Iraqi J Pharm Sci, Vol.21(1) 2012 Treatment of idiopathic male infertility 14 Effect of L-carnitine, Multivitamins and their Combination in the Treatment of Idiopathic Male Infertility # Jehan J. Aram * , Kassim J. Al-Shamma **,1 and Ansam N. Al-Hassani * * College of Pharmacy, Hawler Medical University, Erbil, Iraq. ** College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract The aim of the present study is to investigate and compare the efficacy of L-carnitine, multivitamins and their combination therapies on semen characteristics in idiopathic male infertility. Idiophathic infertile patients were randomly divided into three groups who had received three different treatment regimens for three months: group A (45 patients) has received 2 grams daily of L-carnitine alone; group B (55 patients) had received the combination of L-carnitine (2 grams daily) plus one tablet daily of multivitamins (Stresstabs ® ); and group C (29 patients) had received one tablet daily of multivitamins alone. The study was started on 1/11/2009 and completed on 31/3/2010 and performed at Rizgari Teaching Hospital in Hawler City/Erbil Governorate. Thirty fertile male volunteers were used as a control group as well. Seminal fluid analysis has been done before treatment and then monthly after treatment for three month. The results of the present study clearly demonstrated that the combination therapy of L – carnitine and multivitamins was more efficient than each drug therapy alone and this evidenced by improvement and significant increase in the semen parameters; sperm concentration (72%), sperm count (79%), actively motile sperm (29%), and progressive motile sperm count (125%) when compared to pretreatment values than either L – carnitine or multivitamins therapy alone (53%, 60%, 19% and 84%), (21%, 30%, Non Significant, 34%) respectively.The results demonstrated that the combination therapy of L-carnitine and multivitamins was more efficient and produced more significant improvement in semen characteristic than either therapy alone, and that L- carnitine therapy was more efficient than multivitamins.Improvement in these semen parameters can aid in the treatment of idiopathic male infertility. Key words : L-carnitine, multivitamins, infertility. كارَتيٍ وانفيتاييُاث انًتعددة وكالهًا سىيت نعالج يزض عقى انذكىر يجهىل انسبب -تقييى نـ جيهاٌ جالل آراو * ، قاسى جهيم انشًاع **،1 اَساو َاجي انحسُي و * .* كهٛح انصٛذنح ، خايؼح ْٕنٛش انطثٛح ، استٛم ، انؼشاق كهٛح انصٛذنح ، خايؼح تغذاد انطثٛح ، تغذاد ، انؼشاق** الخالصة نًرؼذدج أٔكالًْا يؼا ػهٗ كاسَرٍٛ أٔانفٛرايُٛاخ ا -ْذف ْزِ انذساسح ْٕ انرحمك ػٍ ٔ يماسَح كفاءج ػالخاخ نـ إٌ خصائص انسائم انًُٕ٘ )حدى انسائم انًُٕ٘، ذشكٛض انحٛايٍ، ػذد انحٛايٍ، شكم انحٛايٍ، حشكح انحٛايٍ، ػذد انحٛايٍ راخ انحشكح أشٓشح ذحسٍٛ ْزِ انخصائص خالل ثالث إنٗانًرمذيح، ٔػذد انخالٚا انًذٔسج( .فٙ ػمى انزكٕس يدٕٓل انسثة فٙ يحافظح استٛم، ْادفح فٙ ْزِ يٍ انؼالج ٔالكرشاف انطشٚمح انصحٛحح نرمذٚى انثٛاَاخ انًخرثشٚح نرحهٛم انسائم انًُٕ٘ ٔانرٙ ٚرى ػًهٓا فٙ يحافظح استٛم. نًذج األخشثالثح يدايٛغ كم يُٓا اخز ػالج ٚخرهف ػٍ إنٗيشٚض ) ٔانؼمى يدٕٓل انسثة ( تطشٚمح ػشٕائٛح 921انذساسح ذى ذمسٛى يسرًشج: شأشٓثالثح كاسَرٍٛ ٕٚيٛا، -غشاو يٍ نـ 2يشٚض ( ذُأنٕا 54) –أ –انًدًٕػح كاسَرٍٛ يغ حثح ٔاحذج يٍ انفٛرايُٛاخ انًرؼذدج ) سرشٚسراتض -غشاو يٍ نـ 2يشٚض ( ذُأنٕا 44) –ب –انًدًٕػح ® .) سرشٚسراتض يشٚض ( ذُأنٕا حثح ٔاحذج يٍ انفٛرايُٛاخ انًرؼذدج ) 21) –ج –انًدًٕػح ® .) يرطٕع ركش خصة. نمذ ذًد يراتؼح انًشضٗ 03) انكَٕرشٔل ( فٙ ْزِ انذساسح ٔانرٙ ذضًُد أخشٖٔلذ ذى اخز ٔذسدٛم يدًٕػح ( رٔ انًُٕرج t. ْزا ٔلذ ذى اسرخذاو فحص )أشٓشٔذى ذحهٛم انسائم انًُٕ٘ لثم انثذء تأخز انؼالخاخ ٔشٓشٚا خالل اخز انؼالج نًذج ثالثح يٍ انؼالج، ٔلذ اػرثشخ لًٛح احرًانٛح انصذفح أشٓشخٙ نهًماسَح