Concentrations of carotenoids, retinol and {alpha}-tocopherol in plasma and follicular fluid of women undergoing IVF

Florian J. Schweigert1,4, Beate Steinhagen1, Jens Raila1, Anette Siemann2, David Peet2 and Ulrich Buscher3

1 Institute of Nutritional Science, University of Potsdam, 2 Fertility Center of Dr Peet and Dr Sydow, Berlin, and 3 Department of Obstetrics, Charité, Campus Virchow-Klinikum, Berlin, Germany

4 To whom correspondence should be addressed at: Institute of Nutritional Science, University Potsdam, Arthur-Scheunert-Allee 114–116, D-14558 Potsdam-Rehbrücke, Germany. e-mail: fjschwei{at}rz.uni-potsdam.de


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
BACKGROUND: Carotenoids are transferred into follicular fluid where they might serve as antioxidants and/or as precursors of retinoids which might modulate follicular or oocyte functions. METHODS AND RESULTS: In 77 women undergoing IVF differences between plasma and follicular fluid in the levels of carotenoids, retinol and {alpha}-tocopherol were evaluated especially with regard to fertilization success. Concentration of total carotenoids, retinol and {alpha}-tocopherol determined by HPLC in follicular fluid and plasma were 0.06 ± 0.02 versus 0.56 ± 0.23 µmol/l, 1.26 ± 0.52 versus 1.66 ± 0.37 µmol/l and 4.89 ± 2.13 versus 21.0 ± 5.7 µmol/l (mean ± SD) respectively (P < 0.001 for all). Differences between plasma and follicular fluid were greater for {beta}-carotene and lycopene (<20% of plasma concentration) than for lutein and zeaxanthin (>40%). Intrafollicular retinol and retinol-binding protein (RBP) levels represented 58 ± 23% and 60 ± 19% of plasma level. Similar molar ratios of retinol/RBP were observed. While no differences in the mean values of all components investigated were observed in plasma and follicular fluid between women with and without reproductive success, the variability in the concentration was much greater in follicular fluid obtained from women without reproductive success. CONCLUSIONS: It remains to be elucidated, if this is indicative of a disturbed sieving effect of the blood–follicle barrier with possibly negative consequences for oocyte maturation.

Key words: carotenoids/follicular fluid/human fertilization/retinol/tocopherol


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
It is now widely accepted that in addition to endocrine factors, nutrition also affects reproductive performance by modulation of the endocrine environment. Whereas the roles of gonadotrophins and steroids in follicular development have been thoroughly investigated in many species, very limited information is available regarding the importance of micronutrients such as minerals, trace elements and vitamins. Specific micronutrients might modulate endocrine mechanisms by acting like or mimicking steroid hormones. Potential candidates are retinoids as well as their precursors the carotenoids (Hattori et al., 2000Go). Currently, many gene products related to reproduction are known to be modulated by retinoic acid, the product of retinol oxidation. Additionally, the reactivity of the microenvironment during ovulation and implantation in reproduction has been discussed (Rodgers et al., 1995Go; Jozwik et al., 1999Go). Because the follicular fluid is a product of a transfer of blood plasma constituents and the metabolism of the granulosa cells (McNatty, 1978Go), the concentration of specific components of follicular fluid may reflect its transfer across the blood follicular barrier or the influence of the metabolism of the follicular structures.

The importance of retinoids in early embryonic development has been addressed in numerous studies. In general the importance of retinol and its plasma carrier retinol-binding protein (RBP) has been shown in the early steps of implantation (Fazleabas et al., 1994Go) as well as the importance of retinoic acid as a morphogen in embryonic development (Zile, 2001Go). A recent study describing the human follicular fluid proteome by two-dimensional electrophoresis singled out four proteins in follicular fluid as markers of follicular quality, two of them being RBP and transthyretin (TTR) (Anahory et al., 2002Go). This observation supports our earlier studies in cattle in which a clear relationship between follicular quality and intrafollicular retinol levels were observed (Schweigert and Zucker, 1988Go; Brown et al., 2003Go).

