|ORIGINAL RESEARCH REPORT
|Year : 2015 | Volume
| Issue : 1 | Page : 14-19
Preliminary report on plasma homocysteine and hormonal variations in infertile women in Lagos, Nigeria
Vincent Oluseye Osunkalu1, Christian C Makwe2, Oluwatosin Jonadab Akinsola3, Adediran Adewumi1, Oluseun Funke Akomolafe1
1 Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos, Lagos, Nigeria
2 Department of Obstetrics and Gynaecology, College of Medicine, University of Lagos, Lagos, Nigeria
3 Department of Community Health and Primary Care, College of Medicine, University of Lagos, Lagos, Nigeria
|Date of Web Publication||14-Jul-2015|
Dr. Vincent Oluseye Osunkalu
Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos, Lagos
Source of Support: None, Conflict of Interest: None
Background: The relationship between infertility and hormonal variations has been variously documented, but little has been reported on the interactions between hormonal factors, homocysteine (Hcy), and female infertility. Objective: This study aimed to evaluate the relationship between plasma Hcy levels and hormonal variations in infertile women. Materials and Methods: This descriptive cross-sectional study was carried out among eligible infertile and fertile women seeking care at the Lagos University Teaching Hospital, Lagos, Nigeria. The subjects were 100 women referred for management of infertility, and the controls were 50 fertile women who had given birth within the past year. Fasting plasma levels of Hcy were estimated using enzyme immunoassay. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, prolactin, and progesterone were measured using Access 2 (Immunoassay systems-beckman coulter, inc.250S.Kraemer blvd.Brea, ca 92821. U.S.A). Results: Mean plasma Hcy levels for subjects and controls were 9.50 ± 1.88 μmol/L and 9.44 ± 1.85 μmol/L, respectively, with no significant variation (P = 0.952). Plasma Hcy was not significantly associated with infertility and hormone levels. The mean serum levels of LH, FSH, and prolactin were significantly higher among subjects compared to controls. Conclusion: Hormonal variations observed in infertile women did not appear to significantly alter plasma Hcy levels, and hyperhomocysteinemia (HHcy) may not have significantly contributed to female infertility in our environment.
Keywords: Female, homocysteine, hormone parameters, infertility, Nigeria
|How to cite this article:|
Osunkalu VO, Makwe CC, Akinsola OJ, Adewumi A, Akomolafe OF. Preliminary report on plasma homocysteine and hormonal variations in infertile women in Lagos, Nigeria. J Clin Sci 2015;12:14-9
|How to cite this URL:|
Osunkalu VO, Makwe CC, Akinsola OJ, Adewumi A, Akomolafe OF. Preliminary report on plasma homocysteine and hormonal variations in infertile women in Lagos, Nigeria. J Clin Sci [serial online] 2015 [cited 2020 Sep 20];12:14-9. Available from: http://www.jcsjournal.org/text.asp?2015/12/1/14/160761
| Introduction|| |
Infertility is the inability of a couple to achieve pregnancy after 1 year of regular unprotected sexual intercourse. Infertility is described as primary if the couple has never conceived and secondary if they fail to conceive after a previous conception. The prevalence of infertility varies from one community to another and the estimate ranges 10-15%.  In Nigeria, secondary infertility is more prevalent,  and anovulation accounts for 20% of secondary female infertility. 
Several intermediates of the homocysteine (Hcy) pathway are directly or indirectly involved in the synthesis of proteins, synthesis and repair of DNA, and balancing the degree of oxidative stress; they are critical intermediates in gametogenesis. Elevated levels of plasma Hcy may lead to harmful effects such as direct cellular toxicity, endothelial damage, excessive generation of reactive oxygen species (ROS), increased expression of proinflammatory cytokines, altered nitric oxide (NO) bioavailability, inhibition of transmethylation reactions, and aberrant gene expressions. ,,,,
Elevations in plasma levels of Hcy are typically caused either by nutritional deficiencies in folate (vitamin B 9 ), vitamin B 6, and cobalamin (vitamin B 12 ) or by enzyme deficiency in the associated metabolic pathways.  Increasing age,  tobacco smoking, and coffee consumption  are other documented causes of elevated Hcy. A plasma Hcy level greater than 12 μmol/L is considered hyperhomocysteinemia (HHcy). 