تٍٛ انمٛى لثم انؼالج ٔانمٛى انًماتهح نٓا تؼذ شٓش ٔشٓشٍٚ ٔثالثح انضٔ (P راخ )كاسَرٍٛ انًسرخذو -نـ إٌ. نمذ أظٓشخ َرائح ْزا انثحث تصٕسج ٔاضحح 3034إحصائٛح ػُذيا ذكٌٕ لًٛرٓا الم يٍ أًْٛح ٚحسٍ ذشكٛض انحٛايٍ، ػذد انحٛايٍ، َسثح انحٛايٍ انًرحشكح تصٕسج فؼانح، أٌيسرًشج يًكٍ أشٓشٛا نًذج ثالثح غشاو ٕٚي 2تدشػح ٔػذد انحٛايٍ راخ انحشكح انًرمذيح ػُذ انشخال انزٍٚ ٚؼإٌَ يٍ انؼمى ) لهح ذشكٛض انحٛايٍ أ لهح سشػرٓا أ كالًْا( يدٕٓل انسثة، كاسَرٍٛ ٔ انفٛرايُٛاخ انًرؼذدج نًذج انؼالج َفسٓا ْٕ اكثش كفاءج ) حصٕل صٚادج يًٓح احصاءٚاً فٙ -ٔاٌ انؼالج انز٘ ٚدًغ تٍٛ نـ % تانرؼالة يماسَح يغ انمٛى انًماتهح نٓا لثم انؼالج ( يٍ 924%، 21%، 21%، 22تًمذاس أػالِخصائص انسائم انًُٕ٘ انًزكٕسج %، غٛش يٓى 03%، 29ٚاً فٙ خصائص انسائم انًُٕ٘ انًزكٕسج اػالِ تًمذاس كاسَرٍٛ نٕحذِ ) حصٕل صٚادج يًٓح احصاء -ػالج نـ ٚساػذ فٙ أٌ% تانرؼالة يماسَح يغ انمٛى انًماتهح نٓا لثم انؼالج ( نرحسٍٛ خصائص انسائم انًُٕ٘ ْزِ ٔيٍ انًًكٍ 05احصاءٚاً، يٍ ػالج يدًٕػح انفٛرايُٛاخ أفضمٚكٌٕ أٌٍ كاسَرٍٛ يٍ انًًك -نزنك، ػالج نـ تاإلضافحػالج ػمى انشخال يدٕٓل انسثة. َظاو انؼالج انز٘ ذى ذطثٛمّ فٙ ْزِ انذساسح فٙ ذحسٍٛ خصائص انسائم انًُٕ٘ ػالج ػمى انشخال يدٕٓل انسثة. تاسرخذاوانًرؼذدج كارَتيٍ ، قيتاييُاث ، عقى . -انكهًاث انًفتاحيت : نـ # Based on oral presentation in the eighth scientific conference of the College of Pharmacy /University of Baghdad held in 23-24 February 2011. 1Corresponding author E- mail : drkassim_alshamaa@yahoo.com Received : 22/3/2011 Accepted : 15/11/2011 Iraqi J Pharm Sci, Vol.21(1) 2012 Treatment of idiopathic male infertility 15 Introduction Male infertility means the male is unable to impregnate the female because of male factors.It represents 25-40% of the total causes of infertility (1) .Although the etiology of male infertility is not clear in most cases, different treatment options have been suggested to increase sperm count and motility (2) . These treatment options include general, medical and surgical management. Medical treatment includes gonadotrophins, androgens, corticosteroids, antibiotics, alpha1- sympathomimetics, anticholinergics, antiestrogens, aromatase inhibitors, and alternative therapy which is considered the safest among the treatment options and include antioxidants (multivitamins) and L-carnitine (3) . Carnitine is a zwitterionic amino acid (3- Hydroxy-4-trimethylamino-butyic acid). It is found in different food items and derived endogenously from lysine and methionine (2) . L-carnitine is an essential cofactor that could accelerate lipid metabolism and has a pivotal role in mitochondrial β-oxidation of long-chain fatty acids for cellular energy production (4,5) . L-carnitine and L-acetylcarnitine are highly concentrated in the epididymis and play a crucial role in sperm metabolism and maturation (6) .The redox system in the spermatozoa regulates the processes that are crucial for fertilization (7) , but increased reactive oxygen species (ROS) observed in semen of infertile men might cause cellular damage and this have brought about the widespread use of antioxidants (8) . Many vitamins such as vitamin C, vitamin E, vitamin B12, and many other antioxidants were used to improve sperm quality for the treatment of idiopathic oligoasthenozoospermia (9) . In addition, sperm concentration was increased in a number of studies on subfertile men after treatment with zinc and folic acid (10, 11) . The aim of the present study is to investigate and compare the efficacy of L-carnitine, multivitamins and their combination therapies on semen characteristics (seminal fluid volume, sperm concentration, sperm count, sperm morphology, sperm motility, progressive motile sperm count and round cells count) in idiopathic male infertility. Patients and Methods