polymorphism in infertility and endometriosis-associated infertility

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Infertility is a very common medical problem that affects approximately 15–20% of couples who attempt pregnancy. Overall, infertility is due to female factors in ...
European Journal of Obstetrics & Gynecology and Reproductive Biology 151 (2010) 66–69

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Luteinizing hormone b-subunit gene (LHb) polymorphism in infertility and endometriosis-associated infertility ˆ ngela M.B. Souza, Fernanda A. Mafra, Bianca Bianco, Denise M. Christofolini, A Karina Zulli, Caio P. Barbosa * Division of Gynecological Pathology and Human Reproduction, Department of Gynecology and Obstetrics, ABC School of Medicine, Avenida Prı´ncipe de Gales 821, CEP: 09060-650, Santo Andre´, SP, Brazil

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 November 2009 Received in revised form 22 January 2010 Accepted 30 March 2010

Objective: To establish the frequency of LHb G1502A polymorphism in infertile women with endometriosis, infertile women without endometriosis and a control group. Study design: Case–control study including 110 infertile women with endometriosis, 84 infertile women without endometriosis and a control group consisting 209 healthy fertile women recruited from the ABC School of Medicine. The LHb G1502A polymorphism was studied by RPLP-PCR (restriction fragment length polymorphism-polymerase chain reaction). Results: Genotypes GG, GA and AA of the LHb G1502A polymorphism presented frequencies of 54.6%, 31.8% and 13.6%, respectively, in the women with endometriosis (p = 0.0398); of 52.4%, 38.1% and 9.5% (p = 0.0123), respectively, in the infertile women without endometriosis; and of 68.9%, 21.5% and 9.6%, respectively, in the control group. In patients with minimal/mild endometriosis and moderate/severe endometriosis, the GG, GA and AA genotype frequencies were, respectively, 47.3%, 36.4% and 16.3% (p = 0.0118); and 61.8%, 27.3% and 10.9% (p = 0.5975). Considering the alleles, allele G was present in 70.5% of the patients with endometriosis, 71.4%% of the infertile women without endometriosis and in 79.7% of the controls, whereas allele A was present in 29.5%, 28.6% and 20.3%, respectively, in the infertile women with endometriosis (p = 0.0121), infertile women without endometriosis (p = 0.0409) and controls. Alleles G and A presented frequencies of 65.5% and 34.5% and 75.5% and 24.5%, respectively, in minimal/mild endometriosis (p = 0.0026) and moderate/severe endometriosis (p = 0.4062). Conclusion: The data suggest that LHb G1502A polymorphism may be involved in the predisposition to infertility and minimal/mild endometriosis-associated infertility, although endometriosis might be only a coincidental finding along with infertility. ß 2010 Elsevier Ireland Ltd. All rights reserved.

Keywords: Endometriosis Infertility Polymorphism Luteinizing hormone LHb gene

1. Introduction Infertility is a very common medical problem that affects approximately 15–20% of couples who attempt pregnancy. Overall, infertility is due to female factors in 58%, to male factors in 25%, and is unexplained in 17% of cases. Many factors have been implicated in female infertility, although some causes are well established, including age, tubal state, and frequency of ovulation, while others remain controversial, such as luteal phase deficiency, endometriosis, and cervical and immunologic factors [1]. Endometriosis is a common disease, defined as the growth of endometrial tissue outside the uterine cavity, that often results in a vast array of gynecological problems including dyspareunia, dysmenorrhea, pelvic pain and infertility. Endometriosis affects

* Corresponding author. Tel.: +55 11 4438 7299; fax: +55 11 4438 7299. E-mail address: [email protected] (C.P. Barbosa). 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.03.022

3–10% of women in their reproductive years and 20–50% of women with infertility [2–4]. Susceptibility to endometriosis depends on a complex interaction of immunologic, genetic and hormonal factors [5]. Many aspects of female reproductive function are strongly influenced by genetic factors, and numerous studies have attempted to identify susceptibility genes for disorders affecting female fertility such as endometriosis [1,5–7]. Luteinizing hormone is important in the development of follicle growth, stimulation of steroidogenesis, and maturation of the oocyte. In women, it promotes ovulation and luteinization of the ovarian follicle and stimulates the production of androgen that in turn serves as a substrate for follicular estradiol synthesis in the ovaries. Luteinizing hormone may also play a role in maintaining the progesterone production of the corpus luteum. Abnormal LH secretion induces anovulation, luteal insufficiency, and premature oocyte maturation, leading to menstrual disorders, polycystic ovary syndrome (PCOS), recurrent miscarriage and infertility [8].

