- Email: [email protected]
Prospective, randomized trial of metformin and vitamins for the reduction of plasma homocysteine in insulin-resistant polycystic ovary syndrome
Prospective, randomized trial of metformin and vitamins for the reduction of plasma homocysteine in insulin-resistant polycystic ovary syndrome One hundred and two women with insulin-resistant polycystic ovary syndrome were randomized to treatment with a vitamin B preparation, metformin, or both, in conjunction with standard infertility treatment. Plasma homocysteine levels were significantly reduced by both B vitamins and metformin, but to a greater degree by B vitamins, and higher pregnancy rates were associated with vitamin B treatment. (Fertil Steril威 2007;88: 227–30. ©2007 by American Society for Reproductive Medicine.)
The majority of women with polycystic ovary syndrome (PCOS) are insulin-resistant with compensatory hyperinsulinemia, which is linked to elevated homocysteine levels via suppression of cystathione beta-synthase. Homocysteine (Hcy) metabolism is also affected by the bioavailability of folic acid, methyl group donors, and B vitamins (1– 4). Elevated Hcy is linked to pregnancy complications and increased pregnancy loss (5). Treatment of infertility in the patient with PCOS now commonly incorporates insulin-reducing measures such as exercise, weight loss, and drugs such as metformin or rosiglitazone, which have improved treatment results in terms of both higher pregnancy rates (PRs) and lower miscarriage rates. Metformin, while improving insulin resistance, was shown in certain clinical situations to increase serum Hcy levels by reducing levels of folic acid and vitamin B12 (6 – 8). We prospectively examined the effect of B vitamins and/or metformin on plasma Hcy levels in insulin-resistant patients with PCOS wishing to conceive, and we observed the effects on reproductive outcomes. One hundred and two infertile patients diagnosed with insulin-resistant PCOS and who desired pregnancy were recruited for this study over a period of 14 months. Diagnostic criteria for PCOS included at least two of the three Rotterdam criteria (9). Eighteen patients were scheduled for induction of ovulation, and 84 patients for IVF-intracytoplasmic sperm injection (ICSI) because of additional infertility factors. Insulin resistance was determined by a static fasting glucose and insulin measurement, calculating the homeostasis model assessment (HOMA) index (glucose [mmol/L] · insulin [mIU/L]/22.5) and the logarithmic transformation of the HOMA index (log10 HOMA). The upper limit of normal was constructed by calculating the mean Received August 1, 2006; revised October 8, 2006; accepted November 16, 2006. Laboratory costs were partly supported by Solgar Israel, Ltd., Netanya, Israel. Reprint requests: Morey Schachter, M.D., In Vitro Fertilization and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, 70300 Zerifin, Israel (FAX: ⫹972-8-9779003; E-mail: [email protected]).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.11.071
⫹ 2 SDs of a normal control group, for each variable, which reflect accepted reference values (10 –12). These 102 patients were randomized before treatment, and after giving informed consent, assigned to one of four groups by opening sealed envelopes containing computergenerated random assignation numbers. Group one (control) underwent infertility treatment only. Group two underwent infertility treatment and metformin 1,700 mg per day (two divided doses of 850-mg tablets; Glucomin, Teva, Israel), started simultaneously with gonadotropin injections. Group three underwent infertility treatment and vitamin B treatment, including 50 mg B6, 400 g folic acid, 500 g B12 (as cobalamin), 1 g trimethylglycine (betaine), and 6 mg pyridoxal-5-phosphate per day (Homocysteine Modulators; Solgar, Leonia, New Jersey) started simultaneously with gonadotropin injections. Group four underwent infertility treatment and both metformin (glucomin 1,700 mg/day, as in group 2) and vitamin B treatment (Homocysteine Modulators, as in group 3). The control and metformin patients (groups 1 and 2) were treated with a standard daily dose of 0.4 mg folic acid (folic acid; CTS, Kiryat Gat, Israel). Induction of ovulation was carried out with the use of standard low-dose