1 IVF Centre, Department of Reproductive Medicine, 2 Department of Clinical Chemistry, Academic Hospital Vrije Universiteit, de Boelelaan 1117, 1081 HV, Amsterdam, 3 Department of Clinical Epidemiology and Biostatistics, University Medical Centre, University of Amsterdam, Amsterdam, The Netherlands, 4 Andrology Laboratory E3, Reproductive Biology Unit, Department of Obstetrics and Gynaecology, Tygerberg Hospital and University of Stellenbosch, Tygerberg 7505, and 5 Biostatistics, MRC, Cape Town, South Africa
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Abstract |
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Key words: fertilization failure/IVF/pregnancy/spermatozoa/TRAP
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Introduction |
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Before starting this expensive, time-consuming and often stressful treatment, considerable effort should be devoted to identifying those patients who can actually benefit from IVF treatment. This policy should lead to stricter inclusion criteria, resulting in a reduction of the number of cancelled IVF treatment cycles due to total fertilization failure (TFF). In order to do this, clinicians require variables that are simple to measure, minimally invasive and inexpensive, yet have reliable predictive value.
Traditional semen variables such as sperm count, motility and morphology, as described in the World Health Organization (WHO, 1987) manual, are unreliable predictors of a successful outcome in either assisted reproduction treatment, IVFembryo transfer or intracytoplasmic sperm injection (ICSI) (Vawda et al., 1996). As a result, alternative tests of sperm function have been developed, ranging from the zona-free hamster oocyte penetration assay to computer- assisted semen analyses (CASA). Most of the recently described tests have proved to be either too complicated, too time-consuming or too unreliable for routine use (Biljan et al., 1996
). In contrast to these complicated tests, a recent study cites the prognostic value of the number of progressively motile spermatozoa following Percoll separation when predicting IVF outcome (Hammadeh et al., 1997
).
Sperm morphology, as evaluated by the WHO criteria (WHO, 1992), is a classical semen parameter. Recently, it has been adjusted to conform with the strict Tygerberg criteria as introduced by others (Menkveld et al., 1990). This was because the original WHO (World Health Organization, 1987
) morphology criteria had only a limited predictive value for oocyte fertilization in vitro (Duncan et al., 1993
). Using strict criteria for the evaluation of sperm morphology, it was shown (Kruger et al., 1988
) that there was suboptimal fertilization when normal sperm morphology was
14%. The lowest levels of fertilization were observed at values of <4%.
However, this view was not universally shared. As for morphological assessment, the stricter criteria for normal sperm morphology were reported to be lacking in accuracy. Oocyte fertilization and pregnancy rates in a group of men with 14% normal morphology were not significantly different from those in a group with >14% normal morphology (Morgentaler et al., 1995
). In addition to the strict criteria, other modified morphological parameters have been developed, such as cytoplasmic residues (Gomez et al., 1996
) and acrosome index (AI) (Menkveld et al., 1996
). Semen samples containing a low percentage of spermatozoa with intact acrosomes were also associated with TFF (Chan et al., 1999
).
A high cytoplasmic residue score seems to reflect an important factor in the development of oxidative stress-related pathology (Gomez et al., 1996). It appears that the disruption of spermatogenesis leads to the retention of excess residual cytoplasm by the differentiating spermatozoa. This in turn leads to enhanced reactive oxygen generation causing peroxidative damage that results in loss of sperm function through effects on the fluidity and integrity of the plasma membrane. As a consequence, the spermatozoa cannot participate in the membrane fusion events associated with fertilization (Rao et al., 1989
; Gomez et al., 1996
).
Recent studies have indicated that the aetiology of impaired fertility can be ascribed to reactive oxygen species (ROS) originating either from certain sperm cell populations or from leukocytes (Aitken et al., 1992; Kovalski et al., 1992
; Smith et al., 1996
). Although the importance of seminal plasma in the protection of spermatozoa is well known (Kovalski et al., 1992
), only a few studies have properly investigated its antioxidant properties (Lewis et al., 1995
; Smith et al., 1996
; Jozwik et al., 1997
; Rhemrev et al., 2000
), particularly in relation to low-molecular weight antioxidants such as vitamin C and uric acid (Thiele et al., 1995
; Lewis et al., 1997
).
Some studies showed an impaired, non-enzymatic antioxidant capacity in the seminal plasma of infertile men, while others found an erratic distribution between fertile and non-fertile populations (Lewis et al., 1995; Smith et al., 1996
; Jozwik et al., 1997
).
