days).     

Assessment of serum luteinizing hormone during ovarian stimulation with gonadortrophins

An immunoradiometrte assay (IRMA), using monoclonal antibodieswith high affinity for human luteinizing hormone (HLH), wasevaluated for quantitative measurement of serum LH after humanchorionic gonadotrophin (HCG) administration in patients undergoingstimulation of multiple folh'cular development. Compared toa radioimmunoassay (RIA) commonly used to monitor serum LH,LH IRMA was more effective by several orders of magnitude indiscriminating between HLH and HCG and showed no crossreactivityat HCG concentrations normally found in serum after hormonetreatment. Assays of serum samples obtained from 10 patientsreceiving HCG as part of an HMG/HCG protocol to induce ovulationfor IVF/GIFT also demonstrated that RIA values were greatlyaffected by exogenous HCG. It was estimated that 17–32%of serum HCG was measured as serum LH in RIA. In contrast, determinationsof serum LH by IRMA was not biased by exogenous HCG. Data fromIRMA indicated that eight of the 10 patients showed a significantrise in LH secretion, relative to mean baselines, at either12 or 36 h after adminstration. In one patient the rise hadalready occurred before HCG administration. When an LH riseoccurred, either before or after HCG injection, mean valueswere 2to 9fold higher than those of baseline levels. Assumingthat LH rises > 12 mlU/ml may relate to an endogenous surgeof LH, none of the patients showed a surge prior to HCG administration.On the contrary, the occurrence of an ‘LH surge’after HCG was apparent in four patients. These data demonstratethe application of monoclonal antibodies incorporated in anIRMA to study the occurrence of endogenous LH surges duringstimulation of follicular development by gonadotrophins.     

Characteristics of urinary luteinizing hormone (LH) during the induction of LH surges of different magnitude in blood

Urinary luteinizing hormone (LH) testing has been proposed asa reliable method for the prediction of ovulation but its accuracyhas been challenged by some studies. To check how accuratelythe oscillations of urinary LH reflected the plasma changes,surges of LH of different magnitude and duration were artificiallyinduced in plasma and the hormone was measured simultaneouslyin urine. Post-menopausal women (n = 16) were stimulated during1 week with a combination of transdermal oestradiol (400 µg)and i.m. progesterone (25 mg on day 4, 50 mg on day 5) to obtainan LH discharge comparable with the pre-ovulatory LH peak. Ashort and moderate peak of LH was induced by the i.v. injectionof 100 µg gonadotrophin-releasing hormone (GnRH) in sixpre-menopausal women, whereas an LH discharge of higher amplitudeand longer duration was induced by a single dose of 0.3 mg s.c.buserelin. The total urine production of the day was fractionatedinto 8 h periods. LH was measured by a commercial radioimmunoassay.Unambiguous peaks of LH were detected in the urine of all thewomen stimulated with either oestradiol plus progesterone orbuserelin, but in only three out of the six women receivingGnRH. The urine LH reproduced the plasma changes of the hormonewith short delay since the peaks were mostly detected in thesame time fraction in which the serum discharge occurred.     

Blind, olfactory bulbectomized female rats do not have daily luteinizing hormone surges

Previous studies from other laboratories have shown that female hamsters on short photoperiod become acyclic and have daily LH surges. These effects are eliminated if the animals are pinealectomized (PX) before being placed on the short photoperiod. Reiter and colleagues have shown that pre-pubertally blinded (BL) and olfactory bulbectomized (BX) female rats also have irregular estrous cycles, and this effect is also eliminated by PX [Endocr. Rev., 1 (1983) 109]. The main question addressed by the present study was whether the BL + BX rats also have daily LH surges. Twenty-five-day-old female Sprague-Dawley rats were divided into 5 groups: LD 14:10 sham (control); BL + BX; BL + BX + PX; LD 6:18 sham; and LD 6:18 BX. Ten weeks following surgery, all animals were sampled (0.5 ml) every 5 h for 2 days from an indwelling atrial catheter. Daily vaginal smears indicated that the BL + BX group were in estrus much less frequently than controls (15.8 +/- 1.8 vs 27.3 +/- 1.5% of days cornified cells, 10 rats/group smeared for more than 23 days each) and in general had longer, irregular cycles. The other 3 groups all had smear patterns similar to controls. All 5 groups had LH surges on the day of proestrus (greater than 200 ng/ml maximum value), but no group had LH surges on 2 sequential days or an LH surge on any other day of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of ovulation by low-dose mifepristone (RU 486).