protocols, starting with 75 IU/day recombinant FSH (Gonal-F; Serono, Geneva, Switzerland) and 5,000 IU hCG (Pregnyl, Organon, the Netherlands). The IVF-ICSI cycles were performed with midluteal triptorelin, 0.1 mg/day (Decapeptyl; Ferring, Ceasarea, Israel) and 150 IU/day (starting dose) of hMG (Menogon, Ferring, Denmark) with hCG, as previously mentioned. All subsequent IVF and ICSI laboratory procedures and ETs were performed as described by Strassburger et al. (13). Only clinical pregnancies (visualized by ultrasound 3– 4 weeks after hCG injection) were counted. Ongoing pregnancies were those that progressed beyond 12 gestational weeks. Glucose, insulin, and Hcy were measured from plasma separated and frozen immediately. Homocysteine was measured as total plasma L-homocysteine, determined using a fluorescence polarization immunoassay by IMX analysis
Fertility and Sterility姞 Vol. 88, No. 1, July 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
227
(Axis-Shield; Abbott Diagnostics, Oslo, Norway). All intra-assay coefficients of variation (CVs) were 2.7%– 4.7%, and all interassay CVs were 2.2%– 6.2%. Fasting plasma glucose, insulin, and Hcy were measured before treatment, and after three cycles of treatment, or after a positive pregnancy test, whenever this was achieved. Overall analysis of all patients (n ⫽ 102) showed an average age of 28.8 ⫾ 0.4 years, and an average body mass index of 27.3 ⫾ 0.5 kg/m2. Average fasting insulin was 22.1 ⫾ 1.1 U/ml (normal, ⬍19 U/ml), and the average log HOMA was 0.63 ⫾ 0.01 (normal, ⬍0.59), with no difference found among treatment groups randomized. Average baseline plasma Hcy was 11.2 ⫾ 0.6 mol/L (95th percentile in our infertile patients with normal ovaries was 11 mol/L). Homocysteine levels were significantly correlated to insulin-resistance indices, including fasting insulin, HOMA, and log HOMA (P⬍.001 for all, F ⫽ 176 –284, by analysis of variance [ANOVA]). Homocysteine levels were reduced after treatment in all groups, from 7%–32% (Table 1). Differences between treatment groups were found to be statistically significant (P⬍.001, F ⫽ 5.3, ANOVA), although no one treatment group was found to be significantly better in terms of Hcy reduction than other treatment groups, versus the control group (Table 1). Vitamin treatment, with or without metformin, resulted in a reduction of Hcy of 24.7% (⫾4%) versus a reduction of 10.9% (⫾6%) for the nonvitamin treatment groups (control and metformin-only) (P⫽.02, F ⫽ 9.5, ANOVA). No differences were seen in levels of fasting glucose or insulin after treatment. The overall three-cycle cumulative PR for all groups was 69.6% (71/102). There were no statistically significant differences between treatment groups in PRs or ongoing PRs, although the highest PRs and ongoing PRs were seen in the vitamin-only and vitamin-metformin groups (75%–77% and 61%– 67%, respectively).
Homocysteine levels are positively correlated to oxidative stress in vascular endothelium, activation of platelets (14), impairment of blood flow (15), and stimulation of vascular smooth muscle proliferation (16). Thus, elevated Hcy may impair implantation by interfering with endometrial blood flow and vascular integrity, and was documented to increase the probability of early pregnancy loss (17). Both impaired implantation and increased rates of miscarriage are more frequent in PCOS, even after controlling for ovulatory abnormalities, increased LH, and hyperandrogenism, which might be due in part to elevated Hcy in these patients. Our previous study (18) and that of others (19) found an association between insulin resistance and elevated Hcy in women with PCOS. Several studies examined the effect of both insulin-reducing medications such as metformin or rosiglitazone, and vitamin preparations, on Hcy levels in insulin-resistant patients or patients with or PCOS. Kilicdag et al. (6), Vrbikova et al. (8), and Wulffele et al. (20) reported that treatment of patients with PCOS or Diabetes Mellitus II (DM II) with metformin increased the average level of Hcy by 1.6 –3.3 mol/L. These results contrast with those found in this study, which found decreases in Hcy levels of 12% (approximately 2 mol/L) after treatment with metformin for periods of 6 –16 weeks. The only difference in these studies was the intake