1 Département de Médecine de la Reproduction, Hôpital Edouard Herriot, place d'Arsonval, 69373 Lyon cedex 08, 2 INSERM U418, Hôpital Debrousse, 29 Rue Soeur Bouvier, 69322 Lyon cedex 05, 3 Centre Commun de Quantimétrie, 8 avenue Rockefeller, 69373 Lyon cedex 08 and 4 Laboratoire de Virologie, Faculté de Médecine, Université Joseph Fourier de Grenoble, Avenue Gresivaudan, 38706 La Tronche, France
5 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France. Email: mehdi.benchaib{at}sante.univ-lyon1.fr
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Abstract |
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Key words: flow cytometry/IVF/pregnancy/sperm DNA fragmentation/sperm DNA methylation
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Introduction |
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After zygote formation, the parental alleles maintain their identity so that one allele eventually becomes preferentially expressed. Methylation of CpG dinucleotides is proposed to be one mechanism for differentially marking the parental chromosomes, since methylation can be stably inherited in somatic cells yet can be removed and reset in the next generation according to the parent of origin (Razin and Cedar, 1994; Jaenisch, 1997
). The developmental stages prior to blastocyst formation are of particular importance in genomic imprinting (Solter, 1998
). Genome-wide, oocyte DNA tends to be hypomethylated while sperm DNA tends to be hypermethylated (Monk et al., 1987
). During preimplantation development, the overall level of methylation decreases. Most methylation moieties present on the original parental chromosomes are removed from the DNA by the morula stage, giving rise to a predominantly unmethylated genome which remains this way at least through blastulation. A wave of de novo methylation follows, leading to an overall increase in genome methylation levels as the newly implanted embryo develops and differentiates. Disruption of global methylation patterns is lethal to mammals (Li et al., 1992
).
Because male and female pronuclei do not exhibit the same evolution during the first stages post-fertilization (Bouniol-Baly et al., 1997; Haines et al., 2001
), the methylation level of male and female gametes may influence the development potential of embryos (Mayer et al., 2000
). Thus abnormal methylation level in one or other gamete could explain some implantation failures whereas these gametes were apparently normal. We had evoked this deleterious effect concerning abnormal methylation level in male gametes in a previous work (Benchaib et al., 2003a
), on a small group of patients. The influence of sperm DNA methylation on pregnancy was shown on mice by use of 5-aza-2'-deoxycytidine, which incorporated into DNA and led to decreased DNA methylation (Kelly et al., 2003
). Moreover, in altered sperm the imprinting genes seemed to be more altered (Marques et al., 2004
).
Recently the conditions of assisted reproduction techniques were suspected of increasing the frequency of some pathologies in relation to genomic imprinting in, for example, Angelman syndrome and BeckwithWiedemann syndrome (Gosden et al., 2003). One explanation could be the loss of gene imprinting during preimplantation development under certain culture conditions (Mann et al., 2004
). Thereby, the methylation status of the paternal genome may represent an important factor.
The aim of the present study was to assess the impact of sperm methylation level in IVF success, in terms of both fertilization and pregnancy rates. This procedure involves immunostaining of m5c and its quantification by flow cytometry, which together provide an objective estimation of global DNA methylation.
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Materials and methods |
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Ovarian stimulation
After 3 weeks of desensitization by GnRH analogues (Decapeptyl®; Ipsen), ovarian stimulation was achieved by recombinant FSH (Gonal F®; Serono; or Puregon®; Organon), and monitored by endovaginal echography and plasma estradiol. When the follicles reached a correct diameter, 36 h before oocyte retrieval, 10 000 IU of hCG (Organon) was administered. The oocyte retrieval was carried out under general anaesthesia by a vaginal ultrasonographic-guided aspiration.