Carotenoids have recently gained attention because they function similarly to vitamin E as very potent antioxidants by acting with reactive oxygen species (ROS) (Sies and Stahl, 1995Go). In addition to this, specific carotenoids such as {beta}-carotene, {alpha}-carotene and {beta}-cryptoxanthin can serve as local precursors for retinol (Schweigert, 1998Go). In cattle, changes in the intrafollicular concentration of retinol have been observed dependent on the quality of the follicle (Schweigert and Zucker, 1988Go) and attributed to the local conversion of {beta}-carotene into retinol by granulosa cells (Schweigert et al., 1988Go). The importance of carotenoids in follicular fluid as local antioxidants has recently been discussed with regard to a reduced fertility rate in smoking women undergoing IVF (Palan et al., 1995Go). Information on these specific components with regard to their possible function in follicular development and their possible participation in fertilization and very early embryonic development is limited.

It has been shown that carotenoid composition of follicular fluid is different from that in plasma, but no direct comparisons are available (Palan et al., 1995Go). A prerequisite to interpreting possible specific differences between plasma and follicular fluid and between individual follicles, however, is a better understanding of the transfer of these components from the blood plasma across the blood–follicle barrier into the follicle. This will enable us to differentiate whether differences between plasma and follicular fluid are due to differences in the transfer across the blood–follicle barrier or may be attributed to its metabolism in follicular structures.

The aim of the present study was therefore to evaluate differences between plasma and follicular fluid with regard to levels of carotenoids, retinol and {alpha}-tocopherol, the distribution of carotenoids and {alpha}-tocopherol among the lipoprotein fractions and the presents of RBP and TTR as carriers for retinol. Moreover, the study addressed the question of whether fertilization success is related to the intrafollicular concentration of the investigated components.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Patients
A total of 77 paired samples of follicular fluid and plasma were obtained from women who underwent IVF at the Fertility Clinic of Dr Peet, Berlin, Germany and the Department of Obstetrics, Charité, Virchow-Klinikum of the Humboldt University Berlin, Germany. The study protocol was approved by the local Ethics Committee of the Charité University Hospital in Berlin. Informed consent for experimental use of plasma and follicular fluids was obtained from all patients. The women’s age ranged from 22–41 years (mean ± SD 32.3 ± 4.7). The pregnancy rate was 38%. All 77 paired plasma and follicular fluid samples were analysed for carotenoids, tocopherol and retinol. In a subset of 17 paired samples, lipoprotein distribution and RBP and TTR were investigated.

IVF stimulation protocols
Four different stimulation protocols were administered, two protocols using either a series of FSH (Serono, Unterschleissheim, Germany) or hMG (Organon, Oberschlelssheim, Germany) and two protocols, both with hMG in the follicular phase, either with the GnRH agonist Decapeptyl (GnRH agonist/hMG; Ferring AB, Kiel, Germany) or with clomiphene citrate (CC/hMG; Serono) in the mid-luteal phase for luteal phase down-regulation.

Aspiration of follicular fluid and collection of blood plasma
Transvaginal follicular aspiration was performed under vaginal sonographic guidance 36 h following the administration of hCG. Each ovary was aspirated with a separate needle. We sampled the fluid from the leading follicle on each side only if oocyte aspiration was successful at the first attempt. If flushing of the follicle with medium was necessary for retrieving the oocyte, the content was not used for measurement. Follicles of >16 mm diameter and follicular fluids with a mature oocyte and no blood contamination were centrifuged at 1000 g for 15 min at 4°C to eliminate residual cells.

Blood samples were collected in tubes on the day of oocyte retrieval and centrifuged at 1500 g for 10 min at 4°C. Plasma and follicular fluid samples were stored at –80°C and analysed within one month after collection, a period and temperature at which carotenoids have been reported to be stable (Comstock et al., 1993Go).

Selective precipitation of lipoproteins in plasma and follicular fluid
High density lipoproteins (HDL) were isolated from plasma and follicular fluid using a solution of 10 g dextran sulphate (molecular weight 50 kDa) diluted in 1 mol/l magnesium chloride to precipitate selectively the very-low and low density lipoprotein (VLDL, LDL) fractions. Briefly, plasma and follicular fluid (400 µl) were transferred into micro tubes and 40 µl reagent added to each tube. The tubes were immediately vortexed. After 10 min incubation at room temperature in the dark the sample was centrifuged for 30 min at 1500 g. The precipitate (VLDL/LDL) was resuspended and analysed (Vogel et al., 1996Go).

