X/99/$03.00/0 Vol. 84, No. 5 The Journal of Clinical Endocrinology & Metabolism Copyright 1999 by The Endocrine Society

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1 X/99/$03.00/0 Vol. 84, No. 5 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society A New Contributing Factor to Polycystic Ovary Syndrome: The Genetic Variant of Luteinizing Hormone* JUHA S. TAPANAINEN, RIITTA KOIVUNEN, BART C. J. M. FAUSER, ANN E. TAYLOR, RICHARD N. CLAYTON, MADHURIMA RAJKOWA, DAVINIA WHITE, STEPHEN FRANKS, LEENA ANTTILA, KIM S. I. PETTERSSON, AND ILPO T. HUHTANIEMI Department of Obstetrics and Gynecology, University of Oulu (J.S.T., R.K.), Oulu, Finland; the Department of Obstetrics and Gynecology, Dijkzigt Academic Hospital and Erasmus University (B.C.J.M.F.), 3015 GD Rotterdam, The Netherlands; Reproductive Endocrine Unit, Massachusetts General Hospital (A.E.T.), Boston, Massachusetts 02114; the Department of Medicine, School of Postgraduate Medicine, Keele University (R.N.C., M.R.), Hartshill, Stoke-on-Trent, United Kingdom ST4 6QG; the Department of Reproductive Science and Medicine, Imperial College School of Medicine, St. Mary s Hospital (D.W., S.F.), London, United Kingdom; and the Departments of Obstetrics and Gynecology (L.A.), Biotechnology (K.S.I.P.), and Physiology (I.T.H.), University of Turku, Turku, Finland ABSTRACT Although the etiology of polycystic ovary syndrome (PCOS) is still unclear, LH is considered to play a central role in its pathogenesis. An immunologically anomalous form of LH, with two point mutations in the LH gene, has been recently described. This genetic variant of LH (v-lh), of wide geographic distribution, is functionally different from wild-type (wt) LH. To assess the role of the v-lh in PCOS, we analyzed its frequency in groups of PCOS patients from Finland, The Netherlands, the United Kingdom, and the United States. The LH status was determined by two immunofluorometric assays from a total of 1466 subjects. The carrier frequency of the v-lh allele in the whole study population was 18.5%, being highest (28.9%) in Finland and lowest (11.2%) in The Netherlands. In the individual countries, the frequency of v-lh was similar in obese and nonobese controls, but in The Netherlands and Finland, it was 5- to 7-fold lower in obese PCOS subjects compared with that in the other groups (2 4.5% vs. Received August 26, Revision received October 28, Accepted January 22, Address all correspondence and requests for reprints to: Dr. Juha S. Tapanainen, Department of Obstetrics and Gynecology, Oulu University Hospital, FIN Oulu, Finland. juha.tapanainen@ oulu.fi. * This work was supported by grants from the Sigrid Jusélius Foundation and the Academy of Finland %; P 0.05). A similar tendency was found in the United States (5.7% vs %), but not in the United Kingdom. The overall high prevalence of v-lh in healthy women and women with PCOS suggests that it is compatible with fertility. The similar frequency of v-lh in healthy nonobese and obese women indicates that obesity per se is not related to the variant. In contrast, the lower frequency of v-lh in obese PCOS patients suggests that v-lh somehow protects obese women from developing symptomatic PCOS. However, the regional differences in this finding between patients with apparently similar diagnostic criteria emphasizes the multifactorial nature of this syndrome, and that its pathogenesis may vary according to the genetic background. Although the definitive role of v-lh in PCOS remains to be proven, its determination may improve the prediction of risk of PCOS, especially in obese women. (J Clin Endocrinol Metab 84: , 1999) POLYCYSTIC ovary syndrome (PCOS) is the most common ovarian disorder, with typical features of obesity, anovulation, hyperandogenism, hirsutism, and infertility. The prevalence of PCOS varies from 1 8% in the general population depending on the diagnostic criteria (1 3). It is characterized by increased levels of circulating LH, increased