HHcy has been reported as a risk factor in recurrent/early pregnancy losses,  congenital birth defects, , and late pregnancy complications such as preeclampsia/eclampsia, , preterm birth,  intrauterine growth retardation, , low birth weight, placental abruption, and intrauterine fetal death. ,
While a large amount of scientific work has investigated the role of sex steroids on female infertility, there is as yet limited information on the possible involvement of Hcy in the earlier stages of reproductive physiology and infertility. Fewer still are data on the effects of sex hormones on Hcy. A few studies, however, have indicated a decrease in Hcy with the administration of oral estrogen in premenopausal women. 
Hcy levels are reportedly lower in women compared to men, and Hcy is lower during pregnancy and higher during menopause.  Higher estrogen status has been associated with a decreased mean serum total Hcy concentration, independently of nutritional status and muscle mass.  Estrogen modulates thiol amino acid metabolism, particularly methionine metabolism, by interfering with the transsulfuration pathway, thus diminishing Hcy and preventing its accumulation. Estrogen may enhance the net production of glutathione (GSH) and stabilize NO, which contributes to its beneficial effects on the vasculature. 
Though physiological levels of ROS in follicular fluid are necessary for normal oocyte maturation, ovulation, and fertilization, a high concentration of Hcy in follicular fluid may induce excessive oxidative stress, which has a negative effect on ovulation, fertilization, and implantation. ,, Some studies suggest that the Hcy pathway may play an important role in infertile women with ovulation dysfunction ,,,, and unexplained infertility. ,, Despite the huge burden of infertility in Nigeria, there is a paucity of data on the role and/or contribution of Hcy to female infertility in this environment and the effects of hormonal changes on Hcy in infertile females. The aim of this pilot study was to determine the levels of Hcy in infertile women and evaluate a possible association between Hcy and hormonal variations in female infertility in our immediate environment.
| Materials and Methods|| |
This descriptive cross-sectional study was carried out among 150 women attending the outpatient clinic at the Lagos University Teaching Hospital, Lagos, Nigeria after obtaining ethical approval from the Lagos University Teaching Hospital Ethical Research Board and verbal consent from all participants. The subjects were 100 women aged 18-44 years, referred for the management of infertility, and the controls were 50 fertile women who had given birth at least once within the past year with no obstetric complication. Participants with tubal blockage or any documented structural or congenital reproductive anomaly, history of hypertension, cardiovascular disease, or any chronic systemic disorders, and male factor infertility were excluded from the study. Structured questionnaires were administered by the interviewer, and information on the following were obtained: Age, parity, occupation, educational status, last menstrual period and current use of contraceptives, medical history and use of medications, diet history including cigarette smoking, and consumption of caffeinated beverages. Each study participant had his/her weight and height measured to determine the basal metabolic index (BMI).
After overnight fasting, 10 mL of venous blood was collected [3.5 mL of blood was collected in a vial containing 2.5 μg of ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and the rest of the blood was collected into plain tubes]. The samples were transported to the laboratory in cooler boxes immediately after collection. The blood in each Vacutainer ® plain tube was allowed to clot and centrifuged at 500 g for 5 min, and serum was collected and stored at –80°C, awaiting hormonal assay, while the EDTA anticoagulated blood was mixed to prevent clotting. The plasma from the EDTA sample was used for the estimation of Hcy, based on enzyme immunoassay technique using the DRG-homocysteine kit from DRG International Inc. (Mountainside, NJ, USA), with a detection range of 2-50 μmol/L and a minimum detection level of 0.19 μmol/L. he Biotek ELx 800 absorbance microplate reader (serial no- 205808) manufactured by BioTek Laboratories, Inc. Shoreline, Washington, USA was used to read the absorbance. The serum sample was used to estimate luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, progesterone, and estradiol. A serum sample was collected on the third day of each woman's menstrual cycle (early follicular phase) for the estimation of LH, FSH, estradiol, and prolactin. A serum sample was also collected on the 21 st day of the menstrual cycle (or midluteal phase) for estimation of progesterone. Serum prolactin, LH, FSH, estradiol, and progesterone were estimated using the Access 2 Immunoassay System by Beckman Coulter, USA.