RBP and TTR immunoblot analysis
To assess the presence of RBP and TTR, we performed SDS–polyacrylamide gel electrophoresis (PAGE) immunoblot analysis. For this purpose, aliquots of plasma and follicular fluid were separated on 12% SDS–PAGE according to Laemmli (Laemmli, 1970Go). The separated proteins were electroblotted onto a PVDF membrane and Tris-buffered saline (TBS), 0.1% Tween 20 (TBST) containing 5% defatted milk was used to block nonspecific binding sites on the blot. The membrane was then incubated with peroxidase-coupled rabbit anti-human retinol-binding protein (Dako Diagnostica, Hamburg, Germany; 1:300 diluted in TBS containing 0.05% Tween 20) or primary rabbit anti-human transthyretin (Dako; 1:300 diluted in 0.05% TBST) and secondary peroxidase-coupled swine anti-rabbit IgG (Dako; Catalog No. A 0002; 1:1000 diluted in 0.05% TBST) for 1 h at room temperature. Antibody binding was visualized using the Luminol reaction (BM Chemiluminescence Blotting Substrate, Roche Diagnostics GmbH, Mannheim, Germany). Images were processed using the MultImager (Bio-Rad, Munich, Germany) and the Multi-Analyst software 1.0 (Bio-Rad).

Quantitative analysis of RBP in plasma and follicular fluid
Plasma and follicular RBP was quantitatively determined by an ELISA method developed in our laboratory. Polystyrene microtitre plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were incubated for 2 h at 37°C with rabbit anti-human RBP IgG (Dako; 50 µl/well of 1.2 µg/ml in coating buffer: 50 mmol/l carbonate buffer (pH 9.6). The wells were washed 4 times with 200 µl aliquots of washing buffer (PBS/Tween) consisting of 10 mmol/l phosphate buffer with 150 mmol/l NaCl and 0.05% Tween 20 (pH 7.4), shaken dry, wrapped in plastic, and stored at 4°C overnight. After four washings with PBS/Tween, the wells were filled with RBP standard solution (N Protein Standard/Standard SL OQIM 13, Dade Behring GmbH, Marburg, Germany) or diluted plasma (1:5000; 50 µl/well) and follicular fluid (1:5000; 50 µl/well) respectively, and incubated for 2 h at 37°C. After four further washings, the plates were filled with peroxidase- coupled anti-RBP IgG (Dako; 1:2000 diluted in PBS/Tween, 50 µl/well) and incubated for 1 h at 37°C. After four final washings, colour was developed using O-phenylenediamine dihydrochloride (OPD, Sigma, 100 µl/well of 3.7 mmol/l solution in 50 mmol/l disodium phosphate-25 mmol/l citric acid buffer pH 5.2 containing 0.012% H2O2) for 30 min at room temperature. The reaction was stopped by the addition of 1 mol/l H2SO4 (50 µl/well) and absorbance at 490 nm was measured with a spectrophotometer (Microplate Reader, Bio-Rad). Samples were analysed in triplicate. The standard curve obtained at each plate was used to calculate the RBP concentrations in the samples. The curve obtained by serial dilution of plasma and urine was parallel to the standard curve, indicating that the antigen measured was RBP. The sensitivity of the assay was 10 nmol/l, defined as the minimal concentration of RBP that produces an absorbance greater than ten standard deviations of the blank readings. The intra-assay coefficient of variation for urine and plasma samples was 2.9% and the interassay variation was 4.2%. The recovery of addition of known amounts of RBP to normal control serum and follicular fluid varied between 92 and 105%.