On the basis of the above-mentioned literature, several semen variables were selected to be tested in a prospective study of 87 patients attending the IVF centre, prior to their IVF treatment. This enabled these tests to be correlated with the resulting IVF and embryo transfer outcome. The roles of the AI and the cytoplasmic residues were determined and studied. Next, the total radical trapping antioxidant parameter (TRAP) of seminal plasma from the participating individuals was assessed. In addition, the concentrations of two non-enzymatic antioxidants ascorbic and uric acids were determined and related to the other semen variables and to the IVFembryo transfer outcome.
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Materials and methods |
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The population studied consisted of 87 consecutive couples undergoing IVF treatment at the IVF centre of the Division of Reproductive Endocrinology and Fertility of the Vrije Universiteit Hospital in Amsterdam. Couples from other clinics participating in our transport IVF programme were also enrolled in this study. Participating clinics used male and female inclusion criteria according to the guidelines of our IVF centre. In our transport IVF programme that has run for 14 years, both ovarian stimulation and oocyte retrieval are performed elsewhere. The follicular aspirates are then transported to our IVF Centre where, following oocyte isolation, an IVF procedure is carried out. This has resulted in comparable TFF (±10%) and pregnancy rates (±19%), in both groups, during the past 5 years.
Details of the indications for IVF treatment, the definition of unexplained infertility and the stimulation schemes used have already been described in previous studies (Roseboom et al., 1995).
Laboratory investigations
Oocytes were isolated from the follicular aspirate and cultured in Petri dishes, in 40 µl droplets in Earle's medium containing 1% human serum albumin (HSA) under oil (CLB, Amsterdam, the Netherlands) at 36.8°C under 5% CO2. The semen was processed using a modified Percoll 40/90 discontinuous density gradient centrifugation (Rhemrev et al., 1989). Between 38 and 42 h after human chorionic gonadotrophin (HCG) administration, insemination was carried out using 40 000 progressively motile spermatozoa. Gametes and embryos were cultured in incubators at 36.8°C under 5% CO2 in air. Oocyte fertilization was estimated in terms of the number of pronuclei at 18 h after the moment of insemination. Oocytes with two or more pronuclei were included in the study as fertilized.
Semen analysis and preparation
The semen sample required for the IVF procedure was collected before noon on the day of ovum retrieval at the IVF centre. The semen sample was produced by masturbation, and collected in a sterile polystyrene jar. A period of at least 30 min was allowed for liquefaction. Semen analyses were performed on the basis of the WHO (World Health Organization 1992) guidelines. The volume of the semen samples was determined using sterile disposable 5 or 10 ml pipettes. Sperm counts and motility assessments were performed using Makler counting chambers at 36°C. The fresh 15 µl aliquots were examined at a magnification of x200 using phase-contrast microscopy. Motility grades were rated on a semi-subjective scale (a to d) and classified into three groups: number of progressively motile spermatozoa per ml (a+b); number of non-progressively motile spermatozoa per ml (c); and number of non-motile spermatozoa per ml (d). Based on the motility grading, the following motility variables were determined: number of progressively motile spermatozoa per ml; number of motile spermatozoa per ml; and number of non-motile spermatozoa per ml. An additional analysis of the post-wash semen sample was performed. The variable analysed in the isolated fraction was the total number of progressively motile spermatozoa (TPMCpost-wash).
The Makler chamber was used in the evaluations of concentration and motility, the accuracy and repeatability of which was in line with a recently published comparative study (Auger et al., 2000).
Several other variables were subsequently determined, namely: percentage of normal sperm morphology (`strict criteria'); percentage of spermatozoa with a normal acrosome (AI); percentage of spermatozoa with an abnormal cytoplasmic residue (`cytoplasmic residues'); the concentration of ascorbic acid (`ascorbic acid'); the concentration of dehydroascorbic acid (DHA); vitamin Ctotal (= ascorbic acid + DHA); and the concentration of uric acid (uric acid), as well as `fast TRAP' and `slow TRAP'.
Sperm morphology evaluation according to strict criteria
Sperm morphology was evaluated, using Papanicolaou-stained smears, according to strict criteria (Menkveld et al., 1990), and blinded to the IVF outcomes investigated.
