1 Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Boston, MA, USA, 2 Department of Biotechnology and 3 Department of Physiology, University of Turku, Turku, Finland
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Abstract |
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Key words: breast cancer/genetic polymorphisms/infertility/LH/ovarian cancer
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Introduction |
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Materials and methods |
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Study 1 cases were drawn from women with a family history of ovarian cancer recruited through a familial ovarian cancer (FOC) clinic established at the BWH for the purpose of counselling and screening (Muto et al., 1993). Methods for this study have been described previously (Cramer et al., 1994
). Briefly, women with a confirmed family history of at least one primary or two second degree relatives with ovarian cancer were eligible if they were residents of greater Boston, premenopausal, <50 years, and not currently pregnant, breastfeeding, or using oral contraceptives. In the original study, 106 unrelated women with family histories of ovarian cancer were selected and plasma specimens were still available on 101 FOC cases for assessment of LH variant. Study 2 cases were selected from women with ductal BCIS. Cases were <50 years, resided in greater Boston, premenopausal, not pregnant, breastfeeding, or taking exogenous hormones during the preceding 3 months, and had never had chemotherapy, hormonal therapy, or radiation to the pelvis. From this study, there were 94 cases who participated and had plasma specimens still available. In both studies, we selected control women from the general population using annually published lists of residents of Massachusetts. We similarly restricted control selection to women <50 years who were not pregnant, breastfeeding, or using oral contraceptives, and were matched controls to cases by age and precinct of residence. We had the additional requirements that the controls should not have a family history of ovarian cancer or should not have breast cancer or BCIS. Women were randomly sampled to match the age and residency distribution of the FOC or BCIS cases as previously described (Cramer et al., 1994
; Bohlke et al., 1998
). From both studies, plasma was available on 154 subjects.
Information about demographic variables, medical history and family history was collected by personal interviews. Details of the reproductive history collected included cycle irregularity defined as >10 days variation in cycle length, menstrual pain and symptoms, history of endometriosis or ovarian cysts diagnosed by a physician, and infertility defined as difficulty conceiving lasting for 2 years. Blood samples were obtained during one of the first 4 days of the menstrual cycle. The blood was drawn in heparinized tubes, separated into plasma, erythrocyte, and buffy coat specimens and stored at -70°C before assay. The hormonal component of this study involved the measurement of gonadotrophins, including follicle stimulating hormone (FSH) and luteinizing hormone (LH), and oestradiol. Oestradiol was measured by a solid phase radioimmunoassay (Coat-A-Count®; Diagnostic Products Corp., Los Angeles, CA, USA) using unextracted plasma and a highly specific oestradiol antibody. LH and FSH were measured using an immunoradiometric assay (Coat-A-Count®) with the World Health Organization (WHO) International Reference Preparations (IRP) for human menopausal gonadotrophin as the standard. Intra- and interassay coefficients of variation were less than 10 and 15% respectively, for all three assays. Between the two study periods the standard for FSH changed from the first to the second IRP leading to lower measured FSH in the second compared to the first study.
Plasma that had been banked from the studies was retrieved and sent to one of the authors (I.T.H.) for determination of v-LH status as previously described (Pettersson et al., 1992). LH genotypes were inferred from the ratio of two immunofluorometric assays (DELFIA, Wallac OY, Turku, Finland) with different combinations of monoclonal antibodies (mAb). The reference method (assay 2) used two LHß-specific mAB that recognized wild type and v-LH with similar stoichiometries. In the other assay (assay 1), one of the mAB used recognized only the intact LH
/ß dimer, but not v-LH. The ratio of LH values measured by assay 1/assay 2 allowed classification of the samples into three categories: (i) >0.9 (normal ratio), the subject had two normal LHß alleles; (ii) 0.20.9 (low ratio), the subject was heterozygous for the mutant LHß gene; (iii) <0.15 (`zero' ratio), the subject was homozygous for the mutant LH gene. The intra- and interassay variations of assay 1 and 2 were <4 and 5% respectively, at LH concentrations at and above the lowest standard concentration of 0.6 IU/l of the WHO IRP (80/552). Two subjects from the FOC study (one case and one control) had indeterminate assay results and were excluded from this analysis.