Analysis of carotenoids, retinol and {alpha}-tocopherol in plasma, follicular fluid and lipoprotein fractions
For separation and quantification of carotenoids (lutein, zeaxanthin, {alpha}-carotene, {beta}-carotene, {beta}-cryptoxanthin, lycopene) {alpha}-tocopherol and retinol a gradient reversed-phase HPLC-system was used. Briefly, 200 µl of ethanol were added to 100 µl plasma diluted with 100 µl H2O or to 200 µl follicular fluid. After vortexing for 30 s, plasma or follicular fluid were extracted twice with n-hexane, 1 ml each time stabilized with 0.05 % butylated hydroxytoluene (BHT), vortexed for 10 min and centrifuged for 10 min at 1500 g. The supernatants were removed, pooled, and evaporated under nitrogen and reconstituted in 200 µl isopropanol and injected into the HPLC-system (Waters, Eschborn, Germany). For separation and quantification of compounds a C30 carotenoid column (5 µm, 250 x 4.6 mm; YMC, Wilmington, USA) in line with a C18 pre-column (Luna, Phenomenex, Germany) with a solvent system consisting of solvent A with methanol (Roth Chemicals Germany):water (90:10 v:v, with 0.4 g/l ammonium acetate in H2O) and solvent B with methanol:methyl-tert-butyl-ether (Sigma Deisenhofen, Germany):water (8:90:2 v:v:v, with 0.1 g/l ammonium acetate in H2O) was applied as described in detail (Macias and Schweigert, 2001Go). Results for {alpha}-tocopherol and {beta}-carotene were compared with standard reference material 968a [National Institute of Standards Technology (NIST), Gaithersburg, MD, USA]. Coefficient of variability over time using control plasma was 0.9% for lutein, 1.6% for zeaxanthin, 2.6% for {alpha}-carotene, 2.0% for {beta}-carotene, 1.3% for {beta}-cryptoxanthin, 0.9% for lycopene and 1.9% for {alpha}-tocopherol. The recovery rate was 96% for lutein, 95% for zeaxanthin, 92% for {alpha}-carotene, 93% for {beta}-carotene, 96% for {beta}-cryptoxanthin, 90% for lycopene, 96% for retinol and 95% for {alpha}-tocopherol. Carotenoid standards of lutein, zeaxanthin, canthaxanthin, {beta}-cryptoxanthin, lycopene were a kind gift of Hoffmann-La Roche, Switzerland, {alpha}-carotene, {beta}-carotene, {alpha}-tocopherol, retinol and retinyl palmitate were from Sigma (Deisenhofen, Germany).

Statistical procedures
Values are expressed as means and standard deviation (SD). In every patient a plasma/follicular fluid concentration ratio was calculated for each of the carotenoids, {alpha}-tocopherol, retinol and RBP expressed as a percentage of the plasma concentration. Statistical analysis of the data was performed by applying the Student t-test and the Spearman correlation coefficient. Differences were considered significant if P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
In both plasma and follicular fluid, similar carotenoids were present, but always at lower concentrations in follicular fluid compared with plasma (Table I). In all 77 cases the intrafollicular concentration of the investigated carotenoids were lower compared with its level in plasma (P < 0.001). Similarly to the concentrations of carotenoids, retinol and {alpha}-tocopherol, the concentrations of RBP (Table II) was always lower in follicular fluid than in plasma (P < 0.001).


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Table I. Concentrations of carotenoids, retinol and {alpha}-tocopherol (mean ± SD; n = 77) in plasma and ratios of follicular fluid (FF) to plasma (P) concentrations (mean ± SD; n = 77)
 

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Table II. Concentrations of retinol (µmol/l) and RBP (µmol/l) in a subset of 17 corresponding plasma and follicular fluid samples (mean ± SD)
 
Table I depicts the follicular fluid to plasma concentration ratio for individual carotenoids, retinol, and {alpha}-tocopherol. The ratio for individual carotenoids in the follicular fluid differed from 13 to 44% with lowest levels for the more polar carotenoids and highest levels in the less polar ones. The ratio of {alpha}-tocopherol was in between these values. The transfer ratio of retinol and RBP in a subset of 17 samples was very similar with 58 ± 23 versus 60 ± 20% for retinol and RBP respectively (Table II). As a consequence of this, the retinol/RBP ratio was similar in plasma and follicular fluid (Table II).

No differences in the concentrations of plasma carotenoids, retinol and {alpha}-tocopherol between women with successful fertilization and non-successful fertilization were observed (data not shown). The same was true for the concentration of these components in follicular fluid of the two groups. Despite similar mean levels of all components in follicular fluid of women with and without reproductive success, the variation in the ratio of FF/P of investigated components in women without reproductive success was at least twice that of those getting pregnant (Table III). This difference between follicular fluid of women with and without reproductive success was further emphasized by the very close positive correlation of plasma and follicular fluid levels of carotenoids in women getting pregnant, compared with those without reproductive successes (Table III). For retinol and tocopherol a lower overall level of correlation was observed, with differences for retinol but not tocopherol between follicular fluid from women with and without reproductive success.