Data entry and analysis were performed using the software SPSS version 20 (SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was used to determine data normality at a value of P > 0.05. Outcome measures of interest were dichotomized using the 95 th percentile and the 5 th percentile of control values as high and low cutoff values, respectively. Strength of association was evaluated using the Chi-square test, Fisher's exact test, and a logistic regression model, and the mean difference of continuous variables was determined by the student's t-test. A P value of < 0.05 was considered statistically significant.
| Results|| |
A total of 150 participants were recruited for this study comprising 100 infertile women (subjects) and 50 fertile women (controls). Of the 100 subjects, 34% had primary and 66% had secondary infertility. Mean age and BMI of the subjects were higher compared to controls (P = 0.002 and 0.031, respectively). Mean fasting Hcy was slightly higher in the subjects compared to controls, but this difference was not statistically significant (P = 0.952). Mean values of serum FSH, LH, and prolactin were higher in subjects compared to controls, and all these differences were statistically significant (P < 0.01). There were no statistically significant differences in the mean serum estradiol and progesterone values in both groups [Table 1].
|Table 1: Bivariate analysis of mean plasma Hcy and hormonal parameters between subjects and controls |
Click here to view
More infertile subjects were older than 35 years of age compared to the fertile controls (66 of 100 vs 19 of 50; P = 0.001). The percentage of infertile subjects with BMI above 30 kg/m 2 was significantly higher compared to controls (16% vs 10%; P = 0.027). The 95 th percentile value for Hcy within the apparently healthy control group was determined to be 12 μmol/L. However, the percentage of infertile subjects and fertile subjects with plasma Hcy above the 95 th percentile did not differ significantly (12% vs 10%; P = 0.708). Elevated levels of LH, FSH, and estradiol were closely associated with infertility in the subjects (P = 0.000), while low progesterone levels below the 5 th percentile of control was seen in 25% (n = 100) of the infertile subjects compared to 6% (n = 50) of the apparently fertile control group [Table 2].
|Table 2: Comparing variations in the distribution pattern of plasma Hcy and hormonal parameters between infertile subjects and fertile control population |
Click here to view
Neither primary nor secondary infertility was observed to be associated with elevated Hcy. Of the 100 subjects studied, 82% had a form of hormonal derangement. LH, FSH, and prolactin were elevated above the 95 th percentile in 65%, 66%, and 15% of the infertile subjects, respectively, while lower values of estradiol and progesterone below the 5 th percentile of control values were observed in 24% and 25% of infertile subjects, respectively. A higher percentage of subjects with low serum levels of progesterone was associated with lower levels of Hcy (100% vs 0%; P = 0.048). Though a higher proportion of subjects with low serum estradiol had elevated Hcy compared to subjects with higher serum levels of estradiol (13.3% vs 9.4%), statistical significance was not found (P = 0.316). However, serum hormone levels of LH, FSH, and prolactin were not significantly associated with varying plasma Hcy levels (P > 0.05) [Table 3].
|Table 3: Association between Hcy and hormonal parameters in fertile and infertile women |
Click here to view
Infertility was found to be significantly associated with increasing levels of FSH [odds ratio (OR) =1.933; P = 0.00; 95% confidence interval (CI): 1.437, 2.602] and prolactin (OR = 1.155, P = 0.037; 95% CI: 1.009, 1.259). A lower value of BMI was found to be protective against infertility (OR = 0.795; P = 0.011; 95% CI: 0.666, 0.949). However, Hcy was not a significant factor for infertility among these subjects (P = 0.581). The model was a good fit, as the Hosmer-Lemeshow equation was not significant (P = 0.289) [Table 4].
|Table 4: Logistic regression analysis of factors influencing infertility among subjects |
Click here to view
| Discussion|| |
Among the 100 infertile women studied, secondary infertility occurred more frequently than did primary infertility. This finding is similar to published reports on the pattern of infertility in Nigeria. ,
Researchers in Bergen Norway Norway reported that Hcy levels higher than 10.7 μmol/L increased a woman's chance of a miscarriage by up to 38%.  This may imply a closer association between Hcy and secondary subfertility. Though a higher percentage of subjects with secondary infertility had Hcy in the upper centile of the control value compared to women with primary infertility (13% vs 3%), a statistical significance was not reached.