Sperm preparation for assisted reproduction
Sperm were prepared using a discontinuous PureSperm® gradient (Nicadon, Sweden). The gradient consisted of three layers of 1 ml of PureSperm: 90, 70 and 50%. On the 50% layer was deposited 1 ml of semen. The gradient was then centrifuged at 300 g for 20 min. After centrifugation, the 90% layer was collected and washed with 5 ml of Ferticult Flushing medium (FertiPro N.V., Belgium) at 600 g for 10 min. The pellet was then resuspended in IVF Medium (Scandinavian IVF, Sweden). The viability in selected sperm was >90% in all the samples.
Embryo culture and classification
Sixteen to 18 h after insemination or microinjection, the oocytes were assessed for fertilization two-pronuclear stage. Forty-eight hours after oocyte retrieval, the embryos were classified according to their morphology. Classification was as follows: grade A, no fragmentation and four regular cells; grade B, <25% fragmentation; grade C, between 25 and 50% fragmentation; grade D, >50% fragmentation (Ebner et al., 2001). The transfer of the embryos took place either at 48 h, or at 72 h, or at the blastocyst stage. When the transfer was made at 48 or 72 h, the supernumerary embryos were cryopreserved if their morphological states allowed it (grade A or B). If they were not cryopreserved, they were cultivated in sequential medium until the blastocyst stage was reached, and if one or more good quality blastocysts were obtained, those were then cryopreserved. When a transfer at the blastocyst stage was programmed, embryos were cultivated on sequential medium: P-1 Medium (Irvine Scientific, USA) for the first 2 days, Blastocyst Medium (Irvine Scientific) for the last days of culture. After the transfer, the remaining good morphology blastocysts were cryopreserved. A clinical pregnancy was assessed by the succession of three positive plasma
-hCG determinations and ultrasound detection of a fetal heart beat.
Semen sample preparation for DNA methylation study
The detection of DNA methylation level was performed on the spare sperm suspension that was used for the assisted reproduction procedure (the volume was between 50 and 60 µl of selected motile sperm suspension). All patients had previously given their informed consent for the study.
The first protocol described was changed in order to allow an immunostaining with suspension cells (Benchaib et al., 2003a). For fixation, ethanol (70%) (Merck, Germany) at 20;°C for 20 min was used. Cell pellets were washed twice in phosphate-buffered saline with Tween 0.5% (Sigma, USA) (PBS-T) for 5 min at 500 g. For sperm DNA decondensation, cells were incubated at room temperature in 1 mol/l hydrochloric acid (HCl)Tris buffer, pH 9.5 (Merck), containing 25 mmol/l dithiothreitol (DTT; Sigma) for 20 min. The cells were then washed twice in PBS-T.
To ensure that methylated DNA was accessible to anti-m5c antibody, the sperm DNA was denatured with HCl (6 N) for 15 min. The cell pellets were washed with Tris (1 mol/l, pH 9) (Sigma), then with PBS-T. Pellets were then incubated with mouse anti-m5c antibody (14) diluted 1:10 in PBS-T for 20 min at room temperature and washed twice with PBS-T. Controls consisted of cells incubated with buffer instead of the primary antibody.
Anti-mouse antibodies coupled with fluorescein isothiocyanate were incubated with the pellet for 30 min. The cells were then washed twice in PBS-T, and conserved at +4°C in a dark chamber until quantification with flow cytometer. The immunostaining was confirmed by the visualization of the immunofluorescence in the head of sperm with an epifluorescence microscope.