Mifepristone (RU 486) is a potent antigestagen and antiglucocorticoid which when given at a dose of 25-600 mg disrupts folliculogenesis, inhibits ovulation and induces menses in healthy women. This study reports the effects of much lower doses of mifepristone than used previously, given for the duration of a complete menstrual cycle. Healthy female volunteers (n = 11) with regular menstrual cycles were given mifepristone at a daily dose of 5 mg (n = 6) or 2 mg (n = 5) for 30 days, beginning immediately after an ovulatory placebo cycle. Mifepristone prevented menstruation for the duration of the treatment period, with recurrence of menses 15-29 days after replacement of mifepristone with placebo. Daily mifepristone given in either 5 mg or 2 mg doses inhibited ovulation, as indicated by the lack of a rise in urinary pregnanediol excretion. The excretion of oestrone glucuronide in urine rose during treatment, suggesting ovarian follicular development. Inhibition of ovulation appeared to result from a failure of the positive feedback effect of oestradiol on the hypothalamo-pituitary axis, as no surges of luteinizing hormone were seen despite pre-ovulatory levels of oestrone glucuronide being measured during exposure to mifepristone. The cycle immediately following treatment was shorter than the pre-treatment cycle, with lower peak levels of pregnanediol glucuronide, suggesting an inadequate luteal phase. Recovery from the effects of mifepristone treatment was more rapid after 2 mg than after 5 mg and one subject conceived in the immediate post-treatment phase, indicating adequate ovulation and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)     

The urinary calcium/creatinine ratio as a measure of urinary calcium excretion

Urine specimens were collected from 26 normal subjects, 10 patients with proven primary hyperparathyroidism, and eight patients with hypercalcaemia due to other causes. After overnight urine concentration, an oral water load was given to induce a diuresis and provide urine specimens with a relatively wide range of creatinine concentration for each subject. In normal subjects the urinary calcium/creatinine ratio was found to be independent of urine concentration. In eight out of 10 patients with primary hyperparathyroidism and in two out of eight patients with hyper-calcaemia due to other causes, the urinary calcium/creatinine ratio was found to be high when the creatinine concentration was low, but usually normal when the creatinine concentration was high. The results suggest that if the urinary calcium/creatinine ratio of random urine specimens is used as a ;screening' procedure to detect hypercalciuria the latter cannot be excluded if the urinary creatinine concentration is more than 40 mg per 100 ml.     

The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay

Human gonadotrophins undergo metabolic transformations which result in the presence of several smaller, structurally and immunologically related forms of gonadotrophins in the urine. For luteinizing hormone (LH), a beta core fragment (LHbeta cf) has been isolated from the pituitary and characterized. The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physicochemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of LHbeta cf have a different structure, probably in the carbohydrate moieties. This communication characterizes the expression of LHbeta cf in the urine of both reproductive and post-reproductive age women and in men, employing assays highly specific for the pituitary form of the fragment. It was found that LHbeta cf is the predominant LH associated molecular form in the urine during peri-ovulatory period, peaking 1-3 days later than intact LH and reaching a concentration of approximately 600 fmol/mg creatinine, 7-fold higher than either LH or LH free beta subunit. Corresponding concentrations of human chorionic gonadotrophin (HCG) beta cf were <1% that of LHbeta cf. LHbeta cf cross-reaction with some LH or LHbeta monoclonal antibodies may well interfere with the accurate estimation of the day of the LH surge when urinary tests are utilized.      

Influence of missense mutation and silent mutation of LHbeta-subunit gene in Japanese patients with ovulatory disorders

The frequency of variant LHbeta containing two point mutations (T(986)-C and T(1008)-C) and its relationship to reproductive disorders differ widely between ethnic groups. In a Japanese population, variant luteinizing hormone (LH) correlates with ovulatory disorders. Here we examined the relationship between two missense mutations and five silent mutations (C(894)-T, G(1018)-C, C(1036)-A, C(1098)-T and C(1423)-T) in the LHbeta gene, and ovulatory disorders. We studied 43 patients with ovulatory disorders, 79 patients with normal ovulatory cycles, and 23 healthy men who agreed to join our DNA analysis. PCR-amplified LHbeta-subunit gene sequences were compared with a base sequence of wild-type LH reported after direct sequencing. The highest frequency (0.945) of novel allele was observed at the position of the C(1036)-A transition. No homozygotes for wild-type LHbeta (C(1036)) were identified. The frequency of novel allele in patients with polycystic ovary syndrome, endometriosis, premature ovarian failure and luteal insufficiency was significantly different from that of healthy women. The frequencies of novel alleles (C(894)-T, C(1098)-T and C(1423)-T) in patients with ovulatory disorders were significantly higher than those with normal ovulatory cycles. The mean incidence of point mutation in patients with ovulatory disorders was higher than in those with normal ovulatory cycles. Among patients with variant LH, five silent mutations were identified in 87.5% of patients with ovulatory disorders, whereas only a few silent mutations were identified in patients with normal ovulatory cycles. In a Japanese population, five silent mutations of variant LH could have influenced two missense mutations and/or other unknown missense mutations, causing ovulatory disorders.