In contrast, studies conducted among women with unexplained infertility in Pakistan  and India  showed statistically significant increase in plasma Hcy when compared to healthy controls. The observed difference may be attributed to greater dietary intake of vitamins, as a high proportion of Pakistani women are vegetarians,  hence the likelihood of vitamin B 12 deficiency. The lack of evidence for nutritional deficiency as a possible factor in female infertility in this environment is further supported by the lack of association between Hcy and the red cell indices of subjects in this study. In Nigeria, an earlier study on plasma Hcy showed that most women regularly use vitamin supplements; especially those containing folate and cobalamin.  In this study, Hcy was associated with age greater than 35 years in both fertile and infertile women. Similar age-associated increases in plasma Hcy have been reported among healthy Taiwanese subjects. 
In this study, the mean age of infertile women was relatively higher than that of the healthy controls. This statistically significant difference in mean age may partly explain the elevated mean serum FSH and LH in infertile women when compared to fertile controls. In women of reproductive age, the ovarian reserve declines with advancing age. It is estimated that 81% of the variations in ovarian reserve are due to age.  Fertility rates decrease with the depletion of ovarian follicles, especially around the age of 37 years when the rate of oocyte depletion accelerates. , The age-related depletion of ovarian follicles leads to diminished ovarian function and ovarian steroid production, particularly estradiol. Estradiol and inhibin B are known to suppress pituitary FSH secretion, and low serum estradiol and inhibin B lead to elevated serum FSH.  In females, elevated serum FSH suggests poor ovarian reserve and diminished fertility, and the day 3 serum FSH level can reliably predict ovarian reserve. Despite the elevated serum FSH and LH in infertile women, there were no statistically significant differences in the mean values of estradiol and progesterone. In this study, the measured gonadotrophins (FSH and LH) and ovarian steroids (estradiol and progesterone) did not show significant association with serum Hcy levels in both infertile and fertile women. Findings from this study did not corroborate earlier reports by Christodoulakos et al. (2006) or Sbarouni et al. (2005), in both of which the plasma Hcy levels dropped with increasing estrogen level. , If the theory implicating estrogen in the pathway of Hcy metabolism were applicable, it would therefore be expected that infertile women with lower values of serum estrogen have a proportionately higher plasma Hcy level. This association did not reach statistical significance in this study (P = 0.316).
The mean serum prolactin of infertile women was also statistically higher than that of the healthy controls. Hyperprolactinemia has the potential to adversely affect fertility by impairing gonadotrophin-releasing hormone (GnRH) pulsatility and, thereby, ovarian function. The finding of elevated prolactin among infertile women in this study is consistent with similar studies that showed an association between hyperprolactinemia and infertility. In addition, there was no association between hyperprolactinemia and serum Hcy levels in this study (P = 0.224).
| Conclusion|| |
The inability to demonstrate a statistically significant association between plasma Hcy and hormonal derangement in infertile women in this study may imply that HHcy does not significantly contribute to female infertility in this environment.
Source of funding
No financial support exists.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Orhue A, Aziken M. Experience with a comprehensive university hospital-based infertility program in Nigeria. Int J Gynaecol Obstet 2008;101:11-5.
Wasiu Eniola Olooto, Adebayo Adetola Amballi, Taiwo Abayomi Banjo. A review of female infertility; important etiological factors and management. J Microbiol Biotech Res 2012:2:379-85.
Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocys-teinemia and its impact on endothelial function. Curr Drug Metab 2005;6:27-36.
Zhang X, Li H, Jin H, Ebin Z, Brodsky S, Goligorsky MS. Effects of homocysteine on endothelial nitric oxide production. Am J Physiol Renal Physiol 2000;279:F671-8.