Modified sperm morphology variables
In addition to the evaluation of normal sperm morphology percentages according to the strict criteria, two other sperm variables were evaluated, namely `cytoplasmic residues' and AI.
Acrosome index
Sperm acrosomal morphology was evaluated by light microscopy at the same time as the sperm morphology evaluation was carried out. The method, which has been described in detail elsewhere (Menkveld and Kruger, 1996; Menkveld et al., 1996
), is based on acrosomal size and shape, as well as on staining characteristics. The results were expressed as percentage normal acrosomes, the AI. The evaluation of acrosome morphology uses the same principles that are used in the evaluation of normal sperm morphology, according to strict criteria. For an acrosome to be regarded as normal it must have a smooth normal oval shape, with the same dimensions as those in normal spermatozoa. Acrosomes must be well defined and should comprise about 4070% of the normally sized sperm head. A spermatozoon can only be classified as normal if the acrosome is classified as normal. As with the routine sperm morphology evaluation, at least 100 spermatozoa were examined. The sperm head may have an abnormal or a normal shaped post-acrosomal region, but the other sperm regions must be strictly normal. The inclusion of sperm with abnormal post-acrosomal regions will clearly result in a higher AI value, as compared with the traditional normal sperm morphology AI value.
Cytoplasmic residues
The identification and evaluation (of size) of cytoplasmic residues on spermatozoa was performed on Papanicolaou-stained semen smears. This was carried out as part of the routine sperm morphology evaluation process described elsewhere (Menkveld and Kruger, 1996), and in accordance with WHO (1992) criteria. Morphologically normal spermatozoa must have an amount of cytoplasmic material that does not exceed the size of a normal sperm head by >30%. The cytoplasmic material is usually situated at the base of the sperm head, and may stain blue-green or red. At least 100 spermatozoa were evaluated, and the number of spermatozoa containing cytoplasmic residues was expressed as a percentage of the total number of spermatozoa evaluated (`cytoplasmic residues').
TRAP measurements
TRAP measurements were performed by means of the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS; Sigma, Zwijndrecht, The Netherlands) assay (Miller et al., 1993). In this assay, ABTS radicals are produced by oxidation with 2,2'-azo-bis(2-amidinopropane)HCl (ABAP; Polysciences Inc., Warrinton, USA). The subsequent addition of an oxygen-radical scavenger results in the dose-dependent trapping of ABTS radicals, producing a proportional decrease in absorbance. ABTS radicals were produced by mixing 10 ml 2.25 mmol/l ABTS, 10 ml 20 mmol/l ABAP and 80 ml phosphate-buffered saline (PBS) (50 mmol/l phosphate, 0.9% NaCl, pH 7.4) and incubating at 70°C for 20 min. The resulting green solution of ABTS radicals, with an absorbance of approximately 0.75 at 734 nm, was cooled on ice. TRAP measurements were performed by adding 10 µl of the diluted seminal sample to 990 µl of the ABTS radical-solution at 37°C. The decrease in absorbance at 734 nm was determined and compared with the absorbance decrease of a blank, i.e. PBS. HAMF-10 does not have a TRAP value. In order to quantify the TRAP, the vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; Aldrich, Zwijndrecht, The Netherlands), dissolved in ethanol (96%), was used as a reference compound. The decrease in absorbance upon adding 0.202.0 mmol/l Trolox (incremental increases of 0.20 mmol/l) was determined. For each 1.0 mmol/l Trolox, the mean (± SEM) absorbance decreased by 0.280 ± 0.005 units. This value was used to calculate the TRAP of seminal plasma, expressed as Trolox equivalent antioxidant capacity (TEAC). The TEAC of seminal plasma is expressed in mmol/l:
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All seminal plasma samples were assayed as 1:10 dilutions. The assay was linear over the range of dilutions tested (van Overveld et al., 2000). The decrease in absorbance was determined at t = 10 s and t = 300 s after the addition of a sample. The TEAC values calculated from the absorbance decrease at these intervals are defined as `fast TRAP' (t = 10 s) and `total TRAP' (t = 300 s) respectively (Rhemrev et al., 2000
). Additionally, `slow TRAP' was expressed by:
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Ascorbic acid, DHA and uric acid determination
Uric acid concentration (`uric acid') was determined using a colorimetric enzyme assay (Boehringer Mannheim GmbH, Mannheim, Germany). In order to estimate the concentrations of ascorbic acid (`ascorbic acid') and `DHA' (an oxidation product of ascorbic acid), seminal aliquots of the supernatant (after Percoll 40/90 gradient centrifugation) were collected and mixed with an equal volume of 5% metaphosphoric acid. The concentrations were subsequently determined according to a published method (Speek et al., 1984).