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Mutations in the human LHb gene have been reported and linked to infertility [1,9]. Liao et al. [1] studied 40 infertile women with menstrual disorders, polycystic ovary syndrome, and endometriosis; 12 women with idiopathic infertility and a control group consisting of 212 healthy fertile women to LHb G1502A polymorphism by RFLP-PCR (restriction fragment length polymorphism-polymerase chain reaction). The results showed that two infertile women with endometriosis presented LHb G1502A polymorphism and the authors suggested an association between these mutations and infertility, especially endometriosis-associated infertility. Thus, the aim of the present study was to establish the frequency of LHb G1502A polymorphism in infertile women with endometriosis, infertile women without endometriosis and a control group. 2. Materials and methods 2.1. Patients One hundred and ten women with endometriosis and with a history of infertility were screened from the Endometriosis Outpatient clinic of the ABC School of Medicine. Endometriosis was diagnosed by laparoscopy and classified based on histological criteria, according to the guidelines of the American Society for Reproductive Medicine [10]. Eighty-four infertile women without endometriosis were screened at the Human Reproduction Service of FMABC. The causes of their infertility included: tubo-peritoneal factor (n = 15), uterine factor (n = 9), ovulatory factor (n = 26), cervical factor (n = 2), unexplained infertility (n = 27), recurrent pregnancy loss (n = 1) and ovulatory and tubo-peritoneal factors (n = 4). The control group was composed of 209 fertile women who underwent tubal ligation, which allows confirmation of the absence of endometriosis and infertility, also screened at the Family Planning Outpatient clinic. The cause of infertility was investigated according to the minimum preliminary procedure for infertile couples: hormonal and biochemistry profile, serum testing, testing for sexually transmitted diseases, imaging examinations, investigation of genetic and/or immunologic abnormalities, hysterosalpingography, hysteroscopy, laparoscopy (performed in all women up to 36 years old and also in patients over 36 whenever there were symptoms or abnormalities on the imaging examinations), and semen analysis. Patients with endometriosis who did not achieve pregnancy after at least six natural or induced cycles following laparoscopy were considered infertile. All women whose partner presented male infertility factors were excluded from the study. Clinical data and peripheral blood samples were collected only after disclosure of the objectives of the study and signed informed consent from the participants, as approved by the local ethics committee. 2.2. Genotyping of polymorphism LHb G1502A Peripheral blood was collected from each patient and control in an EDTA-containing tube. Genomic DNA was extracted from peripheral blood lymphocytes using the Illustra blood genomicPrep Mini Spin Kit, according to the manufacturer’s instructions (GE Healthcare Life Sciences, USA). PCR was carried out in a total volume of 25 mL reaction mixture, containing 10 reaction buffer (500 mM KCl, 100 mM Tris–Cl; pH 8.3), 2.5 mM MgCl2, 0.8 mM dNTP, 2.0 U Taq polymerase, and 50 nM of LHb sense primer (50 -AGTCTGAGACCTGTGGGGTCAGCTT30 ) and antisense primer (50 -GGAGGATCCGGGTGTCAGGGCTCCA30 ). PCR generated a 395 base-pair (bp) fragment. The cycling profile consisted of denaturation at 93 8C for 30 s, annealing at

Fig. 1. PCR product corresponding to LHb G1502A polymorphism: 100 bp (M), GG genotype (1), GA genotype (2) and AA genotype (3).

63 8C for 45 s, and extension at 72 8C for 30 s, except for the first cycle, when denaturation was extended to 5 min. The PCR product was digested with 5 U of Eco0109I restriction enzyme, and the reaction mixture was incubated at 37 8C for 2 h. The digestion product was subjected to electrophoresis on a gel containing 2% agarose stained with ethidium bromide. In a normal LH sequence (genotype GG), Eco0109I digestion generates two separate bands of 269 bp and 126 bp, respectively. In the presence of the heterozygous genotype (GA), Eco0109I digestion generates three separate bands of 395 bp, 269 bp, and 126 bp, respectively. In the presence of the AA mutation at codon 102, the enzyme recognition site is abolished, so that only one band of 395 bp is seen (Fig. 1). 2.3. Statistical analysis Allele and genotype frequencies were compared between groups using the x2-test. Statistical tests of significance and x2 analysis were carried out using the SPSS for Windows 8.0 software (SPSS, Inc., Chicago, IL). All p-values were two-tailed and 95% confidence intervals (CIs) were calculated. A p-value < 0.05 was considered statistically significant. 3. Results A total of 110 infertile patients with endometriosis (mean age: 34.5  4.3 years), 84 infertile patients without endometriosis (mean age: 35.1  5.2 years) and 209 fertile women without a history of endometriosis (mean age: 39.7  4.8 years) who composed the control group were studied. In the group with endometriosis, 30.9% (34/110) had endometriosis stage I, 19.1% (21/110) endometriosis stage II, 16.4% (18/110) endometriosis stage III, and 33.6% (37/110) endometriosis stage IV. These patients were divided into minimal/ mild endometriosis (stages I and II) (50.0%) and moderate/severe endometriosis (stages III and IV) (50.0%). The frequencies of genotypes GG, GA and AA of the LHb G1502A polymorphism in the patients with endometriosis were 54.6% (60/ 110), 31.8% (35/110) and 13.6% (15/110), respectively, p = 0.0398. Among the women with minimal/mild endometriosis, 47.3% (26/

