Lack of association of the common immunologically anomalous LH with endometriosis

R. Gazvani1,3, P. Pakarinen2, P. Fowler1, S. Logan1 and I. Huhtaniemi1

1 Department of Obstetrics and Gynaecology, University of Aberdeen, Aberdeen Maternity Hospital, Foresterhill, Aberdeen AB25 2ZD, UK and 2 Department of Reproductive Endocrinology, University of Turku, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Subfertile women with endometriosis have been reported to demonstrate impaired follicular growth, ovulatory dysfunction and disturbed LH patterns. In addition, abnormal LH and/or LH receptors have been linked with endometriosis-associated infertility. Carriers of a variant of the ß-subunit of luteinizing hormone (V-LH) are largely healthy; however, differences in their gonadal function such as alterations in gonadal steroidogenesis, ovarian reserve, pubertal development and predisposition to diseases such as infertility and polycystic ovarian disease have been found. METHODS AND RESULTS: To explore the possible relationship between endometriosis and V-LH, we examined its frequency in 230 women undergoing laparoscopic surgery for the investigation of infertility. For the entire study population, 185 (80.4%) were wild type; 42 (18.3%) were heterozygous; and three (1.3%) were homozygous for V-LH. No difference was found between women with (n = 85) and without (n = 145) endometriosis concerning the frequency of the type of LH. CONCLUSION: Our results do not support the hypothesis that the variant form of LH is associated with an altered risk of endometriosis in the population tested.

Key words: ß-subunit/endometriosis/luteinizing hormone/variant


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
LH, like other members of the glycoprotein hormone family, is a heterodimer composed of a common {alpha}-subunit and a unique ß-subunit, which confers biological specificity for the hormone receptor in the target organs (Kurioka et al., 1999Go). LH is important in ovarian follicular growth, the stimulation of ovarian steroidogenesis, oocyte maturation, ovulation, and subsequent luteinization of the follicle. From a clinical perspective, abnormal LH secretion is associated with anovulation, luteal insufficiency, and premature oocyte maturation, leading to menstrual disorders, polycystic ovarian syndrome (PCOS), and infertility (Balen et al., 1993Go; Risma et al., 1995Go).

A common polymorphism in the LH ß-subunit gene, where two point mutations cause alterations in the amino acid sequence (Trp 8 -> Arg and Ile 15 -> Thr) and introduce an extra glycosylation signal to Asn13 has been identified (Furui et al., 1994Go; Pettersson et al., 1994Go; Nilsson et al., 1998Go). Although carriers of this variant gene, common variant LH, (V-LH) are largely healthy, differences in their gonadal function have been found. These include alterations in their gonadal steroidogenesis (Rajkhowa et al., 1995Go), ovarian reserve (Suganuma et al., 1995Go; Takahashi et al., 1999Go), pubertal development (Raivio et al., 1996Go) and predisposition to diseases such as infertility (Furui et al., 1994Go), and PCOS (Rajkhowa et al., 1995Go; Elter et al., 1999Go; Tapanainen et al., 1999Go). In women with menstrual disorders (Ramanujam et al., 1999Go) and men with infertility (Ramanujam et al., 2000Go), no relationship was found with respect to the common V-LH.

Endometriosis is an enigmatic disease associated with pelvic pain and infertility. Although the exact prevalence of endometriosis in the general population is not known, it is a common condition among women of reproductive age (Oral et al., 1996Go). Despite extensive research, the pathophysiology of this disease is still not clearly understood. Nevertheless, the widely accepted retrograde menstruation theory does not explain why some women develop the disease while others do not.

Previous studies have reported ovulatory dysfunction, leading to reduced fertilization rates in women with minimal endometriosis (Cahill et al., 1997Go). Impaired follicular growth and reduced circulating concentrations of estradiol as well as disturbed LH surge patterns were also reported in subfertile women with endometriosis (Cahill and Hull, 2000Go). In addition, abnormal LH and/or LH receptors have been linked with endometriosis and associated infertility (Cheesman et al., 1982Go; Rönnberg et al., 1984Go; Chew et al., 1990Go).