Thaler CD, Epel D. Nitric oxide in oocyte maturation, ovulation, fertilization, cleavage and implantation: A little dab′ll do ya. Curr Pharm Des 2003;9:399-409.
Stühlinger MC, Oka RK, Graf EE, Schmölzer I, Upson BM, Kapoor O, et al
. Endothelial dysfunction induced by hyperhomocyst (e) inemia: Role of asymmetric dimethylarginine. Circulation 2003;108:933-8.
Outinen PA, Sood SK, Pfeifer SI, Pamidi S, Podor TJ, Li J, et al
. Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells. Blood 1999;94:959-67.
Torres-Sánchez L, Chen J, Diaz-Sánchez Y, Palomeque C, Bottiglieri T, López-Cervantes M, et al
. Dietary and genetic determinants of homocysteine levels among Mexican women of reproductive age. Eur J Clin Nutr 2006;60:691-7.
Chou ST, Ko LE, Lim PS, Huang JL, Yang CS. Effect of age and sex on plasma total homocysteine in Taiwanese subjects. Chin J Physiol 2000;43:159-64.
Nygård O, Refsum H, Ueland PM, Stensvold I, Nordrehaug JE, Kvåle G, et al
. Coffee consumption and plasma total homocysteine: The hordaland homocysteine study. Am J Clin Nutr 1997;65:136-43.
Stanger O, Herrmann W, Pietrzik K, Fowler B, Geisel J, Dierkes J, et al
. DACH-LIGA homocystein (German, Austrian and Swiss homocysteine society): Consensus paper on the rational clinical use of homocysteine, folic acid and B-vitamins in cardiovascular and thrombotic diseases: Guidelines and recommendations. Clin Chem Lab Med 2003;41:1392-403.
Nelen WL, Blom HJ, Steegers EA, den Heijer M, Eskes TK. Hyperhomocysteinemia and recurrent early pregnancy loss: A meta-analysis. Fertil Steril 2000;74:1196-9.
Vollset SE, Refsum H, Irgens LM, Emblem BM, Tverdal A, Gjessing HK, et al
. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: The Hordaland Homocysteine study. Am J Clin Nutr 2000;71:962-8.
Verkleij-Hagoort AC, Verlinde M, Ursem NT, Lindemans J, Helbing WA, Ottenkamp J, et al
. Maternal hyperhomocysteinaemia is a risk factor for congenital heart disease. BJOG 2006;113:1412-8.
Makedos G, Papanicolaou A, Hitoglou A, Kalogiannidis I, Makedos A, Vrazioti V, et al
. Homocysteine, folic acid and B12 serum levels in pregnancy complicated with preeclampsia. Arch Gynecol Obstet 2007;275:121-4.
Bibi S, Qureshi, Ahmad M, Qureshi PM, Memon A, Qazi RA. Hyperhomocysteinaemia, vascular related pregnancy complications and the response to vitamin supplementation in pregnant women of Pakistan. J Pak Med Assoc 2010;60:741-5.
Dhobale M, Chavan P, Kulkarni A, Mehendale S, Pisal H, Joshi S. Reduced folate, increased vitamin B (12) and homocysteine concentrations in women delivering preterm. Ann Nutr Metab 2012;61:7-14.
Pandey K, Dubay P, Bhagoliwal A, Gupta N, Tyagi G. Hyperhomocysteinemia as a risk factor for IUGR. J Obstet Gynaecol India 2012;62:406-8.
Furness D, Fenech M, Dekker G, Khong TY, Roberts C, Hague W. Folate, vitamin B12, vitamin B6 and homocysteine: Impact on pregnancy outcome. Matern Child Nutr 2013;9:155-66.
Bergen NE, Jaddoe VW, Timmermans S, Hofman A, Lindemans J, Russcher H, et al
. Homocysteine and folate concentrations in early pregnancy and the risk of adverse pregnancy outcomes: The Generation R Study. BJOG 2012;119:739-51.
de la Calle M, Usandizaga R, Sancha M, Magdaleno F, Herranz A, Cabrillo E. Homocysteine, folic acid and B-group vitamins in obstetrics and gynaecology. Eur J Obstet Gynecol Reprod Biol 2003;107:125-34.