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Table 1 Genotype and allele frequencies of the LHb G1502A polymorphism in infertile women with endometriosis, infertile women without endometriosis and controls. pa

OR (95%CI)b

G (%)

A (%)

15 (13.6)

0.0398

1.85 (1.15–2.97)

155 (70.5)

20 (36.4)

9 (16.3)

0.0118

2.47 (1.35–4.52)

34 (61.8)

15 (27.3)

6 (10.9)

0.5975

84

44 (52.4)

32 (38.1)

8 (9.5)

0.0123

209

144 (68.9)

45 (21.5)

20 (9.6)

Subjects

n

Distribution of genotypes GA (%)

AA (%)

Infertile women with endometriosis Minimal/mild endometriosis Moderate/severe endometriosis Infertile women without endometriosis Controls

110

60 (54.6)

35 (31.8)

55

26 (47.3)

55

GG (%)

pa

OR (95% CI)

65 (29.5)

0.0121

1.64 (1.13–2.39)

72 (65.5)

38 (34.5)

0.0026

2.07 (1.31–3.27)

1.37 (0.74–2.54)

83 (75.5)

27 (24.5)

0.4062

1.27 (0.78–2.09)

2.01 (1.20–3.38)

120 (71.4)

48 (28.6)

0.0409

1.57 (1.04–2.36)

333 (79.7)

85 (20.3)

Alleles

OR = odds ratio; CI = confidence interval. a Versus controls. b GA + AA versus GG genotype.

55) presented the normal homozygous genotype GG, 36.4% (20/55) the heterozygous genotype GA, and 16.3% (9/55) the mutated homozygous genotype AA, p = 0.0118. In the patients with moderate/severe endometriosis, the frequencies of genotypes GG, GA and AA were 61.8% (34/55), 27.3% (15/55) and 10.9% (6/ 55), respectively; p = 0.5975. In the infertile women without endometriosis, genotypes GG, GA and AA were observed in 52.4% (44/84), 38.1% (32/84) and 9.5% (8/84), p = 0.0123. In the control group, 68.9% (144/209) presented the normal homozygous genotype GG, 21.5% (45/209) the heterozygous genotype GA, and 9.6% (20/209) the homozygous mutated genotype AA (Table 1). Considering the alleles, allele G was present in 70.5% (155/220) of the patients with endometriosis, 71.4% (120/168) of the infertile women without endometriosis and in 79.7% (333/418) of the controls, whereas allele A was present in 29.5% (65/220), 28.6% (48/168) and 20.3% (85/418), respectively, in the patients with endometriosis (p = 0.0121), infertile women without endometriosis (0.0409) and controls. Of the women with minimal/mild endometriosis, 65.5% (72/110) presented allele G and 34.5% (38/ 110) presented allele A (p = 0.0026). In the patients with moderate/ severe endometriosis, the frequencies of alleles G and A were 75.5% (83/110) and 24.5% (27/110), respectively (p = 0.4062) (Table 1). 4. Comments Infertility can be attributed to reproductive disorders in both men and women. It affects about 20% of couples [11]. Many factors have been implicated in female infertility, especially endometriosis, and there are no specific genetic markers that could imply the pathogenesis of female infertility and/or endometriosis. SNPs (single nucleotide polymorphisms) are common in the human genome and often provide correlative evidence for the involvement of specific genes in human disease [12]. SNPs that affect the function of crucial components related to reproduction could have profound effects on the human fertility. LH belongs to the family of glycoprotein hormones. Structurally, LH is a heterodimer consisting of two dissimilar subunits: the a-subunit and the hormone-specific b-subunit. The intact heterodimer structure is required for biological activity [13]. G1502A, a common genetic variant of the LHb gene, results in the amino acid substitution of serine for glycine at position 102, and this substitution may have a potent effect on LH function. Because glycine and valine are important components in the formation of hydrophobic regions in a protein and serine has a polar side chain, the replacement of glycine by serine at position 102 introduces a hydrophilic force in the molecule. This could affect the normal conformation and function of LH and thus contribute to the pathogenesis of human infertility [1,14]. Furthermore, Ronnberg et al. [15] demonstrated that, in patients with endometriosis, the LH receptor concentrations in