ovarian androgen production, hyperinsulinemia, and multiple cysts in the ovaries related to arrested follicular development. Despite extensive research, the etiology and basic mechanisms underlying PCOS are still largely unknown. New theories have been introduced, and the interest in the genetics of PCOS has increased considerably during recent years (4). We have previously discovered in Finland a genetic variant of LH (v-lh) that contains two missense point mutations in the LH gene (Trp 8 Arg and Ile 15 Thr) (5, 6). Subsequently, a similar LH form was described from Japan (7), and very recently it was reported to be a common polymorphism with world-wide distribution (8). The two mutations, rather than representing neutral polymorphisms, alter the biological function of LH. v-lh has elevated in vitro bioactivity compared to that of wild-type LH (wt-lh), but its half-time in the circulation is significantly shorter (9). Whether the opposite alterations in the in vitro and in vivo bioactivities of v-lh reflect higher or lower overall bioactivity still remains obscure. Nevertheless, the evidence is emerging that LH action in carriers of the v-lh allele differs from that in individuals with wt-lh. v-lh shows association with various clinical conditions, such as elevated serum estradiol, testosterone, and sex hormone-binding globulin in the follicular phase of the menstrual cycle (10); menstrual disorders (7, 11); PCOS (10); and slow pace of puberty (12). In addition, v-lh is widely distributed in different ethnic groups, varying in frequency from 0% in Kotas from South India to an extremely high 53.5% in aboriginal Australians (6). 1711

2 1712 TAPANAINEN ET AL. JCE&M 1999 Vol 84 No 5 As PCOS patients often have high LH levels, and altered LH action is involved in the pathogenesis of this disease (13, 14), we decided to study the prevalence and distribution of v-lh in a large population of women with PCOS. We studied retrospectively the prevalence of v-lh in nonobese and obese PCOS patients in three patient cohorts from Europe and one from the United States and compared the results with those in healthy control women from the same populations. Subjects and Methods Subjects A total of 1466 serum samples from women in Finland, The Netherlands, the United Kingdom, and the United States (Caucasians from Boston, MA) were analyzed for v-lh. For the comparison between healthy and PCOS subjects, 363 women fulfilled the criteria of PCOS, 79 women had polycystic ovaries without other symptoms of PCOS, and 944 healthy women of similar age with the PCOS subjects served as controls. The age of the subjects varied between yr. A body mass index (BMI; kilograms per meter squared) more than 27 was classified as obese and a BMI of 27 or less was classified as nonobese. The diagnostics of PCOS were polycystic ovaries by transvaginal ultrasound ( 8 follicles of 2 8 mm diameter in 1 plane section), oligoamenorrhea (intermenstrual interval, 35 days), and hirsutism (Ferriman-Gallwey score, 7), or raised serum testosterone ( 2.7 nmol/l). The relative percentages of obese control and PCOS subjects were different in the 4 countries (P ; Table 1); therefore, the data based on BMI were not combined. Hormone assay Two immunofluorometric assays (DELFIA, Wallac Oy, Turku, Finland) with different combinations of monoclonal antibodies (Mab) were used to determine the LH phenotypes. The reference method (assay 2) used two LH -specific Mab that recognize wt and v-lh with similar stoichiometries (8). The other assay (assay 1) uses Mab that recognize only the intact LH / -dimer and the -subunit, but not v-lh (5). The ratio of LH measured by assay 1/assay 2 allowed classification of the samples into three categories: 1) more than 0.9 (normal ratio), the subject has two normal LH alleles; 2) (low ratio), the subject is heterozygous for the mutant LH gene; and 3) less than 0.15 (zero ratio), the subject is homozygous for the mutant LH gene (9). The intra- and interassay