Sbarouni E, Kyriakides ZS, Kremastinos DT. The effect of hormone replacement therapy and simvastatin on plasma homocysteine. See comment in PubMed Commons below J Womens Health (Larchmt) 2005;14:154-8.
Dimitrova KR, DeGroot K, Myers AK, Kim YD. Estrogen and homocysteine. Cardiovasc Res 2002;53:577-88.
Bennett M. Vitamin B12 deficiency, infertility and recurrent fetal loss. J Reprod Med 2001;46:209-12.
Berker B, Kaya C, Aytac R, Satiroglu H. Homocysteine concentrations in follicular fluid are associated with poor oocyte and embryo qualities in polycystic ovary syndrome patients undergoing assisted reproduction. Hum Reprod 2009;24:2293-302.
Ebisch IM, Peters WH, Thomas CM, Wetzels AM, Peer PG, Steegers-Theunissen RP. Homocysteine, glutathione and related thiols affect fertility parameters in the (sub) fertile couple. Hum Reprod 2006;21:1725-33.
Laanpere M, Altmäe S, Stavreus-Evers A, Nilsson TK, Yngve A, Salumets A. Folate-mediated one-carbon metabolism and its effect on female fertility and pregnancy viability. Nutr Rev 2010;68:99-113.
Ocal P, Ersoylu B, Cepni I, Guralp O, Atakul N, Irez T, et al
. The association between homocysteine in the follicular fluid with embryo quality and pregnancy rate in assisted reproductive techniques. J Assist Reprod Genet 2012;29:299-304.
Badawy A, State O, El Gawad SSh, El Aziz OA. Plasma homocysteine and polycystic ovary syndrome: The missed link. Eur J Obstet Gynecol Reprod Biol 2007;131:68-72.
Yilmaz N, Pektas M, Tonguc E, Kilic S, Gulerman C, Gungor T, et al
. The correlation of plasma homocysteine with insulin resistance in polycystic ovary syndrome. J Obstet Gynaecol Res 2008;34:384-91.
Salehpour S, Manzor-Al-Ajdad O, Samani EN, Abadi A. Evaluation of homocysteine levels in patients with polycystic ovarian syndrome. Int J Fertil Steril 2011;4:168-71.
D′Uva M, Di Micco P, Strina I, Alviggi C, Iannuzzo M, Ranieri A, et al
. Hyperhomocysteinemia in women with unexplained sterility or recurrent early pregnancy loss from Southern Italy: A preliminary report. Thromb J 2007;5:10.
Adegbola O, Akindele MO. The pattern and challenges of infertility management in Lagos, Nigeria. Afr Health Sci 2013;13:1126-9.
Kamath SK, Hussain EA, Amin D, Mortillaro E, West B, Peterson CT, et al
. Cardiovascular disease risk factors in 2 distinct ethnic groups: Indian and Pakistani compared with American premenopausal women. Am J Clin Nutr 1999;69:621-31.
Osunkalu V, Onajole A, Odeyemi K, Ogunnowo B, Sekoni A, Ayoola G, et al
. Homocysteine and folate levels as indicators of cerebrovascular accident. J Blood Med 2010;1:131-4.
Wallace WH, Kelsey TW. Human ovarian reserve from conception to the menopause. PLoS One 2010;5:e8772.
Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: Implications for forecasting menopause. Hum Reprod 1992;7:1342-6.
Gougeon A, Ecochard R, Thalabard JC. Age-related changes of the population of human ovarian follicles: Increase in the disappearance rate of non-growing and early-growing follicles in aging women. Biol Reprod 1994;50:653-63.
Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia DE, Soules MR. Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: A study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J Clin Endocrinol Metab 1996;81:2742-5.
Christodoulakos GE, Lambrinoudaki IV, Rizos DA, Alexandrou A, Kountouris AV, Creatsas GC. Endogenous sex steroids and circulating homocysteine in healthy Greek postmenopausal women. Hormones (Athens) 2006;5:35-41.
[Table 1], [Table 2], [Table 3], [Table 4]