IVF and Infertility Unit, Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Tel Aviv University, Zerifin 70300, Israel
1 To whom correspondence should be addressed. e-mail: ivfdoc{at}asaf.health.gov.il
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Abstract |
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Key words: homocysteine/insulin resistance/metabolic syndrome/polycystic ovary syndrome
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Introduction |
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Insulin resistance (IR) or ingestion of a diet with a high insulinaemic index will tend to increase plasma Hcy (Giltay et al., 1998b; Nygard et al., 1998
; Bar-On et al., 2000
; Meigs et al., 2001
).
Patients with non-insulin-dependent diabetes mellitus (NIDDM), or hypertension and IR, especially when associated with nephropathy, have increased Hcy levels (Henning et al., 1999; Stabler et al., 1999
; Sheu et al., 2000
). Insulin sensitivity was found to be inversely related to plasma homocysteine in pregnant women with pre-eclampsia (Rajkovic et al., 1997
; Laivuori et al., 1999
). Elevated levels of plasma Hcy have been shown to be a significant long-term risk factor for atherogenesis and chronic vascular damage (Nygard et al., 1995
; El-Khairy et al., 1999
), especially in situations where insulin levels are increased (Das et al., 1999
; Knekt et al., 2001
; McFarlane et al., 2001
).
Polycystic ovary syndrome (PCOS) is a common endocrinopathy involving ovulatory disturbances, hyperandrogenism, infertility and an increased miscarriage rate. Obesity and hyperinsulinaemia are also frequently encountered in PCOS (Dunaif et al., 1992; Gennarelli et al., 2000
; Iuorno and Nestler, 2001
). Late complications of PCOS commonly include NIDDM, hypertension, dyslipidaemia, atherosclerosis and vascular disease (Talbott et al., 1995
; Robinson et al., 1996
; Dahlgren and Janson, 2000
). Thus it seems logical to hypothesize that elevated Hcy levels could be another feature of PCOS, being both associated with IR, reproductive failure (Craig et al., 2002
) and late vascular complications. This study was designed to examine the relationship between Hcy and IR in infertile patients with or without PCOS.
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Materials and methods |
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Ninety of these patients were candidates for treatment by ovulation induction with gonadotrophins, and 66 were candidates for IVF/ICSI treatment, due to additional infertility diagnoses.
Serum prolactin, thyroid hormone and 17-hydroxy-progesterone determinations were done on all patients and were within normal limits.
One hundred patients attending our infertility unit for treatment during the same time period, due to isolated male factor or tubal factor infertility, were randomly chosen during the 7-month recruitment period as a control group. These patients had regular menstrual periods, normal ovulatory cycles, had no ultrasound or clinical signs of PCOS, and were not selected by any other clinical sign including body weight. These patients had the same height, weight and menstrual blood tests as the PCOS group.
Laboratory tests
All patients were weighed on two separate days and had height measured. Baseline day 3 LH and FSH were measured in a spontaneous or progestogen-induced menses before treatment was initiated. Plasma glucose, insulin and homocysteine were measured after an overnight fast, during menses. Patients treated with ovulation induction (n = 66) had no medical treatment at this time, whereas patients entering an IVF cycle (n = 90) were either initiating at that point injection of Triptorelin 3.75 mg i.m. (Decapeptyl CRTM 3.75; Ferring, Germany) or had already been treated for 810 days with intranasal Nafarelin 600 µg/day (SynarelTM; Delpharm, France). Patients had not started folic acid supplementation or metformin treatment at the time of homocysteine testing. Patients then went on to stimulation with gonadotrophin injections and folic acid supplementation (800 µg/day) to ovulation or IVF after oocyte retrieval, fertilization and embryo transfer. One patient in the IVF group was dropped from the study at this point as she was receiving phenytoin for epilepsy (known to interfere with plasma homocysteine determination).
Glucose and insulin were measured from plasma separated and frozen immediately after venipuncture, and analysed in blocks of 25. Plasma glucose was measured in duplicate by the glucose oxidase method (Beckman, USA). Plasma immunoreactive insulin was measured using the Phadeseph RIA kit (Pharmacia, Sweden). The intra- and inter-assay coefficients of variation (CV) for glucose and insulin were 2.2 and 3.5% (glucose) and 4.7 and 6.2% (insulin) respectively.
Homocysteine was measured as total homocysteine from plasma separated and frozen immediately after venipuncture, and analysed in triplicates in blocks of 25. Homocysteine exists in plasma mostly as oxidized homocysteine bound to albumin, although reduced homocysteine and disulphide homocysteine forms also exist, therefore in this study total L-homocysteine was determined using Fluorescence Polarization Immunoassay (FPIA) by IMX analysis (Abbott Diagnostics, Axis-Shield, Norway). Briefly, bound oxidized and disulphide homocysteine is reduced, then enzymatically converted to S-adenosyl-L-homocysteine, which is then labelled and measured by immunoassay. The intra-assay CV was 2.7% or lower, and the inter-assay CV was 3.75.2% over the spectrum of concentrations determined in our population.