Statistical analyses on hormonal and epidemiological variables were performed using standard SAS programmes for t-tests, 2 or Fisher's exact test, and linear regression analyses (Statistical Analysis System Institute Inc., SAS Campus Dr., Cary, NC, USA). For assessing the effects of several study variables on an outcome variable, generalized linear modelling or multiple linear regression was used.
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Results |
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Discussion |
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We found the overall frequency of heterozygosity for v-LH allele to be 17.6% which matches closely previous estimates of the frequency of v-LH carriers in Northern European populations (Nilsson et al., 1997). The high frequency of the variant has been interpreted to indicate that v-LH probably doesn't impact on male or female fertility but published results are discrepant. A study of Japanese women suggested that the variant does occur with higher frequency in women who have ovulatory problems (Takahaski et al., 1999), while another study (Ramanujam et al., 2000
) found no association between male infertility and LH variants. Among the combined controls in our study, who were selected from the general population rather than infertility clinics, we found a higher frequency of self-reported delay-to-conception associated with v-LH. Medical records were not available to clarify the nature of the fertility problems in women with v-LH. Overall, among v-LH carriers or homozygotes, the proportion of nulliparous women was not significantly increased suggesting that v-LH may be related to subfertility rather than sterility. Of potential interest is the fact that the 16 subjects with v-LH who reported infertility had apparently lower BMI (23.3 kg/m2) compared to the BMI (25.3 kg/m2) of those women with v-LH who did not report infertility (not significant). Although this difference was not significant, it contrasts with nearly identical BMI in the women with and without infertility who were wild type, 23.3 and 23.9 kg/m2 respectively.
There is also some controversy whether v-LH influences menstrual regularity or ovarian conditions such as the polycystic ovarian syndrome (PCOS). An initial report suggested that the frequency of v-LH was increased among women with PCOS, especially those who were obese (Rajkhowa et al., 1995). However, this was not confirmed in a subsequent report involving a different population (Tapanainen et al., 1999
). In a study of women with a variety of menstrual disorders, including PCOS, no association with v-LH was observed (Ramanujam et al., 1999
). In the current study, we had no medically confirmed cases of PCOS but we believe it is of interest that women with v-LH had no higher occurrence of irregular menses and were apparently less likely to report physician-diagnosed ovarian cysts and menstrual weight gain. The latter finding might be compatible with a greater occurrence of anovulatory cycles suggested by the association we found with infertility.
It is difficult to predict a priori what effects v-LH might have on ovarian function compared to the wild type since its bioactivity appears to be increased in vitro but its half-life in circulation may be shorter (Haavisto et al., 1995; Suganuma et al., 1996
). A previous study suggested that follicular phase oestradiol, testosterone, and sex hormone binding globulin concentrations may be higher in women heterozygous for v-LH (Tapanainen et al., 1999
). In this study of early follicular phase hormone concentrations, we found that women who were heterozygous for v-LH compared to wild types had higher early follicular phase LH as measured by the assays done in Boston, after adjustment for age, study, case or control status, BMI, and smoking.
Chance must be regarded as a possible explanation for some of our findings including the observation that women with v-LH were apparently more likely to be depressed and less likely to be smokers compared to the women who were wild type. A finding which was not statistically significant was that women heterozygous or homozygous for v-LH were apparently less likely to have a family history of prostate cancer. This observation may be of interest in view of a report that delayed puberty was more likely to occur in boys who carry v-LH (Raivio et al., 1996). A conclusion of that report was that v-LH might be involved in the regulation of the growth hormone and insulin-like growth factor I axis during childhood, which may also influence prostate cancer risk (Chan et al., 1998
).
In summary, among premenopausal women with either a family history of ovarian cancer or BCIS, we found no evidence of a greater frequency of v-LH that might suggest an association with ovarian or breast cancer. However, our study suggests that v-LH may impact on ovarian function. In particular, women who possess v-LH may have sub-fertility, especially those with normal or below normal BMI. Further studies of the effect of v-LH on ovarian (and testicular) function may be of value in sorting out the long-appreciated link between body mass and fertility.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 28, 2000; accepted on June 19, 2000.