by our control and metformin groups of 0.4 mg/day of folic acid, which could account for some of the difference (6%–12%). Kilicdag et al. (7) conducted another study in 60 patients with PCOS, administering metformin alone, metformin and B-group vitamins, or metformin and folic acid. Their results were a decrease in Hcy in the folic acid and vitamin B supplementation groups of 8.3% and 21%, respectively. Our findings are in agreement with theirs, suggesting that supplementation of B-group vitamins, folic acid, and methyl donors such as trimethylglycine (betaine) could have positive effects on patients with PCOS treated with metformin. The reduction in Hcy levels of 6% in the control group could be the result of the
TABLE 1 Changes in homocysteine levels before and after treatment, and cumulative PRs by groups. Hcy 1 (mol/L)
Group All (n ⫽ 102) Control (n ⫽ 23) Metformin (n ⫽ 28) Vitamins (n ⫽ 24) Metformin and vitamins (n ⫽ 27)
11.2 ⫾ 0.62 10.3 ⫾ 0.9 10.5 ⫾ 0.9 12.2 ⫾ 0.7 11.9 ⫾ 1.9
Hcy 2 (mol/L)
⌬Hcy
Cum PR
Ongoing PR
8.68 ⫾ 0.44 0.175 ⫾ 0.02 69.6% (71/102) 9.45 ⫾ 0.9 0.07 ⫾ 0.03 61.0% (14/23) 8.6 ⫾ 0.7 0.12 ⫾ 0.04 64.0% (18/28) 7.85 ⫾ 0.34 0.32 ⫾ 0.03 75.0% (18/24) 8.85 ⫾ 1.2 0.18 ⫾ 0.05 77.0% (21/27)
63.3% (45/71) 50.0% (7/14) 61.0% (11/18) 61.0% (13/18) 67.0% (14/21)
Note: Hcy 1 ⫽ homocysteine levels before treatment. Hcy 2 ⫽ homocysteine levels after treatment. ⌬Hcy ⫽ reduction in homocysteine levels between first and second measurement. Cum PR ⫽ cumulative three-cycle PR. Ongoing PR ⫽ clinical pregnancies progressing beyond 12 gestational weeks. Schachter. Homocysteine reduction modalities in PCOS. Fertil Steril 2007.
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Vol. 88, No. 1, July 2007
addition of 0.4 mg folic acid, or the effect of increased E2 during the treatment cycle, as previously reported by Lox and Prien (21). Part of the reduction in all treatment groups might be attributed to this effect of the increase in E2. Metformin intake may reduce vitamin B12 levels, which could increase Hcy in treated individuals. On the other hand, reduction in insulin levels by metformin could allow for increased trans-sulfuration of Hcy, and thus reduce Hcy levels. The addition of B vitamins was formulated to tip the balance in favor of a net reductive effect on Hcy. Our results suggest that the combination of B vitamins and metformin would lead to the best results in terms of ongoing pregnancies, because of the reduction of Hcy by all of these supplements, together with the additional teratogenic protection conferred by folate and B12, and the reduction in miscarriage rate for patients with PCOS afforded by metformin (22) and B vitamins (17). Although methyltetrahydrofolate reductase enzyme deficiencies and baseline vitamin deficiencies were not screened for in this patient group before the study was initiated, previous studies (23, 24) did not find significant frequencies of these disorders in patients with PCOS, and thus selection bias is unlikely. We conclude that infertile women with insulin-resistant PCOS have elevated levels of Hcy, and this could be responsible in part for the decreased implantation rates and increased miscarriage rates of patients with PCOS, even with effective induction of ovulation or IVF. The supplementation of B-group vitamins and/or metformin is effective in reducing Hcy; the combination of both could allow for each factor’s benefits to be borne out, improving reproductive results. Acknowledgments: The authors are grateful to Solgar (New Jersey) for the donation of Homocysteine Modulators Vegicaps, and to Alex Maor, Ph.D. (Solgar Israel, Ltd.), for his assistance and follow-up of the research work.
Morey Schachter, M.D.a Arieh Raziel, M.D.a Devorah Strassburger, Ph.D.a Carmela Rotem, M.Sc.b,c Raphael Ron-El, M.D.a Shevach Friedler, M.D.a a In Vitro Fertilization and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, Zerifin; b Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva; and c Research and Development, Solgar Israel, Ltd., Netanya, Israel REFERENCES 1. McCarty MF. Insulin secretion as a potential determinant of homocysteine levels. Med Hypotheses 2000;55:454 –5. 2. House JD, Jacobs RL, Stead LM, Brosnan ME, Brosnan JT. Regulation of homocysteine metabolism. Adv Enzyme Regul 1999;39:69 –91.