IR may be determined by a number of different methods (Del Prato, 1999; Katz et al., 2000
; Stumvoll et al., 2000
; McAuley et al., 2001
); we chose to use those which utilize static fasting glucose and insulin measurements, which are highly correlated to dynamic measurements after glucose loading (Matthews et al., 1985
; Legro et al., 1998
; Bonora et al., 2000
; Brun et al., 2000
; Fukushima et al., 2000
; Hanson et al., 2000
; Mather et al., 2001
). These include fasting insulin, the glucose:insulin ratio [glucose (mmol/l)/insulin (mIU/l)], the HOMA (homeostasis model assessment) index [glucose (mmol/l)*insulin (mIU/l)/22.5], or the logarithmic transformation of the HOMA index (log10HOMA). Correlations between clinical or biochemical parameters and IR were calculated using all of the above-mentioned indices. The upper limit of normal was constructed by calculating the mean + 2SD of the normal control group, for each variable. These calculations accurately reflected the accepted values in the above-mentioned reference publications.
Statistics
Statistics were calculated with the aid of the SPSSTM computerized statistical package (SPSS for Windows, Version 6.1, SPSS Inc. USA), utilizing Fishers exact test to compare differences in rates, and Students t-test to compare differences between parametric data sets. Pearsons correlation coefficients were used to calculate correlation between paired data sets. Significance of correlation and the relative contribution of each variable were calculated by single or multiple regression analyses respectively. Significance was set as P < 0.05.
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Results |
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The PCOS group was stratified by logHOMA: 102 patients had a logHOMA 0.59, (designated PCOS-IR) and 53 had a logHOMA <0.59 (designated PCOS-NIR). Elevated logHOMA was found to be significantly associated with a higher body mass index (BMI), a higher fasting glucose level, all IR indices, and elevated plasma homocysteine (Table I).
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Body weight and other variables
PCOS patients were found to be more obese than our control (normal ovary) patients. When patients were stratified by BMI, 45.2% (70/155) of the PCOS patients had a BMI 27 kg/m2, whereas only 22% (22/100) of the control group had a BMI
27 (
2 = 14.1, P = 0.002) (Table II). Elevated BMI was significantly associated with IR in PCOS patients (BMI in PCOS-IR: 28.6 ± 6.4; BMI in PCOS-NIR: 23.9 ± 3.4; P = 0.002) (Table I).
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Homocysteine as related to other variables
Homocysteine levels were significantly elevated in PCOS patients [11.5 ± 7.4 µmol/l (all PCOS), 12.4 ± 8.4 µmol/l (PCOS-IR) versus 7.4 ± 2.1 µmol/l (controls), P < 0.001] (Table I). Elevated homocysteine was noted in both lean and obese PCOS, as opposed to normal ovary subjects (11.1 and 12.1 versus 7.17 and 8.25 µmol/l for lean and obese PCOS versus lean and obese controls respectively) (Table II).
The 95th percentile for homocysteine in our control group was 11.0 µmol/l: the normal range is stated to be 511 µmol/l (Bar-On et al., 2000; Chambers et al., 2001
). When a level of 11.0 µmol/l was used as a threshold, 34.1% (53/155) of the PCOS patients had Hcy values >95th percentile of the controls (P < 0.0001) (Table III). Of the 53 NIR-PCOS patients, only 13 (24.5%) had an elevated (>11 µmol/l) Hcy level, whereas 43 of the 102 (42%) IR-PCOS patients had an elevated Hcy level (
2 = 4.69, P = 0.04) (Table III), demonstrating the differential effect of IR on homocysteine.
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Correlation between variables
Correlation between clinical or biochemical criteria and homocysteine was examined on a paired-data basis. No correlation was found between age and PCOS, age and IR, or age and homocysteine levels. No correlation between LH or LH:FSH ratios and IR, or homocysteine levels, was found. However, all indices of IR (insulin alone, glucose:insulin ratio, HOMA index, and logHOMA) were significantly correlated to homocysteine levels. Figure 1 shows paired-data analysis of the correlation between homocysteine levels and the logarithmic transformation of the HOMA index in all patient groups: r = 0.30, R2 = 0.091, P = 0.005 (ANOVA linear regression: mean square = 0.71, F = 7.98). All other IR indices, i.e. fasting insulin, HOMA index and glucose:insulin ratio were also similarly significantly correlated on single (paired) regression analysis with Hcy levels (r = 0.26, r = 0.24, r = 0.29, all P < 0.01 respectively).
