The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, VA 23507-1627, USA
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Abstract |
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Key words: acridine orange/DNA fragmentation/DNA stability/intrauterine insemination/sperm
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Introduction |
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The overall success of IUI varies, with pregnancy rates between 5 and 66% per cycle (Allen et al., 1985). Several prognostic factors for IUI outcome have been proposed, including the age of the woman (Campana et al., 1996
; Stone et al., 1999
; Hendin et al., 2000
), endometrial thickness and follicle number by the time of ovulation (Tomlinson et al., 1996
; Stone et al., 1999
; Khalil et al., 2001
), aetiology and duration of infertility (Tomlinson et al., 1996
; Hendin et al., 2000
; Khalil et al., 2001
), presence and type of ovarian stimulation (Khalil et al., 2001
), time and number of inseminations (Silverberg et al., 1992
; Ragni et al., 1999
; Khalil et al., 2001
), percentage of sperm with normal morphology (Lindheim et al., 1996
; Ombelet et al., 1997
), type and percentage of sperm motility (Tomlinson et al., 1996
; Shulman et al., 1998
; Stone et al., 1999
; Hendin et al., 2000
) and total number of motile sperm inseminated (van der Westerlaken et al., 1998
; Khalil et al., 2001
).
We planned this prospective study to investigate whether the degree of sperm DNA fragmentation and stability, as well as conventional semen parameters, can predict IUI outcome. Sperm DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL), which identifies sperm with fragmented DNA. In addition, stability of sperm DNA was tested by acridine orange staining under both acid and acid + heat denaturing conditions.
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Materials and methods |
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Cycle management
Semen was obtained from male partners of couples who were undergoing IUI for treatment of infertility. Only couples who agreed to participate and signed a consent form were included in the study, which comprised a total of 154 cycles out of 119 couples. Cycles were either natural or stimulated. Clomiphene citrate and/or gonadotrophins (FSH or hMG) were used for controlled ovarian stimulation. For clomiphene citrate-stimulated cycles, 100 mg clomiphene citrate was given between days 3 and 7. For clomiphene citrate + gonadotrophin stimulation, 100 mg clomiphene citrate was given between days 3 and 7, followed by 150 IU of gonadotrophins added by day 9. For cycles managed by gonadotrophins only, stimulation was started on day 3 with 75150 IU daily. Follicle maturation was monitored by serial transvaginal ultrasonography and plasma estradiol (E2) levels. Timing of spontaneous pre-ovulatory LH surge was monitored by urine LH kits in 24 cycles, whereas the remaining 130 received either 10 000 IU of hCG or 250 µg of recombinant hCG when the diameter of leading follicle(s) was >18 mm. A single IUI was performed 36 h after hCG injection. Luteal phase was supported by daily vaginal administration of 50 mg progesterone suppositories. Plasma ß-hCG levels were measured routinely, 2 weeks after IUI. Clinical pregnancy was defined as transvaginal ultrasonographic visualization of intrauterine gestational sac(s).
Semen analysis and handling for IUI
All semen parameters were evaluated for every ejaculate, i.e. for couples who underwent more than one cycle, every single sperm parameter (including morphology and DNA parameters) was assessed for that given ejaculate. Semen samples were allowed to liquefy for 30 min at 37°C, followed by assessment of sperm parameters. Sperm concentration and motion parameters were assessed using the HTM-IVOS semen analyser (version GS 771; Hamilton Thorne Research, Beverly, MA, USA) and manually monitored (Oehninger et al., 1990). Motion parameters were examined by mixing the sperm suspension and loading a 5 µl aliquot into a Makler chamber. The chamber was then transferred to the HTM, where it was maintained at 37°C for 2 min before starting data collection, which was conducted on randomly selected fields. Sperm morphology was assessed using strict criteria after slide staining with Diff-Quik (Dade AG, Dudinger, Switzerland).