Settings: This randomized interventional study has been accomplished cooperatively with the laboratory of the Department of Pharmacology in the College of Pharmacy/ Hawler Medical University(Hawler Medical University is besideRizgari Teaching Hospital) , the Laboratory of Rizgari Teaching Hospital, Dr. Hunar Abdul Qadir's Clinic, and Shai and Luay Laboratories in Hawler city / Erbil Governorate/ Iraqi Kurdistan Region. The practical work of this research has been started on 1/11/2009 and completed on 31/3/2010. The research was authorized and approved by the Board and Ethical Committee of Hawler Medical University, Erbil, Iraq. Subjects and Patients Control group:included 30 fertile male volunteers diagnosed by the urologist, their ages ranged between 21 to 38 years; the results of their seminal fluid analyses were within the normal values and this test was done and recorded for them one time only. Patients' inclusion criteria : were infertile or subfertile young males diagnosed by the urologist with age range between 20 to 42 years, absence of leukocytospermia in the semen samples taken before the start of the treatment period, and absence of endocrinopathies or known etiology or female infertility factors. Protocol: all 129 patients had received their treatments for 3 months. Group A (45 patients) had received 2 grams per day of L-carnitine (Nature’s Bounty, INC./USA) in 4 divided doses. Group B (55 patients) had received 2 grams per day of L-carnitine in 4 divided doses plus 1 tablet daily of multivitamins with zinc (Stresstabs ® Weyth healthcare/ Canada ). Group C (29 patients) had received 1 tablet of Stresstabs ® daily.Seminal fluid analysis was done and recorded for each patient before treatment and monthly during the treatment. Collection of semeninal fluid: Samples were collected following a period of sexual abstinence from 2 to 3 days to no longer than 7 days. Most of the patients had collected their semen samples in the laboratory and analysis was performed within 30 minutes by the same examiner. Average readings were taken. Questionnaire paper contained the following questions were prepared and answered by the patients and the controls. 1. The name of the patient. 2. The age of the patient and his wife. 3. Mobile number. 4. Years of Marriage. 5. Years of infertility. 6. Number of children. 7. Smoker or alcoholic. 8. Past and recent medical history. 9. Past and recent surgical history. 10. Past and present illnesses. Iraqi J Pharm Sci, Vol.21(1) 2012 Treatment of idiopathic male infertility 16 11. Weight of the patient (if obese then look for the cause). 12. The type of the diet. 13. The occupation of the patient. 14. Exposure to toxins like lead, mercury, pesticides, or radiation. 15. Presence of female infertility factors. 16. The date of starting the interventional treatment. 17. The assigned dates of the sperm analysis during the treatment. 18. The assigned date of ending the interventional treatment. Laboratory procedures: seminal fluid analysis done (according to Strasinger and DiLorenzo, 2008 (12) , WHO 2010 (13) ) had included the following: recording the semen color, odor, viscosity, liquefaction, and pH of seminal specimen; measurement of seminal fluid volume, sperm count and concentration, sperm morphology, sperm motility (percentage of active, sluggish, immotile sperms, and progressive motile sperm count, (sperm count x percent active motile count), and round cell count. Statistical analysis: All the seminal fluid and sperm parameters in all of the groups were represented as the mean ± the standard error (SE). Paired sample t-test was applied to compare between the values before