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Discussion |
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Evidence has accrued to indicate that hyperinsulinaemia and/or any or all of the phenotypes of the IR syndromes in the general population may have various deleterious metabolic effects, including causing an increase of plasma Hcy (Meigs et al., 2001). We examined this association in our specific population of patients with PCOS and infertility, by determining the correlation between IR and elevated Hcy. IR was found to be extremely common in this patient group, regardless of the defining criteria for IR, and was practically universal in the obese PCOS subgroup. Three previous studies have found an association between IR and elevated homocysteine in specific patient groups in women of reproductive age. Laivuori et al. (1999
) examined the association between IR and elevated Hcy in pregnant women with pre-eclampsia. Elevated Hcy was related to IR in women with pre-eclampsia, but not in normal controls. It is of interest to note that, as in our study, elevated Hcy was best correlated to fasting insulin (single sample) and not to insulin and/or glucose increase after glucose loading. It is possible that the study group included patients with PCOS, although this was not specified. Another study (Yarali et al., 2000, 2001) looked at the cardiac function of PCOS patients, as a function of IR and Hcy levels. Hcy was significantly elevated in both lean and obese PCOS patients, and was related to IR and not necessarily to body weight. As in our study, Hcy levels were even higher in lean PCOS than obese PCOS patients, and to a degree highly similar to that found in our study (lean PCOS: 12.6 ± 5.6 µmol/l; obese PCOS: 10.0 ± 1.3 µmol/l; control: 8.7 ± 2.7 µmol/l). Sills et al. (2001
) did not find any association between the finding of PCO and plasma Hcy and insulin levels were insignificantly higher with polycystic ovaries. This study differentiated between patients on the basis of ultrasound morphology only, not incorporating other components of the syndrome.
Homocysteine levels are positively correlated to risk of cardiovascular disease and complications, by increasing oxidative stress in vascular endothelium, activation of platelets (Stamler et al., 1993; Coppola et al., 2000
; Mujumdar et al., 2002
), impairment of blood flow (Chambers et al., 2001
), stimulation of vascular smooth muscle proliferation (Van Guldener and Stehouwer, 2000
) and may be one of the signals inducing apoptosis in vascular endothelial cells by activating unfolded protein response (Zhang, 2001
). Thus, elevated Hcy may impair implantation by interfering with endometrial blood flow and vascular integrity, and has been documented to increase the probability of early pregnancy loss (Nelen et al., 2000
; Quere et al., 2001
). 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.
IR (or the IR syndromes, IRS) itself is a risk factor for cardiovascular disease, diabetes, hypertension nephropathy, and dyslipidaemia (Goldstein et al., 2001); all of the long-term implications of these facets of the metabolic syndromes can be aggravated by elevated Hcy. Thus, in metabolic terms, PCOS may possibly be considered another variant of the IRS, or at the very least be considered an early marker of the IRS. As such, the infertility treatment provider should actively search for signs of metabolic dysfunction, including elevated Hcy levels, and endeavour to optimize metabolic factors in order to achieve the best results in the short term (reproductive function) and in the long term (cardiovascular and metabolic functions).
Homocysteine levels are influenced by a number of variables which were not examined in this study, including smoking, renal function, vitamin B status and enzyme dysfunction states. All of the patients in our infertility practice are actively discouraged from smoking; the proportion of smokers among our patients was 9.0% (23/255), divided proportionately between study and control groups; these numbers did not allow for adequate statistical testing. Although renal status was not examined, all of these young women entered treatment in good general health and none had hypertension or oedema. Methyltetrahydrofolate reductase (MTHFR) enzyme deficiencies and vitamin levels were not screened in this patient group, as in the study by Laivuori et al. (1999) and Tsanadis et al. (2001
). Vitamin B12 levels and folic acid levels were examined in the study by Yarali et al., and no significant differences were found between PCOS and controls. As the frequency of this mutation is in the order of 510% in unselected populations, it seems unlikely that MTHFR status could be responsible for the differences between homocysteine in PCOS and controls in this study, although this is currently being determined.
The vascular aspect of PCOS has already been implicated in previous studies on plasminogen activator inhibitor-1 (PAI-1), with its attendant effects on blood flow; the effects of elevated PAI-1 may also be aggravated by elevated homocysteine (Atiomo et al., 1998). Endothelial dysfunction in PCOS was documented both by decreased response to vasodilation (Paradisi et al., 2001
) and by the finding of increased levels of endothelin-1 in insulin-resistant PCOS patients (Diamanti-Kandarakis, 2001
) and increased oxidative stress markers (Sabuncu et al., 2001
). It is possible that these findings are due toin partto increased Hcy levels.
We suggest that the vascularendothelial aspect of insulin-resistant PCOSincluding the additional complicating factor of hyperhomocysteinaemiahas important diagnostic and treatment implications, and warrants further clinical and laboratory investigation. Furthermore, actively treating elevated homocysteine in these patients could increase implantation and decrease pregnancy loss. Prospective studies could address the practical implications of our findings in terms of improved treatment success.
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Submitted on March 12, 2002; resubmitted on November 18, 2002; accepted on January 10, 2003.