Semen sample preparation for DNA fragmentation study
For some patients (n=13), both sperm DNA methylation and DNA fragmentation using TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUDP nick-end labelling] were measured. The two analyses were performed on two different sperm preparations, but both originating from the same ejaculate. All patients had previously given their informed consent for the study. The cells were spread out over sialinized slides. Cell fixation was carried out by a methanol/acetic acid mixture (3 volumes/1 volume) for 20 min. The cells were permeabilized with PBS with 1% of Triton x100 (Sigma). Cells with fragmented DNA were revealed by TUNEL by use of the Apoptag plus Kit (Oncor, France). The cells were all balanced with the balanced buffer and then incubated in a moist chamber at 37°C, for 1 h, with the TdT solution in order to allow DNA elongation. The elongation was revealed by incubation of the cells with anti-digoxigenin antibody coupled to peroxidase, for 30 min, in a dark, moist chamber. The peroxidase was revealed with diaminobenzidine. Counterstaining of the sperm nucleus was performed with Harris's haematoxylin. A positive control was made on positive slides furnished in the Oncor Kit. The cells were observed under a transmission microscope (Zeiss, Germany) with a x100 oil objective. The sperm with fragmented DNA had their nuclei stained brown, whereas the other cell nuclei were blue-grey. On each slide, 500 cells were counted; the percentage of sperm with fragmented DNA were thus determined and the result referred to as the DNA fragmentation index (DFI).
Flow cytometry
The samples were analysed by FACSCalibur (Becton Dickinson, USA). The flow cytometer was equipped with a 488 nm excitation filter and a 530 nm emission filter (the fluorescence 1: FL1) and a 585 nm emission filter (the fluorescence 2: FL2). The values of photomultiplier were linearly set. Green fluorescence (FL1) represents the sperm DNA methylation level, expressed in arbitrary units (AU). As a negative control, sperm were immunostained without the first antibody. Sample tubes were mixed well before commencing acquisition by gently flicking the tube to ensure sample homogeneity with a low level of micro air bubbles. All sample tubes were capped to prevent evaporation. Instrument settings were adjusted so that all events (cells and debris) were observed in the dot-plot diagram. Resolution was of 1024 channels. Forward scatter channel (FSC) detector and side scatter channel (SSC) photodiode were set to linear. Some cellular and non-cellular debris were excluded by adjusting the threshold of the SSC parameter. An area delimiting the region containing the cell population to be sorted was drawn on the dot-plot diagram with the use of the polygonal region tool of the WinMDI 2.8 software (Trotter, 2000). A total of 20 000 events was analysed for each sample.
Statistical evaluation
Statistical analysis was performed with SPSS for Windows software package version 11.5 (SPSS Inc., USA). The 2-test was used to analyse the qualitative parameters. For the comparison of quantitative parameters, Student's t-test (with Levene's test) was used. Spearman's correlation coefficients were also calculated.
The optimal threshold value for the methylation level was determined as the value that permitted maximization of the relative risk (RR) of obtaining a pregnancy. The association between this threshold value and IVF parameters was then assessed. Stepwise logistic regression was performed to calculate the significant odds ratio (OR) equivalent to the RR, in order to identify the prognostic parameters in a multivariate analysis. Statistical differences were considered significant at P<0.05.
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Results |
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5-Methylcytosine immunostaining |
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In order to gauge the accuracy of the technique, the measurements were repeated twice for six patients and the coefficient of variation (CV) was calculated. This was <10% for all cases.
Methylation and sperm parameters
No association was found between methylation level and sperm characteristics (concentration, motility, morphology) (Figure 1). No association was found between sperm DNA methylation level and DFI (r=0.45; not significant) (Figure 1).
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The mean age of the women was 34.2±4.3 years. Tubal alterations were found in 41.2% of the cases, and dysovulation in 17.5% of the cases; 22.2% of the women suffered from endometriosis.
The mean age of men was 35.4±5.5 years. In 19.1% of the cases, the sperm used for the assisted reproduction procedure presented minor anomalies according to World Health Organization (1999) standards. Moderate oligozoospermia (1520x106/ml) was found in 14.3% of cases, moderate asthenozoospermia (2025% grade a=b motility) in 4.8% of cases, and no teratozoospermia was found. These anomalies were isolated. The mean value of sperm DNA methylation was 581±83 AU.
Methylation and fertilization rate
The fertilization rate was not correlated to the sperm DNA methylation level (r=0.1, not significant). Moreover, this fertilization rate was similar whether DNA methylation level was below or above an arbitrary level of 555 AU threshold value (83.5 and 82.7% respectively) (Figure 2).