treatment and their corresponding values after 1, 2, and 3 months of treatment. P values of < 0.05 were considered statistically significant. *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment. Results Twenty idiopathic infertile patients were dropped out during the treatment before we had reached to the above final numbers of participated patients. Group A patients Table 1: Effects of L-carnitine treatment on the mean ± SE of seminal fluid volume, sperm concentration, and sperm count in idiopathic infertile patients(n=45) compared to controls (n=30). Patients/Controls Seminal Fluid Volume (Ml) Sperm Concentration (Million/Ml) Sperm Count (Million/ Ejaculate) Controls 2.63 ± 0.13 63.26 ± 10.08 a 168.96 ± 31.82 a Patients before treatment 2.63 ± 0.20 20.08 ± 1.59 b 51.42 ± 5.41 b 1 Month after treatment 2.67 ± 0.15 24.82 ± 2.42 * 67.23 ± 7.21 * 2 Month after treatment 2.66 ± 0.14 28.82 ± 3.23 * 74.16 ± 8.37 * 3 Month after treatment 2.74 ± 0.17 30.88 ± 2.63 * 82.41 ± 8.78 * *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment. Table 2: Effects of L-carnitine treatment on the mean ± SE of sperm motility (represented as percent of total) in idiopathic infertile patients(n=45) compared to controls (n=30). Patients/Controls Active Motile Sperm (%) Sluggish Motile Sperm (%) Immotile Sperm (%) Progressive Motile Count (Million/Ejaculate) Controls 51.50 ± 1.59 a 31.16 ± 0.94 17.33 ± 1.21 a 91.10 ± 20.97 a Patients before treatment 30.11 ± 2.52 b 33.44 ± 1.85 36.44 ± 3.51 b 18.02 ± 2.40 b 1 Month after treatment 30.66 ± 2.58 35.00 ± 1.74 34.33 ± 3.24 23.88 ± 3.82 2 Month after treatment 33.55 ± 2.42 * 34.44 ± 1.48 32.00 ± 2.94 * 27.42 ± 4.09 * 3 Month after treatment 35.77 ± 2.70 * 34.77 ± 1.55 29.44 ± 3.30 * 33.22 ± 4.77 * *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment. Iraqi J Pharm Sci, Vol.21(1) 2012 Treatment of idiopathic male infertility 17 Group B patients Table 3: Effects of L-carnitine and multivitamins treatment on the mean ± SE of seminal fluid volume, sperm concentration, and sperm count in idiopathic infertile patients(n=55) compared to controls (n=30). Patients/Controls Seminal Fluid Volume (ml) Sperm Concentration (Million/ml) Sperm Count (Million /Ejaculate) Controls 2.63 ± 0.13 63.26 ± 10.08 a 168.96 ± 31.82 a Patients before treatment 2.48 ± 0.14 12.96 ± 1.13 b 30.58 ± 2.86 b 1 Month after treatment 2.52 ± 0.12 18.41 ± 1.65 * 44.98 ± 4.43 * 2 Month after treatment 2.48 ± 0.10 20.07 ± 1.50 * 51.05 ± 4.58 * 3 Month after treatment 2.45 ± 0.08 22.43 ± 1.57 * 54.99 ± 4.30 * *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment Table 4: Effects of L-carnitine and multivitamins treatment on the mean ± SE of sperm motility (represented as percent of total) in idiopathic infertile patients(n=55) compared to controls (n=30). Patients/Controls Active Motile Sperm(%) Sluggish Motile Sperm(%) Immotile Sperm (%) Progressive Motile Count (Million /Ejaculate) Controls 51.50 ± 1.59 a 31.16 ± 0.94 17.33 ± 1.21 a 91.10 ± 20.97 a Patients before treatment 22.81 ± 2.22 b 32.58 ± 1.77 44.60 ± 3.18 b 8.02 ± 1.05 b 1 Month after treatment 25.63 ± 2.20 34.36 ± 1.39 40.18 ± 2.84 13.40 ± 2.03 * 2 Month after treatment 26.63 ± 2.02 * 36.00 ± 1.17 * 37.34 ± 2.42 * 15.15 ± 1.96 * 3 Month after treatment 29.36 ± 2.27 * 35.81 ± 1.46 34.90 ± 3.15 * 18.03 ± 2.19 * *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment Table 5: Effects of multivitamins treatment on the mean ± SE of seminal fluid volume, sperm concentration, and sperm count in idiopathic infertile patients(n=29) compared to controls (n=30). Patients/Controls Seminal Fluid Volume (ml) Sperm Concentration (Million/Ml) Sperm