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Table III. Ratios of follicular fluid (FF) to plasma (P) concentrations (mean ± SD) and Spearman rank correlations between follicular fluid and plasma concentrations of selected carotenoids, retinol and {alpha}-tocopherol
 
When plasma and follicular fluid was subject to selective precipitation of the VLDL and LDL fraction of lipoproteins with dextran sulphate, significant differences were observed between plasma and follicular fluid. While in plasma substantial amounts of carotenoids and {alpha}-tocopherol were precipitated associated with the VLDL/LDL fraction with this method, in follicular fluid compared with plasma only trace amounts of carotenoids were removed by the selective precipitation (data not shown).

The SDS–PAGE immunoblotting analysis of paired plasma and follicular fluid samples (n = 17) showed that RBP was present in follicular fluid as well as in plasma at a comparable molecular weight of 21 kDa (Figure 1). TTR was also detectable in plasma and follicular fluid as a band of about 14 kDa. This protein band represents the TTR monomer subunit, which dissociates from the tetrameric holo-protein during SDS-PAGE.



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Figure 1. Representative SDS–PAGE immunoblotting analysis of RBP (21 kDa) and residual TTR monomer (14 kDa) in human plasma (P1-P3) and follicular fluid (F1-F3).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
In this study the concentrations of six carotenoids, retinol and {alpha}-tocopherol were measured for the first time in paired samples of plasma and follicular fluid from women undergoing IVF. Additionally, the distribution of carotenoids and {alpha}-tocopherol among the lipoprotein fractions as well as the presence of the transport proteins for retinol, RBP and TTR were analysed by comparing plasma and follicular fluid. This direct comparison gives the possibility of evaluation if quantitative and qualitative differences of these components between plasma and follicular fluid are due to selective processes at the level of transfer across the blood-follicular barrier or due to the selective metabolism of specific carotenoids within the follicular compartment.

In accordance with previous investigations in other species (Enk et al., 1986Go; Schweigert and Zucker, 1988Go; Schweigert and Schams, 1993Go; Palan et al., 1995Go), the results of the study show for the first time that the concentration of different carotenoids, retinol and {alpha}-tocopherol in follicular fluids were much lower than those in plasma from women undergoing IVF. Differences in the levels of specific carotenoids and {alpha}-tocopherol might be attributed to the known nutritional influence on plasma carotenoid levels or due to differences in the analytical method. In general, the results of the plasma levels of all investigated components correspond well to published data (Steghens et al., 1997Go; Michaud et al., 1998Go). The only other published study addressing the levels of specific carotenoids in human follicular fluid is also consistent with our results (Palan et al., 1995Go).

With regard to the ratio of individual carotenoids and {alpha}-tocopherol between plasma and follicular fluid, substantial differences could be observed. While the concentration for lutein and zeaxanthin represented >40% of the respective plasma concentration, the values for {beta}-carotene and lycopene were <20%. Based on these observed differences, questions arise concerning causes and possible consequences of these differences in follicular fluid. Do these differences reflecting selective metabolic mechanism within the follicle itself or are they the consequence of a selective transfer of carotenoids, retinoids and {alpha}-tocopherol from blood plasma into follicular fluid? Information concerning carotenoid metabolism in the ovarian follicle are limited to the observation that granulosa cells of bovine follicles can convert {beta}-carotene into retinol (Schweigert et al., 1988Go). In addition to this, {beta}-carotene and other carotenoids as well as {alpha}-tocopherol might function as antioxidants (Palan et al., 1995Go). The importance of oxidative stress markers in follicular fluid in the evaluation of the reproductive potential of oocytes, however, has not been shown (Jozwik et al., 1999Go).

In ovarian follicles, non-vascularized granulosa cells are in direct contact with the follicular fluid, but are separated from thecal blood capillaries by the basement membrane. This barrier behaves like a molecular sieve, which allows passage of protein in inverse proportion to molecular weight, but paradoxically not in inverse proportion to molecular size (Shalgi et al., 1973Go). In addition to this, in mice, this barrier has been found to be both charge- and size selective (Hess et al., 1998Go). Thus, a very specific composition of follicular fluid can be observed in mammals with molecules of lower molecular weight such as albumin being present in follicular fluid and higher molecular proteins are excluded (Andersen et al., 1976Go). In consequence of this observation, VLDL/LDL are excluded from the transfer and only HDL are present in follicular fluid of humans as shown in our study and by others (Simpson et al., 1980Go). Thus, only carotenoids and {alpha}-tocopherol associated with the HDL fraction can be recovered in the follicular fluid as found by selective precipitation of the VLDL/LDL fractions of plasma lipoproteins.