Sperm in samples containing round cells <1x106/ml (n = 123) were washed in human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) supplemented with 0.2% human serum albumin (HSA; Irvine), centrifuged at 400 g for 10 min and re-suspended with 0.6 ml HTF + HSA. On the other hand, sperm in samples containing 1x106/ml round cells (n = 31) were isolated by density gradient separation by centrifuging (DGC, by a single layer of 90% ISolateTM) at 400 g for 20 min. Purified populations of highly motile sperm were recovered, washed in HTF + HSA as described above and re-suspended with 0.6 ml HTF + HSA. An aliquot of 100 µl from the final suspension was taken for acridine orange staining and TUNEL assay. The remaining 500 µl was used for insemination.
Acridine orange staining
For acridine orange staining, two smears were prepared from each sample. The smears were air-dried and then fixed overnight in Carnoys solution (methanol:acetic acid, 3:1). Once air-dried again, one slide was stained for 5 min with freshly prepared acridine orange stain (0.19 mg/ml). The remaining slide was stained similarly after being incubated in tamponade solution (80 mmol/l citric acid + 15 mmol/l Na2HPO4, pH 2.5) at 75°C for 5 min to induce heat-provoked DNA denaturation. Coverslips were then applied and sealed. Slides were evaluated on the same day using a fluorescence microscope (490/530 nm excitation/barrier filter; Nikon, Tokyo, Japan). In all, 300 sperm per smear were evaluated at a magnification of x1000. The duration of evaluation was limited to 40 s per field. Sperm with normal DNA content displayed a distinct green fluorescence whereas sperm with an abnormal DNA content emitted fluorescence in a spectrum varying from yellowgreen to red. The spectrum of fluorescence other than distinct green colour was classified as abnormal, since DNA denaturation had begun. Preincubation percentage of green cells was calculated and referred to as preincubation acridine orange staining (AOpre) as previously defined (Tejada et al., 1984). Acridine orange score (AOS) was thereafter calculated as the absolute value after subtracting post-incubation percentage of green cells (AOpost) from preincubation percentage of green cells for each pair of smears (Duran et al., 1998
). It was noted that some samples exhibited an increased ratio of sperm with native DNA after heat treatment, possibly because of renaturation of already denatured DNA. The intra-observer and inter-observer variability was <7 and <10% respectively.
TUNEL
For TUNEL assay, we used the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH, Mannheim, Germany) that uses fluorescein-dUTP to label sites of DNA fragmentation according to the manufacturers instructions and as published (Duru et al., 2000, 2001
). Final suspensions of sperm were fixed with 4% paraformaldehyde and were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. This was followed by incubation in the dark at 37°C for 1 h in TUNEL reaction mixture containing 0.5 IU/µl of calf thymus terminal deoxynucleotidyl transferase, fluorescein-dUTP and propidium iodide. Negative (omitting the enzyme terminal transferase) and positive (using deoxyribonuclease I, 1 mg/ml for 20 min at room temperature) controls were performed in each experiment. A total of 300 cells was randomly analysed per slide. Each sperm was assigned to contain normal (red nuclear fluorescence, due to propidium iodide) or fragmented DNA (intense green nuclear fluorescence). Final percentage of sperm with fragmented DNA is referred to as TUNEL (%). The intra-observer and inter-observer variability was <8 and <7% respectively.
Statistical analysis
Statistical analysis was performed using SPSSTM 9.0 on a personal computer. Parametric (Students t-) and non-parametric (Mann Whitney U-) tests were applied to compare variables as appropriate. Pearson correlation coefficients were calculated for several parameters. Several logistic regression models were built to evaluate the contribution of various parameters to the outcome, using different sets of data.
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Results |
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Significant correlations between various sperm-related parameters are listed in Table II. Of those, correlations between morphology and DNA parameters are noteworthy.