variations of assays 1 and 2 were less than 4% and 5%, respectively, at LH concentrations at and above the lowest standard concentration of 0.6 IU/L of the WHO International Reference Preparation 80/552. The serum samples from cycling women were obtained in the follicular phase, and those from subjects with amenorrhea and oligomenorrhea were obtained randomly. Statistical analysis The frequencies of v-lh among the groups were compared using the 2 test. The confidence intervals for the differences of the frequencies of v-lh among the countries were calculated using the Goodman statistic (15). The serum LH concentrations were compared using one-way ANOVA or Student s t test. Results The carrier frequency of the v-lh allele in the whole study population was 18.5%; it was highest (28.9%) in Finland and lowest (11.2%) in The Netherlands (Table 2). In each country studied, the v-lh carrier frequency was similar in obese and nonobese controls, but it was significantly lower in obese PCOS subjects in The Netherlands and Finland, where the proportions of v-lh carriers in obese PCOS subjects were only 2.0% and 4.5%, respectively (Fig. 1). In contrast to these countries, no such difference, or even a tendency, was seen in United Kingdom. In the United States, such a tendency was apparent (Fig. 1). The number of women homozygous for v-lh was only 25 (1.7%), and none of them was obese. The frequencies of v-lh carriers in nonobese and obese women with PCO only from Finland and United Kingdom were 25.4% and 18.7%, respectively. However, the number of these subjects was only 16, which limits the value of the comparison. The PCOS patients had significantly higher serum levels of LH than control subjects [PCOS: 7.5 IU/L; 95% confidence interval (CI), ; controls: 4.4 IU/L; 95% CI, ; P 0.001] measured by assay 2. The subjects with normal and low ratios exhibited similar serum concentrations of LH, but the nonobese control women with zero ratio (homozygous for v-lh) had significantly lower levels of LH than those with wt-lh or low ratio (heterozygous for v-lh; Table 3). TABLE 1. The frequency of nonobese and obese control (A) and PCOS (B) women in different study groups in different countries Controls Controls 27 CI 95% 27 CI 95% A. Finland (n 299) 266 (88.9) (11.1) a Netherlands (n 204) 146 (71.6) (28.4) United Kingdom (n 233) 176 (75.5) (24.5) USA (n 208) 181 (87.0) (13.0) a PCOS PCOS 27 CI 95% 27 CI 95% B. Finland (n 61) 39 (63.9) (36.1) b,c Netherlands (n 117) 68 (58.1) (41.9) b United Kingdom (n 122) 50 (41.0) (59.0) USA (n 63) 28 (44.4) (55.6) The data are expressed as numbers of subjects, with the frequencies in parentheses (percentages). a P compared to Netherlands; P compared to United Kingdom. b P 0.01 compared to United Kingdom. c P 0.05 compared to USA.

3 LH AND THE ETIOLOGY OF PCOS 1713 TABLE 2. The carrier frequency of the wt and v-lh alleles in different countries, as determined by LH immunoassays Finland (n 425) Netherlands (n 347) United Kingdom (n 404) USA (n 290) Normal ratio 302 (71.1) 308 (88.8) 336 (83.2) 249 (85.9) Low ratio 111 (26.1) 38 (10.95) 66 (16.3) 31 (10.7) Zero ratio 12 (2.8) 1 (0.3) 2 (0.5) 10 (3.5) Low zero ratio 123 (28.9) 39 (11.2) 68 (16.8) 41 (14.1) CI CI a CI a CI a Allele frequency 135 (15.9) 40 (5.8) 70 (8.7) 51 (8.8) CI CI a CI a CI a The normal ratio individuals are homozygotes for wt-lh, those with low ratio are heterozygotes, and those with zero ratio are v-lh homozygotes. Percentages are given in parentheses. a P 0.05 compared to Finland. FIG. 1. The frequency (percentage) of v-lh (homozygotes and heterozygotes) in the different countries among controls and PCOS women with BMI of 27 or less or with BMI of more than 27 kg/m 2. The number of subjects is indicated inside the bars. The frequency of v-lh between controls and PCOS women was compared using 2 test: a, P 0.05; b, P 0.01; c, P 0.03; after continuity correction, P 0.07 compared to controls with BMI above 27 kg/m 2. Discussion The present study