It was suggested that lifelong menstrual irregularities were associated with a reduced risk of endometriosis. Women with irregular cycles during their reproductive life have fewer menses and hence are exposed to a lower risk of retrograde menstruation. The same pathogenic mechanism has been suggested to interpret the reduced risk of endometriosis in women with late menarche (Anonymous, 1999Go). If we therefore accept that abnormal LH patterns may be related to menstrual irregularities, this may influence the risk of endometriosis in these women.

Experimental and clinical observations suggest that endometriosis is estrogen dependent, and that estrogens seem to be important for growth and maintenance of endometriosis (DiZerega et al., 1980Go). It has recently been suggested that this V-LH was less potent in stimulating ovarian estrogen production (Lamminen and Huhtaniemi, 2001Go).

Our hypothesis was that an elevated prevalence of this common variant of LH (V-LH) would explain some of the endocrinological and menstrual changes observed in women with endometriosis. We therefore screened women for the common V-LH in relation to the presence of endometriosis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 235 women undergoing laparoscopic surgery for the investigation of infertility at The Grampian University Hospitals were recruited to take part in the study. The study was approved by the local ethics committee and a written informed consent was obtained prior to the operation. None of the patients had been on medication for at least 1 month prior to the laparoscopy and none were on any long acting drugs.

Operative findings were recorded regarding the presence, location, volume and degree of endometriosis. The condition of tubes, ovaries, pouch of Douglas, and bowels were inspected and endometriosis was graded according to the revised American Society for Reproductive Medicine scoring system (ASRM, 1997Go).

The venous blood (10 ml) was allowed to clot and centrifuged at 400 g for 10 min. Serum was separated, divided into a maximum of four aliquots and frozen. The LH status was determined by two solid phase, two-site immunofluorometric assays (IFMA) (Pettersson and Söderholm, 1990Go, 1991Go). Assay 1 recognizes only the wild-type (wt) LH and assay 2 equally wt- and V-LH. Assay 2 was performed using the DELFIA® hLH Spec kit (Wallac Oy, Turku, Finland) with the ß-subunit-specific monoclonal antibodies. Assay 1 was similar in principle, with exception that the capture antibody was a monoclonal antibody recognizing the intact LH {alpha}/ß dimer and the detecting monoclonal antibody an {alpha}-subunit-specific one. Assays were done following the Spec kit protocol. The ratio of LH concentrations of assay 1 to assay 2 indicates the LH status: for wt-LH ß homozygotes, >0.9; for heterozygotes, 0.2–0.9; and for homozygous variants, <0.2 (Nilsson et al., 1997Go).

Statistical analysis
Non-parametric data were described as median (interquartile range – IQR) and parametric data as mean (standard deviation – SD). Mann–Whitney U-test was used for the comparison of medians. Differences between groups were analysed using Pearson's {chi}2-test. The correlation between two variables was assessed by Spearman's test. Significance was accepted where P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Serum samples from 230 of the 235 women were collected and successfully assayed for the LH variant status. In three cases blood samples were not obtained and in two cases endometriosis status was not determined due to technical difficulties. The study group was 85 women (37%) with a diagnosis of endometriosis and the control group was 145 women (63%) with no evidence of the disease. There was no difference between the study and control groups regarding their age, parity and body mass index (BMI) (Table IGo).


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Table I. Patient characteristics and findings
 