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Various sperm parameters of cycles that resulted in conception were compared with those of failed cycles. Some of these parameters are listed in Table III. Briefly, in cycles resulting in pregnancy, the degree of DNA fragmentation after preparation was significantly lower than in those that did not result in pregnancy (7.3 ± 3.5 versus 13.9 ± 10.8 respectively, P = 0.044). No other sperm parameter exhibited a heterogeneous distribution among cycles likewise, although AOpost tended to be higher for cycles resulted in pregnancy.
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Discussion |
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Apoptosis is a major regulatory mechanism during normal spermatogenesis in several species, including humans (Hikim et al., 1995; Lue et al., 1997
; Hikim et al., 1998
). Unlike rat, mouse or hamster testes, human testes exhibit spontaneous occurrence of germ cell apoptosis involving all three classes of germ cell, including spermatogonia, spermatocytes and spermatids (Sinha Hikim and Swerdloff, 1999
). There is evidence showing that the apoptotic machinery is also present in later stages of germ cell development, including sperm. Fas receptors have been demonstrated on ejaculated sperm of both fertile and oligozoospermic samples, the latter showing a higher ratio of positivity (Sakkas et al., 1999
). Furthermore, we have shown that caspase-3 is also present in ejaculated sperm, in relatively small amounts compared with leukocytes (Weng et al., 2002
). However, the presence of the apoptotic machinery does not necessarily mean that it is active and efficient. Besides, it is less likely for apoptosis to be initiated or advanced in mature sperm, because of its unique cytoplasmic and nuclear organization. Nevertheless, these findings suggest that there may at least be an abortive apoptotic process, which extends until the latest stage of spermatogenesis. Fragmented DNA in some sperm may be an indicator of such an abortive apoptotic process.
It has been demonstrated that ROS can lead to oxidative stress in sperm. White blood cells in the ejaculate, as well as immature germ cells and mature sperm, may contribute to production of ROS (Krausz et al., 1992; Ollero et al., 2001
). Independent of the origin of ROS, it is now well established that they cause fragmentation of DNA in sperm (Lopes et al., 1998
; Barroso et al., 2000
; Duru et al., 2000
). Given this fact, it is not surprising to find a correlation between the concentration of round cells and DNA fragmentation in our findings. It is also reflected as a higher percentage of DNA fragmentation among samples prepared by DGC, where the selection of preparation was entirely based upon round cell concentration in semen. Several other researchers have also reported the presence of negative correlations between the extent of sperm DNA fragmentation and morphology, as well as parameters related to motility similar to those we found (Taylor et al., 1999
; Muratori et al., 2000
; Ramos and Wetzels, 2001
; Zini et al., 2001
). The correlation we found between the extent of DNA fragmentation and denaturation, however, is not as high as the ones that have been reported (Sailer et al., 1995
; Aravindan et al., 1997
; Zini et al., 2001
). This may be due to the methodology used, as flow cytometry is more accurate than microscopic evaluation of acridine orange-stained slides. No influence of AOS on IUI outcome has been detected, although the mean AOpost (percentage of sperm with heat- and acid-resistant DNA) tended to be lower among cycles that resulted in failure, than those that resulted in pregnancy (Table III
).
Several studies have shown that sperm DNA quality had robust power to predict fertilization in vitro (Sun et al., 1997; Duran et al., 1998
; Larson et al., 2000
; Chan et al., 2001
). In a recent report, the only parameter that showed a significant difference between pregnant and non-pregnant groups by IVF was the percentage of sperm with DNA damage after preparation, as assessed by in-situ nick translation. It was significantly higher in those patients that did not establish a pregnancy (Tomlinson et al., 2001
). Furthermore, sperm-derived effects have recently been reported to condition human embryo development (Tesarik et al., 2002
). The authors designed the study as a shared donor oocyte programme and included males, whose sperm yielded poor zygote quality consistently in previous attempts. They compared them with control males, who proved normal zygote quality in preceding cycles, with a similar semen sample in terms of basic semen analysis. Zygotes obtained from two consequent ICSI cycles using the sperm of those males with a poor history of zygote quality (patient group) had a significantly lower quality, when compared with the controls, who shared oocytes from the same donor. This difference in zygote quality was also followed by poor embryo quality, cleavage and implantation rates, in the patient group. Although the origin of this paternal effect needs further clarification, sperm DNA quality is clearly one of the possible candidates.