Fertility and Sterility姞
3. Meigs JB, Jacques PF, Selhub J, Singer DE, Nathan DM, Rifai N, et al. Fasting plasma homocysteine levels in the insulin resistance syndrome. Diabetes Care 2001;24:1403–10. 4. Setola E, Monti LD, Galluccio E, Palloshi A, Fragasso G, Paroni R, et al. Insulin resistance and endothelial function are improved after folate and vitamin B12 therapy in patients with metabolic syndrome: relationship between homocysteine levels and hyperinsulinemia. Eur J Endocrinol 2004;151:483–9. 5. Craig LB, Ke RW, Kutteh WH. Increased prevalence of insulin resistance in women with a history of recurrent pregnancy loss. Fertil Steril 2002;78:487–90. 6. Kilicdag EB, Bagis T, Zeyneloglu HB, Tarim E, Aslan E, Haydardedeoglu B, et al. Homocysteine levels in women with polycystic ovary syndrome treated with metfrormin versus rosiglitazone: a randomized study. Hum Reprod 2005;20:894 –9. 7. Kilicdag EB, Bagis T, Tarim E, Aslan E, Erkanli S, Simsek E, et al. Administration of B-group vitamins reduces circulating homocysteine in polycystic ovarian syndrome patients treated with metformin: a randomized trial. Hum Reprod 2005;20:1521– 8. 8. Vrbikova J, Bicikova M, Tallova J, Hill M, Starka L. Homocysteine and steroids levels in metformin treated women with polycystic ovary syndrome. Exp Clin Endocrinol Diabetes 2002;110:74 – 6. 9. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004; 81:19 –25. 10. Brun JF, Raynaud E, Mercier J. Homeostasis model assessment and related simplified evaluations of insulin sensitivity from fasting insulin and glucose. Diabetes Care 2000;23:1037– 8. 11. Mather KJ, Hunt AE, Steinberg HO, Paeadisi G, Hok G, Katz A, et al. Repeatability characteristics of simple indices of insulin resistance: implications for research applications. J Clin Endocrinol Metab 2001;86:5457– 64. 12. Bonora E, Targher G, Alberiche M, Bonadonna RC, Saggiani F, Zenere M, et al. Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity. Diabetes Care 2000;23:57– 63. 13. Strassburger D, Friedler S, Raziel A, Schachter M, Kasterstein E, Ron-El R. Very low sperm count affects the result of intracytoplasmic sperm injection. J Assist Reprod Genet 2000;17:431– 6. 14. Coppola A, Davi G, De-Stefano V, Mancini FP, Cerbone AM, DiMinno G. Homocysteine, coagulation, platelet function and thrombosis. Semin Thromb Hemost 2000;26:243–54. 15. Chambers JC, Ueland PM, Wright M, Dore CJ, Refsum H, Kooner JS. Investigation of relationship between reduced, oxidized, and protein-bound homocysteine and vascular endothelial function in healthy human subjects. Circ Res 2001;89:187–92. 16. Van Guldener C, Stehouwer CD. Hyperhomocysteinemia, vascular pathology and endothelial dysfunction. Semin Thromb Hemost 2000; 26:281–9. 17. Quere I, Mercier E, Bellet H, Janbon C, Mares P, Gris JC. Vitamin supplementation and pregnancy outcome in women with recurrent early pregnancy loss and hyperhomocysteinemia. Fertil Steril 2001; 75:823–5. 18. Schachter M, Raziel A, Friedler S, Strassburger D, Bern O, Ron-El R. Insulin resistance in patients with polycystic ovary syndrome is associated with elevated homocysteine. Hum Reprod 2003;18:721–7. 19. Rouzi AA, Ardawi MS. Plasma homocysteine and insulin resistance in women with polycystic ovary syndrome. Fertil Steril 2005;84(Suppl):S432. 20. Wulffele MG, Kooy A, Lehert P, Bets D, Ogterop JC, Borger van der Burg B, et al. Effects of short-term treatment with metformin on serum concentrations of homocysteine, folate and vitamin B12 in type 2 diabetes mellitus: a randomized placebo controlled trial. J Intern Med 2003;254:455– 63.