Wild-type (wt) LH was found in 185 (80.4%), while heterozygote V-LH was identified in 42 (18.3%) and homozygote V-LH in 3 (1.3%) women. The incidences of LH types did not show any difference between the study and control groups (Table IGo). Two women in the study group, both with minimal endometriosis (AFS Stage-1) and no family history, and one woman in the control group demonstrated homozygous V-LH. There was no correlation between the presence of endometriosis and LH status.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was undertaken to determine whether the recently discovered V-LH was more common in women with endometriosis. Our results suggest no correlation between the common V-LH status and the presence of endometriosis in the population studied. We found that the overall frequency of heterozygosity for V-LH allele closely matched previous estimates of the frequency of V-LH carriers in Northern European populations (Nilsson et al., 1997Go; Cramer et al., 2000Go). Only three out of 230 subjects (1.3%) were homozygous for the V-LH (2.3% of cases and 0.7% of controls). Consequently, the alleic frequencies between the two groups were similar: controls, wt-LH = 0.90, V-LH = 0.1; endometriosis, wt-LH = 0.89. V-LH = 0.11 (Table IGo). Although these numbers are small, a similar prevalence of homozygosity and heterozygosity in cases and controls suggests that the effect of the common V-LH on endometriosis is not more pronounced in homozygous subjects than in heterozygous subjects.

Analyses of the biological properties of the common V-LH indicate that the described mutations may alter the physiological function of LH. In-vitro studies have suggested that the V-LH has elevated bioactivity in homozygous subjects compared with those homozygous for wt-LH (Haavisto et al., 1995Go; Suganuma et al., 1996Go) whereas the in-vivo half-life of the V-LH in circulation was shorter than that for wt-LH (Haavisto et al., 1995Go). In addition, women heterozygous for the V-LH have slightly but significantly higher serum levels of estradiol, testosterone, and sex hormone binding globulin than women without the variant (Rajkhova et al., 1995). This indicates alterations in the bioactivity of the V-LH and in ovarian response to LH stimulation which could be compatible with the findings that V-LH is more potent at the receptor site but has a shorter life span in circulation (Akhmedkhanov et al., 2000Go). It is possible therefore to suggest that the effect of the hormone with shorter half-life can be at least partly compensated by the enhanced promoter activity of the gene, as has also been demonstrated by others (Jiang et al., 1999Go). This may help to explain the apparent similarity in the prevalence of V-LH in women with endometriosis and healthy controls.

In conclusion, the results of our study do not support the hypothesis that this variant form of LH is associated with an altered risk of endometriosis in the population tested. However, since it is possible that only homozygous V-LH may be associated with endometriosis, the power of this study is limited. Since V-LH is suggested to have a marginally suppressive effect on reproduction, it may be gradually vanishing from the gene pool, but the pace appears to be diverse in different populations. In genetic isolates, like Aboriginal Australians, or Finnish Lapps, the pace is slower (Nilsson et al., 1997Go, 1998Go) due to the enrichment of the V-LH allele and of `genetic drift', i.e. inefficient mixing of genes in isolated populations. The population in Scotland, especially in Aberdeen, is a relatively stable one and therefore may be expected to demonstrate differences when compared with populations from the other parts of the UK.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Ms Tarja Laiho for her technical assistance throughout this project.


    Notes
 
3 To whom correspondence should be addressed. E-mail: R.Gazvani{at}lwh-tr.nwest.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Akhmedkhanov, A., Toniolo, P., Zeleniuch-Jacquotte, A., Pettersson, K. and Huhtaniemi, I. (2000) Genetic variant of luteinizing hormone and risk of breast cancer in older women. Cancer Epidemiol. Biomarkers. Prev., 9, 839–842.[Abstract/Free Full Text]

Anonymous (1999) Risk factors for pelvic endometriosis in women with pelvic pain or infertility. Gruppo Italiano per lo Studio dell' endometriosis. Eur. J. Obstet. Gynecol. Reprod. Biol., 83, 195–199.[ISI][Medline]

ASRM (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil. Steril., 67, 817–821.[ISI][Medline]

Balen, A.H., Tan, S.L. and Jacobs, H.S. (1993) Hypersecretion of luteinizing hormone: a significant cause of infertility and miscarriage. Br. J. Obstet. Gynaecol., 100, 1082–1089.[ISI][Medline]

Cahill, D.J. and Hull, M.G. (2000) Pituitary–ovarian dysfunction and endometriosis. Hum. Reprod. Update., 6, 56–66.[Abstract/Free Full Text]