Statistical analysis
The semen variables were initially analysed as continuous variables. The TPMCpost-wash data were log-transformed to satisfy the assumptions of normality. Pearson's correlation coefficients were calculated to assess associations between the semen parameters. Scatter plots were made to assess associations between fertilization [TFF versus at least one fertilized oocyte (= `fertilization') or pregnancy (pregnant versus non-pregnant)] on the one hand, and the variables TPMCpost-wash, `strict criteria', AI, `cytoplasmic residues' and `fast TRAP' on the other hand. The cut-off points of the semen variables were found by visual examination of the scatter plots. The continuous variables were then dichotomized. Odds ratios (OR) were determined at these previously found cut-off points. Logistic regression analyses were used to assess associations between `fertilization' and pregnancy (`pregnancy') as dependent variables and the semen variables (both as continuous and dichotomous variables) as independent variables. Finally, pregnancy rates, in four defined groups were compared.
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Results |
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With regard to the cause of infertility (such as tubal occlusion, endometriosis, ovulatory disorders, sperm antibodies, or unexplained infertility) there was no difference between couples that became pregnant and those with a TFF, or those where an embryo transfer did not result in a pregnancy (data not shown).
Mean (± SD) values of the different newly described semen variables in the TFF versus the `fertilization' group and the pregnant versus non-pregnant group are shown in Tables II and III.
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An additional stepwise logistic regression analysis was performed, with `pregnancy' as the dependent variable and the previously mentioned semen variables (used as dichotomous variables at their cut-off points: `strict criteria' 2%, `cytoplasmic residues' 24%, AI 5%, TPMCpost-wash 1.1x106, `fast TRAP' 1.14 mmol/l) as independent variables.
The likelihood-ratio test showed that AI and `fast TRAP', at their specific, previously mentioned cut-off points, contributed significantly to the prediction of pregnancy (P = 0.024).
Using the previously mentioned cut-off points of the AI (5%) and the `fast TRAP' (1.14 mmol/l), it was subsequently possible to define four groups that showed clear differences in IVF-embryo transfer success (Table IV).
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Significant correlations were seen between `cytoplasmic residues' and both the total amount of vitamin C in semen samples (R = 0.234; P = 0.03) and the total amount of uric acid + vitamin C per semen sample (R = 0.235; P = 0.03). Furthermore, significant correlations were seen between TPMCpost-wash and both vitamin Ctotal (R = 0.358; P = 0.001) and `cytoplasmic residues' (R = 0.454; P = 0.0001). Finally, as shown in a previous publication (Rhemrev et al., 2000), a strong correlation was seen between `ascorbic acid', `uric acid' and `fast TRAP' (R = 0.547; P = 0.001).
The age of the women involved showed no association with either `fertilization' or `pregnancy' (data not shown).
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Discussion |
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`Fast TRAP' as a predictor of IVF-embryo transfer outcome
With respect to `fast TRAP', the current results indicate that if seminal plasma of the spermatozoa used for IVF has a `fast TRAP' <1.14 mmol/l TEAC, the spermatozoa in such a semen sample may be capable of fertilizing oocytes, but a pregnancy will rarely result. Therefore, a low `fast TRAP', as a predictor of IVFembryo transfer outcome underlines the importance of seminal plasma antioxidants in the protection of spermatozoa against oxidative stress. The effect of a compromised `fast TRAP' on IVFembryo transfer outcome can be explained by the adequate radical buffer capacity of progressively motile spermatozoa against lipid peroxidation (Rhemrev et al., 2001), showing that previous oxidative stress might not have damaged the well-protected sperm cell membranes, leaving its receptors intact and thereby maintaining the fertilizing capacity of these sperm cells. On the other hand, it may have caused DNA damage resulting in impairment and/or failure of the embryo development. Thus, whether a compromised `fast TRAP' can be circumvented by ICSI to produce a pregnancy as a result of ART, seems quite unlikely.
`Slow TRAP' did not show any relationship with TFF, nor with the occurrence of a pregnancy. In contrast, other investigators have observed a remarkable correlation between the `slow TRAP' values of various flavenoids and their physiologically relevant antioxidative properties, such as protection against microsomal-lipid peroxidation and free radical-mediated cardiotoxicity (van den Berg et al., 2000).