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21. Lox CD, Prien SD. The homocysteine pathway during the first two weeks of pregnancy following IVF-ET. Fertil Steril 2000;74(Suppl):S205. 22. Jakubowicz DJ, Iuorno MJ, Jakubowicz S, Roberts KA, Nestler JE. Effects of metformin on early pregnancy loss in the polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87:524 –9. 23. Yarali H, Yildirir A, Aybar F, Kabakci G, Bukulmez O, Akgul E, Ota A. Diastolic dysfunction and increased serum homocysteine
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Correspondence
concentrations may contribute to increased cardiovascular risk in patients with polycystic ovary syndrome. Fertil Steril 2001;76: 511– 6. 24. Yilmaz M, Bukan N, Ayvaz G, Karakoc A, Toruner F, Cakir N, et al. The effects of rosiglitazone and metformin on oxidative stress and homocysteine levels in lean patients with polycystic ovary syndrome. Hum Reprod 2005;20:3333– 40.
Vol. 88, No. 1, July 2007
The majority of women with polycystic ovary syndrome (PCOS) are insulin-resistant with compensatory hyperinsulinemia, which is linked to elevated homocysteine levels via suppression of cystathione beta-synthase. Homocysteine (Hcy) metabolism is also affected by the bioavailability of folic acid, methyl group donors, and B vitamins (1– 4). Elevated Hcy is linked to pregnancy complications and increased pregnancy loss (5). Treatment of infertility in the patient with PCOS now commonly incorporates insulin-reducing measures such as exercise, weight loss, and drugs such as metformin or rosiglitazone, which have improved treatment results in terms of both higher pregnancy rates (PRs) and lower miscarriage rates. Metformin, while improving insulin resistance, was shown in certain clinical situations to increase serum Hcy levels by reducing levels of folic acid and vitamin B12 (6 – 8). We prospectively examined the effect of B vitamins and/or metformin on plasma Hcy levels in insulin-resistant patients with PCOS wishing to conceive, and we observed the effects on reproductive outcomes. One hundred and two infertile patients diagnosed with insulin-resistant PCOS and who desired pregnancy were recruited for this study over a period of 14 months. Diagnostic criteria for PCOS included at least two of the three Rotterdam criteria (9). Eighteen patients were scheduled for induction of ovulation, and 84 patients for IVF-intracytoplasmic sperm injection (ICSI) because of additional infertility factors. Insulin resistance was determined by a static fasting glucose and insulin measurement, calculating the homeostasis model assessment (HOMA) index (glucose [mmol/L] · insulin [mIU/L]/22.5) and the logarithmic transformation of the HOMA index (log10 HOMA). The upper limit of normal was constructed by calculating the mean Received August 1, 2006; revised October 8, 2006; accepted November 16, 2006. Laboratory costs were partly supported by Solgar Israel, Ltd., Netanya, Israel. Reprint requests: Morey Schachter, M.D., In Vitro Fertilization and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, 70300 Zerifin, Israel (FAX: ⫹972-8-9779003; E-mail: [email protected]).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.11.071
⫹ 2 SDs of a normal control group, for each variable, which reflect accepted reference values (10 –12). These 102 patients were randomized before treatment, and after giving informed consent, assigned to one of four groups by opening sealed envelopes containing computergenerated random assignation numbers. Group one (control) underwent infertility treatment only. Group two underwent infertility treatment and metformin 1,700 mg per day (two divided doses of 850-mg tablets; Glucomin, Teva, Israel), started simultaneously with gonadotropin injections. Group three underwent infertility treatment and vitamin B treatment, including 50 mg B6, 400 g folic acid, 500 g B12 (as cobalamin), 1 g trimethylglycine (betaine), and 6 mg pyridoxal-5-phosphate per day (Homocysteine Modulators; Solgar, Leonia, New Jersey) started simultaneously with gonadotropin injections. Group four underwent infertility treatment and both metformin (glucomin 1,700 mg/day, as in group 2) and vitamin B treatment (Homocysteine Modulators, as in group 3). The control and metformin patients (groups 1 and 2) were treated with a standard daily dose of 0.4 mg folic acid (folic acid; CTS, Kiryat Gat, Israel). Induction