Count (Million/ Ejaculate) Controls 2.63 ± 0.13 63.26 ± 10.08 a 168.96 ± 31.82 a Patients before treatment 2.43 ± 0.17 20.37 ± 1.11 b 47.68 ± 3.61 b 1 Month after treatment 2.39 ± 0.14 22.37 ± 1.31 * 52.72 ± 4.44 2 Month after treatment 2.46 ± 0.14 23.89 ± 1.78 * 57.68 ± 4.97 * 3 Month after treatment 2.50 ± 0.11 24.72 ± 1.70 * 62.17 ± 5.29 * *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment Table 6: Effects of multivitamins treatment on the mean ± SE of sperm motility (represented as percent of total) in idiopathic infertile patients(n=29) compared to controls (n=30). Patients/ Controls Active Motile Sperm (%) Sluggish Motile Sperm (%) Immotile Sperm (%) Progressive Motile Count (Million/ Ejaculate) Controls 51.50 ± 1.59 31.16 ± 0.94 17.33 ± 1.21 91.10 ± 20.97 a Patients before treatment 46.06 ± 1.47 34.24 ± 1.39 19.68 ± 1.65 21.81 ± 1.66 b 1 Month after treatment 45.68 ± 1.12 33.96 ± 1.39 20.34 ± 1.40 24.29 ± 2.28 2 Months after treatment 46.03 ± 1.43 34.13 ± 1.26 19.82 ± 1.45 26.52 ± 2.58 * 3 Months after treatment 46.89 ± 1.72 33.44 ± 1.21 19.65 ± 1.71 29.29 ± 3.19 * *p<0.05 represents significant difference between before treatment and after treatment. Different letters represent significant difference between controls and the patients before treatment Iraqi J Pharm Sci, Vol.21(1) 2012 Treatment of idiopathic male infertility 18 Table 7: Percentages of increase in sperm concentration, sperm count, actively motile sperm, and progressive motile sperm count measured after 3 months of treatment when compared with their corresponding values before treatment in group A, B, and C patients. Parameter measured Percentage of increase in the semen parameters measured after 3 months when compared with values before treatment Group A patients Group B patients Group C patients Sperm concentration 54% 73% 21% Sperm count 60% 80% 30% Active sperm motility 19% 29% Non Significant (NS) Progressive motile sperm count 84% 125% 34% No significant differences were found between the values of round cell count in the patients of different groups before and after treatment and those of the control subjects. The above results had demonstrated that L-carnitine at a daily dose of 2 gm/day for a treatment period of 3 months can improve sperm concentration, sperm count, percentage of actively motile sperm, and progressive motile sperm count among men with idiopathic oligo- and/or asthenozoospermia, and the combination of L-carnitine and multivitamins for the same period of treatment is more efficient than either L-carnitine or multivitamins alone(in the form of Stresstabs®) for the improvement in these semen parameters and can aid in the treatment of idiopathic male infertility. In addition, L- carnitine may be better than the use of oral multivitamins in the dosage regimens applied in this study in improving semen parameters and treatment of idiopathic male infertility. Moreover, the use of the progressive motile sperm count in the seminal fluid analysis is more indicative for the improvement in semen parameters than the sperm count and percentage of actively motile sperms separately, since it represents the product of multiplying the sperm count by the percentage of actively motile sperm. Discussion Other similar studies have suggested an improvement in semen parameters of men with low sperm motility following treatment with oral carnitine (14, 15, 2) . These observations suggest a role of carnitine or acetylcarnitine as empiric therapy for idiopathic asthenospermia (16) .The significant effect of L-carnitineon increasing sperm concentration, sperm count, and percentage of actively motile sperm in this study could