In addition, to predict fertilization in vitro, sperm DNA stabilityas assessed by sperm chromatin structure assay (SCSA)has been documented as a powerful diagnostic and prognostic tool in a human fertility clinic (Evenson et al., 1999). The SCSA measures susceptibility to DNA denaturation in situ in sperm exposed to acid for 30 s, followed by acridine orange staining. Utilization of flow cytometry in SCSA increases its dependability. Consequently, among couples who were trying to conceive over 12 months, sperm with denatured DNA was the best predictor for whether or not a couple would achieve a pregnancy. Furthermore, SCSA data predicted 39% of miscarriages.
Ours is the first report to claim sperm DNA quality to be a predictor of fertilization and pregnancy in vivo, as a result of IUI for treatment of human infertility. All of the logistic regression analyses performed herein, including the one assessing all cycles as well as others for five different subsets of data, contained a sperm DNA parameter related to either stability or fragmentation (as assessed by AOpost and TUNEL respectively), sometimes being the only male parameter to predict outcome. In addition, no woman inseminated with a sample having >12% of sperm with fragmented DNA achieved a pregnancy. Furthermore, the two patients who miscarried were inseminated with the samples containing the highest degree of DNA fragmentation among all cases who became pregnant (TUNEL = 12 and 10%).
Washing may seem a superior method for sperm preparation for IUI, based on the logistic regression analysis for all cycles. However, since the method of sperm preparation was selected according to the number of round cells in the ejaculate, the result may simply reflect its consequences, indirectly. Thus, based on our findings, one should not conclude washing to be a better sperm preparation technique, with a higher likelihood of pregnancy.
Other parameters we report to predict pregnancy after IUI are compatible with those published in the literature, especially those studies with a larger sample size (Campana et al., 1996; Stone et al., 1999
; Goverde et al., 2000
; Hendin et al., 2000
; Khalil et al., 2001
). The two parameters with the strongest impact were related to female factors, indicating the importance of the number of follicles and age of the woman for successful reproductive outcome. These parameters are indicators of the quality and availability of the female gamete, which is also the major determinant of successful outcome in assisted reproduction treatment. The sample size of our study is also large enough to show the importance of the female gamete for successful reproductive outcome under in-vivo conditions. However, gonadotrophins seem to compensate for the negative effects of advanced maternal age, at least to a certain degree, when used for ovarian stimulation (Table IV
). In this case, the age of the man appears to be a negative confounding variable instead. In some men, fertility may be decreased in the absence of evidence of disruption in testis morphology or semen production, although it is usually maintained up to a very high age. Reasons for this decrease in fertility may include defects in sperm maturation; age-related increases in germ cell mutations, impairment of DNA repair mechanisms and apoptotic processes (Rolf and Nieschlag, 2001
). Our results support the existence as well as importance of age-dependent problems with sperm DNA, especially when female factors are corrected for.
In conclusion, the number of follicles, the age of the woman/man and the quality of sperm DNA may predict IUI outcome. Whether it is a result of suboptimal maturation, apoptosis, an oxidative hazard or other causes, the stability of the sperm DNA, as well as the extent of its fragmentation is an indicator of poor IUI outcome. Further assessment of sperm DNA status and different types of damage is warranted to establish their overall importance in the efficiency of human reproduction. We suggest that tests analysing sperm DNA quality should be a part of the routine semen analysis for patients suffering infertility, regardless of the type of treatment they undergo.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 22, 2001; resubmitted on June 25, 2002; accepted on August 9, 2002.