In the current study, no significant role could be ascribed to either `ascorbic acid', DHA or `uric acid'. These low-molecular weight antioxidants have been presumed to be important (Thiele et al., 1995; Lewis et al., 1997
) in predicting TFF and the occurrence of pregnancy. In the current study, `fast TRAP' and `slow TRAP' were determined using an improved post-addition assay. This assay is less susceptible to artefacts that might lead to misinterpretation of the measured TEAC values, than with other described methods (Miller et al., 1993
; Strube et al., 1997
, van Overveld et al., 2000
). The test can easily be automated and requires only 10 µl plasma. This simple, minimally invasive and inexpensive test is therefore a good candidate for a new routine test for use in IVF laboratories. Moreover, to test the usefulness of `fast TRAP' and `slow TRAP' in IVF, these were determined in semen samples used in IVF procedures and subsequently correlated with the resulting IVF outcomes. Aside from one other study (Jozwik et al., 1997
), no previous studies have evaluated TRAP values from groups receiving the same treatment (Lewis et al., 1995
; Smith et al., 1996
). This renders the conclusions of these other studies difficult to interpret.
The AI as predictor of IVF-embryo transfer outcome
Interestingly, the AI predicts both TFF (OR 18.3) and the occurrence of a pregnancy (OR 7.8) at a cut-off of 5%. The relationship with TFF seems clear, in that an oocyte can only be fertilized when a minimum number of sperm cells with intact acrosomes are present. The correlation between AI, the acrosin content of a semen sample and the fertilization rate in IVF, that is mentioned in the literature (Menkveld et al., 1996) suggests a functional relationship between sperm morphology and some of the major events taking place in the fertilization pathway. The significant correlation of the acrosomal status (evaluated either by the spermac stain procedure (Chan et al., 1999
) or by the method described earlier (Menkveld et al., 1996
) with fertilization in vitro has already been reported in the literature. In line with these studies, we consider that in order to prevent TFF in IVF, couples may benefit from ICSI where there is an AI <5%, using our AI criteria, to circumvent this compromised fertilization pathway. To the best of our knowledge, the correlation between AI and pregnancy outcome from IVF seen in the current study has never been reported previously. Nonetheless, it remains to be proven whether an ICSI procedure in case of an AI <5% will result in higher pregnancy rates.
TPMCpost-wash predicts TFF in IVF
No predictive value could be attributed to TPMCpost-wash and the occurrence of a pregnancy following an embryo transfer, as reported previously (Bollendorf et al., 1996). In contrast, TFF in IVF was best predicted by TPMCpost-wash. It must be taken into account that the sample size is small, i.e. 11 patients in the TFF group versus 76 in the fertilization group, and therefore less suitable for establishing the differences in contribution to the prediction of TFF by the newly described semen variables using logistic regression analysis. Therefore, the current study should be a marker for possible further research.
Determination of TPMCpost-wash was performed using Makler counting chambers. In our laboratory, an overestimation of 25 ± 12% was foundan effect also reported elsewhere in the literature (Seaman et al., 1996). An adjustment of TPMCpost-wash thresholds is therefore required before this procedure is adopted by other laboratories, depending on their own sperm counting procedures. Centres that do not use Makler counting chambers but use, for example, the WHO-favoured Improved Neubauer haemocytometer (WHO, 1992), may have to use a lower cut-off value taking into account this 25% overestimation.
Thus, after adequate adjustment of these thresholds, as mentioned above, we would recommend that `TPMCpost-wash' can be used as a predictive semen variable when counselling infertile couples about the most appropriate ART, i.e. IVF or ICSI. Given that concerns have recently been expressed about injecting genetically defective spermatozoa, conventional IVF remains the preferred approach when appropriate (Johnson, 1998). Thus, the possibility of ICSI should be proposed only where there is an unacceptably high chance of TFF. ICSI is known to be an adequate therapy for circumventing fertilization disorders caused by low sperm cell counts (Lundin et al., 1997
).