be due to or explained by of which is that L-carnitine is an essential cofactor that could accelerate lipid metabolism and has a pivotal role in mitochondrial β-oxidation of long-chain fatty acids for cellular energy production (4,5) . There is also an unknown effect of L-carnitine in Sertoli cell– spermatogenic line interaction, an action on the postmeiotic phases of spermatogenesis (for example, on the chromatin stability or mitochondrial function of spermatocytes or spermatids), or an improvement in the quality of the epididymal microenvironment, reducing gamete phagocytosis at this level while increasing ejaculated spermatozoa (17) . In addition, carnitine administration increases prostaglandin E2 concentration (18) , which affects sperm count (19) . Moreover, carnitine protects cell membrane and DNA against damage induced by free oxygen radicals. It also prevents protein oxidation and lactate oxidative damage (20, 9) . Hence, it acts as an “anti-aging” substance, protecting against damage induced by free oxygen radicals (17) .In the same manner, there was a significant increase in the progressive motile sperm count, since it is a product of sperm count and the percentage of active motile sperm. A number of studies have suggested a beneficial role of antioxidants or anti-ROS drugs (vitamin E, vitamin C and GSH), including improved sperm quality and increasing fertilizing capacity (3) .Vitamin E is one of the major membrane protectants against ROS and lipid peroxidation (21) . Because vitamin E is a chain- breaking antioxidant and not a scavenging antioxidant, it would be expected to offer protection to membrane components without influencing ROS generation (22) . Vitamin E, on the other hand, acted as an effective antioxidant and significantly enhanced the capacity of human spermatozoa for sperm- oocyte fusion. Vitamin B12 and folic acid are important in cellular replication, especially for the synthesis of RNA and DNA, and deficiency states have been associated with decreased sperm count and motility (23) . Vitamins B1, B2, B3, B5, and B6 are important in many metabolic pathways in cells in general (and sperm in particular), and any deficiency in these vitamins can lead to defects in sperm metabolism and normal Iraqi J Pharm Sci, Vol.21(1) 2012 Treatment of idiopathic male infertility 19 functions (24) .Although pregnancy was not a principal end point in many studies (15, 14) , including this study, as it is difficult to avoid the many confounding variables acting on naturally induced fertilization and subsequent pregnancy, one pregnancy was recorded in group B patients after 2 months of treatment. In conclusion the present study had demonstrated that L-carnitine at a daily dose of 2 gm/day for a treatment period of 3 months can improve sperm concentration, sperm count, percentage of actively motile sperm, and progressive motile sperm count among men with idiopathic oligo- and/or asthenozoospermia, and the combination of L- carnitine and multivitamins for the same period of treatment is more efficient than either L-carnitine or multivitamins alone References 1. Yao M.W.M., and Schust D.J. Infertility. Berek J.S, editor. Novak's Gynecology. 13 th Edition. USA: Lippincott Williams and Wilkins.2002; pp: 973-1048. 2. Peivandi S., Karimpour A., and Moslemizadeh N. Effects of L-carnitine on infertile men's spermogram: a randomized clinical trial. J ReprodFertil;2010; 10(4): 41-44. 3. Schiff J.D., Ramirez M.L., and Bar-Chama N. Medical and surgical management of male infertility. EndocrinolMetabClin N Am; 2007; 36: 313-331. 4. JeulinC., and Lewin L.M. 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