of ovulation was carried out with the use of standard low-dose protocols, starting with 75 IU/day recombinant FSH (Gonal-F; Serono, Geneva, Switzerland) and 5,000 IU hCG (Pregnyl, Organon, the Netherlands). The IVF-ICSI cycles were performed with midluteal triptorelin, 0.1 mg/day (Decapeptyl; Ferring, Ceasarea, Israel) and 150 IU/day (starting dose) of hMG (Menogon, Ferring, Denmark) with hCG, as previously mentioned. All subsequent IVF and ICSI laboratory procedures and ETs were performed as described by Strassburger et al. (13). Only clinical pregnancies (visualized by ultrasound 3– 4 weeks after hCG injection) were counted. Ongoing pregnancies were those that progressed beyond 12 gestational weeks. Glucose, insulin, and Hcy were measured from plasma separated and frozen immediately. Homocysteine was measured as total plasma L-homocysteine, determined using a fluorescence polarization immunoassay by IMX analysis
Fertility and Sterility姞 Vol. 88, No. 1, July 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
227
(Axis-Shield; Abbott Diagnostics, Oslo, Norway). All intra-assay coefficients of variation (CVs) were 2.7%– 4.7%, and all interassay CVs were 2.2%– 6.2%. Fasting plasma glucose, insulin, and Hcy were measured before treatment, and after three cycles of treatment, or after a positive pregnancy test, whenever this was achieved. Overall analysis of all patients (n ⫽ 102) showed an average age of 28.8 ⫾ 0.4 years, and an average body mass index of 27.3 ⫾ 0.5 kg/m2. Average fasting insulin was 22.1 ⫾ 1.1 U/ml (normal, ⬍19 U/ml), and the average log HOMA was 0.63 ⫾ 0.01 (normal, ⬍0.59), with no difference found among treatment groups randomized. Average baseline plasma Hcy was 11.2 ⫾ 0.6 mol/L (95th percentile in our infertile patients with normal ovaries was 11 mol/L). Homocysteine levels were significantly correlated to insulin-resistance indices, including fasting insulin, HOMA, and log HOMA (P⬍.001 for all, F ⫽ 176 –284, by analysis of variance [ANOVA]). Homocysteine levels were reduced after treatment in all groups, from 7%–32% (Table 1). Differences between treatment groups were found to be statistically significant (P⬍.001, F ⫽ 5.3, ANOVA), although no one treatment group was found to be significantly better in terms of Hcy reduction than other treatment groups, versus the control group (Table 1). Vitamin treatment, with or without metformin, resulted in a reduction of Hcy of 24.7% (⫾4%) versus a reduction of 10.9% (⫾6%) for the nonvitamin treatment groups (control and metformin-only) (P⫽.02, F ⫽ 9.5, ANOVA). No differences were seen in levels of fasting glucose or insulin after treatment. The overall three-cycle cumulative PR for all groups was 69.6% (71/102). There were no statistically significant differences between treatment groups in PRs or ongoing PRs, although the highest PRs and ongoing PRs were seen in the vitamin-only and vitamin-metformin groups (75%–77% and 61%– 67%, respectively).
Homocysteine levels are positively correlated to oxidative stress in vascular endothelium, activation of platelets (14), impairment of blood flow (15), and stimulation of vascular smooth muscle proliferation (16). Thus, elevated Hcy may impair implantation by interfering with endometrial blood flow and vascular integrity, and was documented to increase the probability of early pregnancy loss (17). Both impaired implantation and increased rates of miscarriage are more frequent in PCOS, even after controlling for ovulatory abnormalities, increased LH, and hyperandrogenism, which might be due in part to elevated Hcy in these patients. Our previous study (18) and that of others (19) found an association between insulin resistance and elevated Hcy in women with PCOS. Several studies examined the effect of both insulin-reducing medications such as metformin or rosiglitazone, and vitamin preparations, on Hcy levels in insulin-resistant patients or patients with or PCOS. Kilicdag et al. (6), Vrbikova et al. (8), and Wulffele et al. (20) reported that treatment of patients with PCOS or Diabetes Mellitus II (DM II) with metformin increased the average level of Hcy by 1.6 –3.3 mol/L. These results contrast with those found in this study, which found decreases in Hcy levels of 12% (approximately 2 mol/L) after treatment with metformin for periods of 6 –16 weeks. The only difference in these studies was the intake by our control