Cahill, D.J., Wardle, P.G., Maile, L.A., Harlow, C.R. and Hull, M.G. (1997) Ovarian dysfunction in endometriosis-associated and unexplained infertility. J. Assist. Reprod., 14, 554–557.[ISI]

Cheesman, K.L., Ben-Nun, Chatterton, R.T. Jr and Cohen, M.R. (1982) Relationship of luteinizing hormone, pregnanediol-3-glucuronide, and estriol-16-glucuronide in urine of infertile women with endometriosis. Fertil. Steril., 38, 542–548.[ISI][Medline]

Chew, P.C., Peh, K.L., Loganath, A., Gunasegaram, R. and Ratnam, S.S. (1990) Elevated peritoneal fluid luteinizing hormone and prolactin concentrations in infertile women with endometriosis. Int. J. Gynaecol. Obstet., 33, 35–39.[ISI][Medline]

Cramer, D.W., Pettersson, K.S., Barbieri, R.L. and Huhtaniemi, I.T. (2000) Reproductive hormones, cancers, and conditions in relation to a common genetic variant of luteinizing hormone. Hum. Reprod., 15, 2103–2107.[Abstract/Free Full Text]

DiZerega, G.S., Barber, D.L. and Hodgen, G.D. (1980) Endometriosis: role of ovarian steroids in initiation, maintenance and suppression. Fertil. Steril., 33, 649–653.[ISI][Medline]

Elter, K., Erel, C.T., Cine, N., Ozbek, U., Hacihanefioglu, B. and Ertungealp, E. (1999) Role of the mutations Trp8 -> Arg and Ile15 -> Thr of the human luteinizing hormone beta-subunit in women with polycystic ovary syndrome. Fertil. Steril., 71, 425–430.[ISI][Medline]

Furui, K., Suganuma, N., Tsukahara, S., Asada, Y., Kikkawa, F., Tanaka, M., Ozawa, T. and Tomoda, Y. (1994) Identification of two point mutations in the gene coding luteinizing hormone (LH) beta-subunit, associated with immunologically anomalous LH variants. Clin. Endocrinol. Metab., 78, 107–113.

Haavisto, A.M., Pettersson, K., Bergendahl, M., Virkamäki, A. and Huhtaniemi, I. (1995) Occurrence and biological properties of a common genetic variant of luteinizing hormone. J. Clin. Endocrinol. Metab., 80, 1257–1263.[Abstract]

Jiang, M., Pakarinen, P., Zhang, F.P., El-Hefnawy, T., Koskimies, P., Pettersson, K. and Huhtaniemi, I. (1999) A common polymorphic allele of the human luteinizing hormone beta-subunit gene: additional mutations and differential function of the promoter sequence. Hum. Mol. Genet., 8, 2037–2046.[Abstract/Free Full Text]

Kurioka, H., Takahashi, K., Irikoma, M., Okada, M., Ozaki, T., Ueda, T. and Miyazaki, K. (1999) Diagnostic difficulty in polycystic ovary syndrome due to an LH-ß-subunit variant. Eur. J. Endocrinol., 140, 235–238.[ISI][Medline]

Lamminen, T. and Huhtaniemi, I. (2001) A common genetic variant of luteinizing hormone; relation to normal and aberrant pituitary-gonadal function. Eur. J. Pharmacology, 414, 1–7.[ISI][Medline]

Nilsson, C., Pettersson, K., Millar, R.P., Coerver, K.A., Matzuk, M.M. and Huhtaniemi, I. (1997) Worldwide frequency of a common genetic variant of luteinizing hormone: an international colloborative research. International Collaborative Research Group. Fertil. Steril., 67, 998–1004.[ISI][Medline]

Nilsson, C., Jiang, M., Pettersson, K., Iitiä, A., Mäkelä, M., Simonsen, H., Easteal, S., Herrera, R.J. and Huhtaniemi, I. (1998) Determination of a common genetic variant of luteinizing hormone using DNA hybridization and immunoassays. Clin. Endocrinol. (Oxford), 49, 369–376.[ISI][Medline]