The important role of TPMCpost-wash cannot be ascribed to a forced lower insemination concentration. Indeed, in most cases enough sperm cells were available for fertilization of the oocytes, i.e. 40 000 per inseminated droplet of 40 µl. Therefore, we consider that the difference in TFF will be caused by the quality of the population used for insemination. These findings are in line with published findings (Aitken et al., 1993) which showed that semen samples with high yields of post-wash progressively motile sperm cells (TPMCpost-wash) possess a low potential for lipid peroxidation, good 24 h survival, and a higher level of spermatozoonoocyte fusion than samples that produce a low yield of progressively motile sperm cells. Consequently, we consider that the Percoll washing procedure is a semen quality test, with TPMCpost-wash as its outcome. Thus, the quality of the spermatozoa inseminated is to a large extent dependent on the quantity of progressively motile spermatozoa isolated by the selection procedure.
Sperm morphology according to strict criteria as a predictor of outcome of IVFembryo transfer
No predictive value could be attributed to sperm morphology for the occurrence of a pregnancy after an embryo transfer procedure. According to one meta-analysis (Coetzee et al., 1998), `strict criteria' showed a significant positive predictive value for `pregnancy' outcome. Other investigators (Aziz et al., 1996
; Figueiredo et al., 1996
; Lundin et al., 1997
), in agreement with the current study, found no significant positive predictive value for `pregnancy' outcome.
Among men whose semen samples show normal morphology <2%, the odds of TFF were substantially higher than in male patients with a normal morphology score 2%. We would therefore suggest that males with a normal morphology score <2% should be offered ICSI, which is known to be a more effective therapy than IVF for individuals with such extremely teratozoospermic semen samples (Lundin et al., 1997
). The validity of the strict criteria used routinely in laboratories remains a matter of debate, this being mainly due to the large variations between results obtained by individual technicians, and which result from the highly subjective nature of this assessment method (Liu and Baker, 1992
). The best results with this method are generally obtained at centres with a reputation for expertise in the assessment of this particular variable (Kruger et al., 1988
; Aziz et al., 1996
; Vawda et al., 1996
; Coetzee et al., 1998
). In order to circumvent this problem in the current study, the sperm smears were evaluated by a recognized expert (R.M.).
Cytoplasmic residues as a predictor of IVF outcome
Unlike previous studies, the current study has correlated `cytoplasmic residues' with `pregnancy' outcome. No predictive value could be attributed to the percentage of cytoplasmic residues and the occurrence of a pregnancy after an embryo transfer procedure.
Whereas, in the current study, for males with 24% of spermatozoa showing cytoplasmic residues, the odds of TFF occurring were 25-fold lower than for males where >24% of spermatozoa had cytoplasmic residues (OR 0.04; 95% CI 0.00.2%). Therefore, if an adequate evaluation of the presence of `cytoplasmic residues' indicates the likelihood of an unacceptable TFF, we would suggest that an ICSI procedure be performed. This conclusion does conflict partly with some reports in the literature, as different studies show abnormalities of the mid-piece to be both negatively related and unrelated to the fertilization rate (Sukcharoen et al., 1995
; Lim et al., 1998
). The predictive power of `cytoplasmic residues' for TFF (Sukcharoen et al., 1995
; Lim et al., 1998
) is very suggestive of ROS involvement in sperm fertilizing potential. This is emphasized by the clear correlation between `cytoplasmic residues' and both vitamin Ctotal and `TPMCpost-wash'. A correlation has also been shown between the presence of excess residual cytoplasm in the mid-piece and enhanced generation of ROS (Gomez et al., 1996
), while others demonstrated a high lipid peroxidative potential in such cells (Rao et al., 1989
). These findings suggest that incomplete extrusion of germ cell cytoplasm during spermatogenesis causes a loss of cell function associated with the induction of oxidative stress. This oxidative stress may damage receptors on the sperm cell membrane, thereby impairing its fertilizing capacity. ICSI may therefore circumvent this compromised fertilization pathway, although its effect on subsequent pregnancies remains to be proven.
In conclusion, TFF was significantly predicted by TPMCpost-wash, `strict criteria', AI and `cytoplasmic residues' (all P < 0.05). AI and `fast TRAP' predict the occurrence of a pregnancy after IVF-embryo transfer. In samples with an AI <5% and a `fast TRAP' <1.14 mmol/l, no pregnancies occurred after IVF.
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Notes |
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6 Present address: Department of Obstetrics and Gynaecology, Bronovo Hospital, Bronovolaan 5, 2597 AX, The Hague, The Netherlands
7 To whom correspondence should be addressed. E-mail: j.vermeiden{at}azvu.nl
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References |
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Submitted on January 15, 2001; accepted on May 16, 2001.