and metformin groups of 0.4 mg/day of folic acid, which could account for some of the difference (6%–12%). Kilicdag et al. (7) conducted another study in 60 patients with PCOS, administering metformin alone, metformin and B-group vitamins, or metformin and folic acid. Their results were a decrease in Hcy in the folic acid and vitamin B supplementation groups of 8.3% and 21%, respectively. Our findings are in agreement with theirs, suggesting that supplementation of B-group vitamins, folic acid, and methyl donors such as trimethylglycine (betaine) could have positive effects on patients with PCOS treated with metformin. The reduction in Hcy levels of 6% in the control group could be the result of the
TABLE 1 Changes in homocysteine levels before and after treatment, and cumulative PRs by groups. Hcy 1 (mol/L)
Group All (n ⫽ 102) Control (n ⫽ 23) Metformin (n ⫽ 28) Vitamins (n ⫽ 24) Metformin and vitamins (n ⫽ 27)
11.2 ⫾ 0.62 10.3 ⫾ 0.9 10.5 ⫾ 0.9 12.2 ⫾ 0.7 11.9 ⫾ 1.9
Hcy 2 (mol/L)
⌬Hcy
Cum PR
Ongoing PR
8.68 ⫾ 0.44 0.175 ⫾ 0.02 69.6% (71/102) 9.45 ⫾ 0.9 0.07 ⫾ 0.03 61.0% (14/23) 8.6 ⫾ 0.7 0.12 ⫾ 0.04 64.0% (18/28) 7.85 ⫾ 0.34 0.32 ⫾ 0.03 75.0% (18/24) 8.85 ⫾ 1.2 0.18 ⫾ 0.05 77.0% (21/27)
63.3% (45/71) 50.0% (7/14) 61.0% (11/18) 61.0% (13/18) 67.0% (14/21)
Note: Hcy 1 ⫽ homocysteine levels before treatment. Hcy 2 ⫽ homocysteine levels after treatment. ⌬Hcy ⫽ reduction in homocysteine levels between first and second measurement. Cum PR ⫽ cumulative three-cycle PR. Ongoing PR ⫽ clinical pregnancies progressing beyond 12 gestational weeks. Schachter. Homocysteine reduction modalities in PCOS. Fertil Steril 2007.
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Schachter et al.
Correspondence
Vol. 88, No. 1, July 2007
addition of 0.4 mg folic acid, or the effect of increased E2 during the treatment cycle, as previously reported by Lox and Prien (21). Part of the reduction in all treatment groups might be attributed to this effect of the increase in E2. Metformin intake may reduce vitamin B12 levels, which could increase Hcy in treated individuals. On the other hand, reduction in insulin levels by metformin could allow for increased trans-sulfuration of Hcy, and thus reduce Hcy levels. The addition of B vitamins was formulated to tip the balance in favor of a net reductive effect on Hcy. Our results suggest that the combination of B vitamins and metformin would lead to the best results in terms of ongoing pregnancies, because of the reduction of Hcy by all of these supplements, together with the additional teratogenic protection conferred by folate and B12, and the reduction in miscarriage rate for patients with PCOS afforded by metformin (22) and B vitamins (17). Although methyltetrahydrofolate reductase enzyme deficiencies and baseline vitamin deficiencies were not screened for in this patient group before the study was initiated, previous studies (23, 24) did not find significant frequencies of these disorders in patients with PCOS, and thus selection bias is unlikely. We conclude that infertile women with insulin-resistant PCOS have elevated levels of Hcy, and this could be responsible in part for the decreased implantation rates and increased miscarriage rates of patients with PCOS, even with effective induction of ovulation or IVF. The supplementation of B-group vitamins and/or metformin is effective in reducing Hcy; the combination of both could allow for each factor’s benefits to be borne out, improving reproductive results. Acknowledgments: The authors are grateful to Solgar (New Jersey) for the donation of Homocysteine Modulators Vegicaps, and to Alex Maor, Ph.D. (Solgar Israel, Ltd.), for his assistance and follow-up of the research work.
Morey Schachter, M.D.a Arieh Raziel, M.D.a Devorah Strassburger, Ph.D.a Carmela Rotem, M.Sc.b,c Raphael Ron-El, M.D.a Shevach Friedler, M.D.a a In Vitro Fertilization and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, Zerifin; b Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva; and c Research and Development, Solgar Israel, Ltd., Netanya, Israel REFERENCES 1. McCarty MF. Insulin secretion as a potential determinant of homocysteine levels. Med Hypotheses 2000;55:454 –5. 2. House JD, Jacobs RL, Stead LM, Brosnan ME, Brosnan JT. Regulation of homocysteine metabolism. Adv Enzyme Regul 1999;39:69 –91.
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Vol. 88, No. 1, July 2007