The differences in the transfer efficiency for individual carotenoids arises from the observation that individual carotenoids are not equally distributed among the lipoprotein fractions. The determining factor in the distribution of carotenoids among the different lipoprotein fractions may be the nature of the carotenoid. The less polar hydrocarbon carotenoids such as {beta}-carotene, {alpha}-carotene, lycopene and also {beta}-cryptoxanthin are primarily associated with the LDL fraction and represent 58–79% of the total amount in plasma. The more polar carotenoids, including zeaxanthin and lutein, are distributed more or less equally between LDL and HDL (Romanchik et al., 1995Go; Ziouzenkova et al., 1996Go). As a consequence of this, non-polar carotenoids are present to a lower extent compared with the others that are primarily associated with the HDL fraction. Therefore, differences in the distribution of the individual carotenoids among the lipoprotein fractions in plasma might determine the rate of transfer into follicular fluid.

It cannot be completely excluded, however, that the intrafollicular concentration of some of the investigated components might be additionally effected by other means. These effects might be a local conversion of carotenoids with provitamin A activity such as {alpha}-carotene, {beta}-carotene and {beta}-cryptoxanthin into vitamin A or carotenoids and {alpha}-tocopherol might serve as antioxidants by quenching of free radicals produced during the metabolism of follicle cells (Sies and Stahl, 1995Go). Lower fertilization rate in women who smoke has been linked to lower levels of carotenoids in plasma and follicular fluid (Palan et al., 1995Go).

It is now well established that vitamin A and its active derivates is essential for development of the mammalian embryo (Ross et al., 2000Go). Both vitamin A deficiency as well as excess results in congenital defects in the embryo. Thus, the transport and metabolism of retinol and its derivates, the retinoids, is tightly regulated by the retinoid binding proteins (Noy, 2000Go). In plasma, the 1:1 molar binding of retinol to RBP ensures the regulated supply of vitamin A in the adult body as well as in the embryo. Approximately 95% of RBP-retinol (holo RBP) is bound to the 55 kDa TTR, that prevents holo-RBP from glomerular filtration and catabolism in the kidney (Blaner, 1989Go). In our study, we demonstrated the presence of RBP and TTR in plasma as well as follicular fluid using SDS–PAGE-immunoblotting analysis. Recently, the detection of RBP and TTR as components of human follicular fluid was also shown by high resolution 2D PAGE (Anahory et al., 2002Go). Therefore, both proteins are probably involved in the transport of retinol into the follicular fluid. Additionally, the expression of RBP has been shown in follicular structures of the bovine ovary, indicating its possible involvement in retinol transport across the basement membrane (Brown et al., 2003Go).

Although we were not able to find any differences in the concentration of carotenoids, retinol and {alpha}-tocopherol in plasma and follicular fluid between women with different fertilization successes, a striking observation was the variability of the FF/P ratio of the investigated components in follicular fluid of women in which no pregnancy was established was double the one observed in those women in whom IVF treatment resulted in pregnancy. This would be supported by the observation that due to the molecular sieve of the blood-follicular fluid barrier in healthy follicles only HDL are present (Jaspard et al., 1996Go). In cases of follicle and oocyte hypermaturity, however, VLDL and LDL particles can be detected. This indicates a disturbance of the molecular sieve causing differences in steroid metabolism that might negatively effect oocyte development (Volpe et al., 1991Go).


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
It might be concluded that the differences in the distribution of the individual carotenoids among the lipoprotein fractions in plasma and the differences in the decrease of individual carotenoids in follicular fluid, may support the hypothesis that differences between plasma and follicular fluid are primarily due to the selective transfer of carotenoids from plasma into follicular fluid. It cannot be excluded, however, that carotenoids and {alpha}-tocopherol in follicular fluid serve important functions as potent antioxidants and/or as local precursors of retinoids, which might be of significance for the maturation and the development of follicular structures and the oocyte. Greater variation in the transfer rate of carotenoids in women with no reproductive success might indicate a disturbed sieving effect of the blood-follicular barrier of yet to be described consequences for the carotenoid and retinoid metabolism of follicles and thus possibly for the maturation of the oocyte.


    Acknowledgements
 
We thank A.Hurtienne and E.Pilz for their analytical support. The study was supported by Deutsche Forschungsgemeinschaft (DFG – Innovationskolleg 26, TP12).


    References
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 Discussion
 Conclusions
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Submitted on January 20, 2003; accepted on February 26, 2003.