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Methylation and pregnancy
Sperm DNA methylation level was 607.7±69.8 and 573.4±86.8 AU for cycles with and without pregnancies following embryos transfer respectively. This difference was not statistically significant. With a threshold value equal to 555 AU, the RR of pregnancy was 1.38 (P<0.05). The pregnancy rates were 8.3% (2/24) and 33.3% (13/39) for sperm DNA methylation <555 and >555 AU respectively (P<0.05) (Figure 3).
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Discussion |
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In this study, we chose to include samples with normal or few altered sperm characteristics according to the World Health Organization standards (i.e. 40% total motility). This is why ICSI was never necessary; thus the group was relatively homogeneous regarding both sperm characteristics and the assisted reproduction technique, represented by conventional IVF. Sperm chromatin methylation analysis was completed using the same selected sperm sample that was used for IVF. No association was found between GML and sperm concentration or sperm motility, but as the sperm characteristics were normal or moderately altered, the results disagreed with the data of Marques et al. (2004), who found a relationship between alteration of gene imprinting and severe oligozoospermia. As sperm DNA fragmentation impacted on pregnancy in assisted reproduction treatment (Benchaib et al., 2003b
; Larson-Cook et al., 2003
), the DNA fragmentation was measured together with GML in a group of 13 patients. This comparison was important because one had to verify that methylcytosine staining did not simply reflect another estimation of DNA packaging that would decrease considerably its interest. However, the lack of correlation between DFI and methylation could be due to the low number of subjects. Figure 1 shows a tendency to negative correlation: one could hypothesize that some sperm with a hypomethylated DNA could be more sensitive to DNA fragmentation. As no relationship was found between GLM and DNA fragmentation, these indices could represent two different sperm factors. Moreover, as discussed earlier, GML is not correlated to sperm characteristics, whereas some studies have found significant correlation between sperm characteristics and DFI (Benchaib et al., 2003b
; Larson-Cook et al., 2003
). So GML seems to be an interesting factor that brings new information concerning DNA quality of sperm.
As observed in our preliminary study (Benchaib et al., 2003a) this analysis could predict the pregnancy outcome: the pregnancy rate was significantly higher for GML above an arbitrary threshold value. On the contrary, sperm GML was not correlated with either the fertilization rate or with the quality of embryos. In fact, the events that are involved in the fertilization process, the embryo quality and the embryo development are all different. Normally, sperm DNA quality would not be expected to play a role in oocyte activation process or in early development, since it is assumed that in humans the first 23 days of development (until the 68-cell stage) are mainly controlled by maternal transcripts. However, this assessment can be nuanced since in some cases, sperm characteristics are so poor that one can expect that all sperm functions are altered, leading to disturbances of both fertilization (by IVF) and embryo development (if ICSI is performed). The relationship between a low sperm GML and an abnormal embryo development is complex. It can be assumed that in the case of sperm DNA hypomethylation, some genes are not repressed, as they normally should be, thus the embryo genome expression shows some degree of assynchronism. This global hypomethylation may also alter the process of cell differentiation, as observed in neoplasic cells (Piyathilake et al., 2003
).
It could be argued that the zygotic paternal genome is demethylated under oocyte control (Mayer et al., 2000), so the status of the male genome methylation is not as important. However, it has been recently shown that embryo development failure could be related to aberrant methylation patterns observed at the 2-cell stage and originating from gamete DNA (Shi and Haaf, 2002
). Although they are not transduced, it is possible that the Alu or L1 repeated sequences, of which methylated cytosines are an important constituent (Yoder et al., 1997
), may play a role in the expression of genes involved in cell differentiation as postulated by Mayer (2000).
In conclusion, our data show that the global status of sperm DNA methylation does not influence the fertilization rate but does influence embryo development, which is impaired if global DNA methylation level is below a threshold value.
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Acknowledgements |
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References |
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Submitted on July 26, 2004; resubmitted on October 10, 2004; accepted on November 24, 2004.
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