ovarian follicles and corpora lutea were lower during the early and late follicular phase and the late luteal phase of the cycle than those seen in control subjects. In patients with severe and extensive endometriosis, the LH receptor concentrations were extremely low. It was speculated that the abnormal LH or LH receptor profiles might cause infertility in patients with endometriosis. In this way, the LH receptor altered by the polymorphism could present a lower sensitivity to the hormone. Even if the LH is at normal levels, due to the conformational abnormalities on receptor it could be understood as a hormonal deficiency—an inappropriate hormonal feedback mechanism. In the present study, the genotype frequencies of the LHb G1502A polymorphism were statistically different between infertile women with endometriosis (p = 0.0398) and infertile women without endometriosis (p = 0.0123) compared to controls. When we studied the patients with minimal/mild and moderate/ severe endometriosis separately, the genotype difference was evident in the group of women with minimal disease (p = 0.0118). When alleles G and A were studied separately, there was a statistically significant difference in the frequency of allele A between the group of women with endometriosis (p = 0.0121), especially minimal/mild endometriosis (p = 0.0026), infertile women without endometriosis (p = 0.0989) and controls. This finding suggests that the LHb G1502A polymorphism is related to minimal/mild endometriosis-associated infertility. During adult life, until menopause, LH together with FSH (follicle-stimulating hormone) regulates the production of the steroid sex hormones estradiol and progesterone by theca cells that surround growing follicles in the ovary [14]. The sensitivity of endometriosis to hormonal stimuli is clearly documented in the literature. Endometriosis is associated with alterations in the hypothalamus–hypophysis–ovary axis, with alterations resulting therefrom in the concentrations of hormones FSH, LH, estradiol and progesterone in serum, peritoneal fluid and follicular fluid of women with endometriosis. The association of endometriosis with alterations in the neuroendocrine axis suggests an abnormality in follicular function, with altered LH concentration, reduced fertilization capacity of the oocyte and altered luteal function [16,17]. It has, in fact, been reported that granulosa cells of the preovulatory follicles of infertile women with endometriosis are less sensitive to luteinizing hormone [18]. A significant reduction in aromatase activity in the follicles of women with endometriosis might explain the smaller production of steroids by the follicle [19]. Taken together, all these data show that the follicles of women with endometriosis present severely impaired steroidogenesis. Obviously, altered steroidogenesis can affect oocyte function, which might explain the ovulatory dysfunction, impaired fertilization and defective implantation, parameters frequently associated to endometriosis [20].

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In conclusion, we believe that the LHb G1502A polymorphism may be involved in the predisposition to infertility and minimal/ mild endometriosis-associated infertility, although endometriosis might be only a coincidental finding along with infertility. However, further studies with much larger samples are needed to evaluate the true role of LHb gene in infertility and endometriosis. Acknowledgments The authors wish to thank Dr Cristiane Gimenes for her help in selecting the patients with endometriosis and the control group and NEPAS (Study, Research and Health Assistance Nucleus of the ABC Faculty of Medicine) for granting student Fernanda Abani Mafra a Scientific Initiation scholarship. References [1] Liao WX, Roy AC, Chan C, Arulkumaran S, Ratnam SS. A new molecular variant of luteinizing hormone associated with female infertility. Fertil Steril 1998;69(1):102–6. [2] Gao X, Outley J, Botteman M, Spalding J, Simon JA, Pashos CL. Economic burden of endometriosis. Fertil Steril 2006;86(6):1561–72. [3] Barbosa CP, de Souza AM, Bianco B, Christofolini DM, Mafra FA, de Lima GR. OC125 immunostaining in endometriotic lesion samples. Arch Gynecol Obstet 2009 [Epub ahead of print]. [4] Barbosa CP, de Souza AM, Bianco B, Christofolini DM, Mafra FA, de Lima GR. Frequency of endometriotic lesions in peritoneum samples of asymptomatic fertile women and correlation with CA125. Sao Paulo Med J 2009 [Epub ahead of print]. [5] Bianco B, Christofolini DM, Mafra FA, Brandes A, Zulli K, Barbosa CP. +1730 G/A polymorphism of the estrogen receptor b gene (ERb) may be an important genetic factor predisposing to endometriosis. Acta Obstet Gynecol Scand 2009;88(12):1397–401.

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