on women with PCOS confirms our previous findings on the common occurrence of the v-lh and its considerable variation among different populations and even within Caucasian populations. The overall high prevalence of v-lh suggests that it is compatible with fertility, and in some conditions it may even be beneficial. This could contribute to the wide variation in its frequency between different populations, i.e. from 0% to more than 50% (6, 8). With regard to PCOS, the most striking finding was the low v-lh frequency of 2 4.5% in obese patients from The Netherlands and Finland, with a similar tendency in the United States. The in vitro bioactivity of v-lh is higher than that of wt-lh, whereas its in vivo half-life is shorter than that of wt-lh (9, 11). It is therefore unclear whether its high activity at the receptor site but shorter half-time in the circulation result in a net increase or decrease in the overall bioactivity

4 1714 TAPANAINEN ET AL. JCE&M 1999 Vol 84 No 5 TABLE 3. Serum concentrations of LH in PCOS subjects and controls with wt-lh (normal assay 1/assay 2 ratio) or with v-lh (low or zero ratio) BMI Controls 27 PCOS 27 CI 95% Controls 27 PCOS 27 CI 95% wt-lh (IU/L) 4.2 (2.2) 8.1 (2.1) a 4.3, (1.9) 6.7 (2.3) 2.5, 1.5 (n 573) (n 139) P (n 139) (n 143) P 0.05 v-lh (IU/L) 4.5 (1.8) 8.6 (1.8) 4.8, (1.7) 7.4 (2.7) 4.3, 1.7 (n 140) (n 27) P (n 29) (n 19) P Low ratio only 4.6 (1.7) 8.2 (1.8) 4.5, (1.7) 7.4 (2.7) 4.3, 1.7 (n 127) (n 23) P (n 29) (n 19) P Zero ratio only 3.9 (1.4) b 10.6 (2.8) 8.5, 4.9 c c c (n 13) (n 4) P 0.05 Values are geometric means (SD). The 95% confidence intervals for the differences of the means between controls and PCOS subjects in the same weight group are calculated. Student s two-tailed t test was used for all paired comparisons, as all variables were normally distributed after log transformation. a P vs. PCOS with BMI greater than 27. b P 0.05 vs. respective wt-lh and heterozygote (low ratio only). c No zero ratio found in obese subjects. of in vivo. It is also possible that the nature of the wt- and v-lh peaks (long vs. short) in the face of unaltered pulse frequency (9) will cause qualitative changes in target cell responses to LH stimulation. The LH action in heterozygotes, with long and short acting hormones in each pulse, may be distinct from that of wt-lh or v-lh alone. In support of this, we have found a variety of differences in LH action between wt-lh and mainly heterozygous individuals, that cannot be easily explained by quantitative differences in LH action. Women heterozygous for v-lh have significantly elevated levels of serum estradiol, testosterone, and sex hormonebinding globulin in the follicular phase (10). Heterozygous boys have a slower progression of puberty (12). Japanese studies report menstrual disturbances in women homozygous for the v-lh allele (7, 11). As v-lh is apparently related to altered LH action, and LH is considered to play a central role in the pathogenesis of PCOS (13, 14), it is intriguing to interpret the present results. Obesity per se was not related to the variant heterozygosity, as the carrier frequency was the same in lean and obese control women. However, all of the homozygous women were nonobese, but their number was only 25. The low frequency of v-lh in obese PCOS patients in 3 of the populations studied suggests that obese women with v-lh may somehow be protected from developing symptomatic PCOS, and those with wt-lh are more likely to develop the disease. Previously, Rajkhowa et al. (11) from the United Kingdom have shown that obese women with PCOS exhibit a higher frequency of v-lh than obese controls. The present study confirmed this finding, which is at variance with the conspicuously low frequency of v-lh in obese PCOS subjects in Finland and The Netherlands, and with a similar trend in the United States. This discrepancy is difficult to explain, but given the multifactorial pathogenesis of PCOS, it is possible that another genetic