Oral, E., Olive, D.L. and Arici, A. (1996) The peritoneal environment in endometriosis. Hum. Reprod. Update, 2, 385–398.[Abstract/Free Full Text]

Pettersson, K. and Söderholm, J.R. (1990) Ultrasensitive two-site immunometric assay of human lutropin by time-resolved fluorometry. Clin. Chem., 36, 1928–1933.[Abstract/Free Full Text]

Pettersson, K. and Söderholm, J.R. (1991) Individual differences in lutropin immunoreactivity revealed by monoclonal antibodies. Clin. Chem., 37, 333–340.[Abstract/Free Full Text]

Pettersson, K., Mäkelä, M.M., Dahlén, P., Lamminen, T., Huoponen, K. and Huhtaniemi, I. (1994) Gene polymorphism found in the LH ß gene of an immunologically anomalous variant of human luteinizing hormone. Eur. J. Endocrinol, . 130 (Suppl. 2), 65.

Raivio, T., Huhtaniemi, I., Anttila, R., Siimes, M.A., Hagenäs, L., Nilsson, C, Pettersson, K. and Dunkel, L. (1996) The role of luteinizing hormone-ß gene polymorphism in the onset and progression of puberty in healthy boys. J. Clin. Endocrinol. Metab., 81, 3278–3282.[Abstract]

Rajkhowa, M., Talbot, J.A., Jones, P.W., Pettersson, K., Haavisto, A.M., Huhtaniemi, I. and Clayton, R.N. (1995) Prevalance of an immunological LH beta-subunit variant in UK population of healthy women and women with polycystic ovary syndrome. Clin. Endocrinol. (Oxford), 43, 297–303.[ISI][Medline]

Ramanujam, L.N., Liao, W.X., Roy, A.C., Loganath, A., Goh, H.H. and Ng, S.C. (1999) Association of molecular variants of luteinizing hormone menstrual disorders. Clin. Endocrinol., 51, 243–246.[ISI][Medline]

Ramanujam, L.N., Liao, W.X., Roy, A.C. and Ng, S.C. (2000) Association of molecular variants of luteinizing hormone with male infertility. Hum. Reprod., 15, 925–928.[Abstract/Free Full Text]

Risma, K.A., Clay, C.M., Nett, T.M., Wagner, T., Yun, J. and Nilson, J.H. (1995) Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc. Natl Acad. Sci., 92, 1322–1326.[Abstract]

Rönnberg, L., Kauppila, A. and Rajaniemi, H. (1984) Luteinizing hormone receptor disorder in endometriosis. Fertil. Steril., 42, 64–68.[ISI][Medline]

Suganuma, N., Furui, K., Furuhashi, M., Asada, Y., Kikkawa, F. and Tomoda, Y. (1995) Screening of the mutations in luteinizing hormone beta-subunit in patients with menstrual disorders. Fertil. Steril., 63, 989–995.[ISI][Medline]

Suganuma, N., Furui, K., Kikkawa, F., Tomoda, Y. and Furuhashi, M. (1996) Effects of the mutations (Trp8 -> Arg and Ile15 -> Thr) in human luteinizing hormone (LH) beta-subunit on LH bioactivity in vitro and in vivo. Endocrinology, 137, 831–838.[Abstract]

Takahashi, K., Ozaki, T., Okada, M., Kurioka, H., Kanasaki, H. and Miyazaki, K. (1999) Increased prevalence of luteinizing hormone beta-subunit variant in patients with premature ovarian failure. Fertil. Steril., 71, 96–101.[ISI][Medline]

Tapanainen, J.S., Koivunen, R., Fauser, B.C., Taylor, A.E., Clayton, R.N., Rajkowa, M., White, D., Franks, S., Anttila, L., Pettersson, K.S. and Huhtaniemi, I.T. (1999) A new contributing factor to polycystic ovary syndrome: the genetic variant of luteinizing hormone. Clin. Endocrinol. Metab., 84, 1711–1715.

Submitted on December 10, 2001; accepted on January 20, 2002.





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