factor(s), enriched in the United Kingdom population, alters the ovarian response to v-lh. Another discrepant finding was the overrepresentation of v-lh homozygotes in the United States population. As genetic isolation in this Caucasian population from the Boston area is unlikely, and all of the other populations studied to date for frequency of the v-lh allele are in Hardy-Weinberg equilibrium (6, 8), it apparently represents chance. The high overall frequency of the v-lh allele in women in general and its low frequency in obese PCOS patients suggest that v-lh has a role in reproductive functions and may somehow contribute to the pathogenesis of PCOS in obese individuals. Although it remains unknown whether the two mutations in v-lh lead to a net increase or decrease in the overall LH action, it is tempting to speculate that it offers advantage or disadvantage to certain individuals depending on their genetic background and environment. Hyperinsulinemia, hyperandrogenism, and dyslipidemia are commonly found in women with PCOS (16, 17). PCOS appears often at a young age, and therefore, we should be able to identify young women at future risk of these hormonal and metabolic changes. As a prognostic or diagnostic approach, the detection of v-lh may allow the discrimination between individuals with high and low risks of PCOS. Acknowledgments We thank Drs. Janet Hall, Kathryn Martin, William Crowley, Nanette Santoro, and Judith Adams for subject recruitment, and Sirkka Pramila, M.Sc., for statistical analysis. References 1. Franks S Polycystic ovary syndrome: a changing perspective. Clin Endocrinol (Oxf). 31: Futterweit W, Mechanick JI Polycystic ovarian disease: etiology, diagnosis, and treatment. Compr Ther. 14: Franks S Polycystic ovary syndrome. N Engl J Med. 333: Franks S, Gharani N, Waterworth D, et al The genetic basis of polycystic ovary syndrome. Hum Reprod. 12: Petterson K, Ding Y-Q, Huhtaniemi I An immunologically anomalous luteinizing hormone variant in a healthy woman. J Clin Endocrinol Metab. 74: Nilsson C, Jiang M, Pettersson K, et al A common genetic variant of the human luteinizing hormone (LH); determination by allele specific oligonucleotide hybridization assay and by variant specific immunoassay. Clin Endocrinol (Oxf). 49: Furui K, Suganuma N, Tsukahara S-I, et al Identification of two point mutations in the gene coding luteinizing hormone (LH) subunit, associated with immunologically anomalous LH variants. J Clin Endocrinol Metab. 78: Nilsson C, Pettersson K, Millar RP, Coerver KA, Matzuk MM, Huhtaniemi I Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. Fertil Steril. 67: Haavisto A-M, Pettersson K, Bergendahl M, Virkamäki A, Huhtaniemi I.

5 LH AND THE ETIOLOGY OF PCOS Occurrence and biological properties of a common genetic variant of luteinizing hormone. J Clin Endocrinol Metab. 80: Rajkhowa M, Talbot JA, Jones PW, et al Prevalence of an immunological LH -subunit variant in a UK population healthy women and women with polycystic ovary syndrome. Clin Endocrinol (Oxf). 43: Suganuma N, Furui K, Furuhashi M Screening of the mutations in luteinizing hormone -subunit in patients with menstrual disorders. Fertil Steril. 336: Raivio T, Huhtaniemi I, Anttila R, et al The role of luteinizing hormone gene polymorphism in the onset and progression of puberty in healthy boys. J Clin Endocrinol Metab. 81: Balen AH Hypersecretion of luteinizing hormone and the polycystic ovary syndrome. Hum Reprod. 8: Balen AH, Conway GS, Kaltsas G, et al Polycystic ovary syndrome: the spectrum of the disorder in 1741 patients. Hum Reprod. 10: Miller R Multiple comparisons. In: Kotz S, Johnson NL, eds. Encyclopedia of statistical sciences. New York: Wiley and Sons; Conway GS, Agrawal R, Betteridge DJ, Jacobs HS Risk factors for coronary heart disease in lean and obese women with the polycystic ovary syndrome. Clin Endocrinol (Oxf). 37: Conway GS, Jacobs HS Clinical implications of hyperinsulinaemia in women. Clin Endocrinol (Oxf). 39: