Department of Obstetrics and Gynaecology, University of Würzburg, Josef-Schneider-Strasse 4, D-97080 Würzburg, Germany
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Abstract |
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Key words: ICSI/male factor infertility/medium/oligoasthenoteratozoospermia/sperm preparation technique
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Introduction |
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Antisperm antibodies associated with the sperm surface can influence male fertility. Treatment options in severe immunological cases include both standard IVF and ICSI (Bates, 1997). It is generally agreed in the literature that ejaculation in medium increases the proportion of antibody-free spermatozoa in semen samples containing anti-sperm antibodies, thereby enhancing the chances of successful fertilization (Elder et al., 1990
; Bollendorf et al., 1994
; Katsoff et al., 1995
).
There are many reasons for IVF or ICSI failure. Sub-optimal sperm chromatin integrity can lead to a disturbed post-embryonic development (Larson et al., 2000). Poor oocyte quality can influence the cleavage rates (Rawe et al., 2000
) as well as impaired sperm parameters. Bacteria and detritus are often present in OAT ejaculates. Early dilution of the semen sample with medium could prevent the binding of bacteria to the sperm surface or tail, thereby improving sperm function. Furthermore, ejaculation into medium containing HEPES buffer and human serum albumin (HSA) could have a protective effect against the action of free oxygen radicals. Concentrations of free oxygen radicals are increased in the seminal plasma of infertile men (De Lamirande and Gagnon, 1995
) leading to sperm membrane and DNA damage.
As it has been shown that HEPES medium is a potent DNA protector (Ermilov et al., 1999), it can be postulated that early incubation of the ejaculate in HEPES buffered medium may enhance the chances of successful fertilization. Therefore, a prospective randomized controlled trial was undertaken to study the effect of ejaculation into buffered medium on fertilization and pregnancy rates after ICSI.
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Material and methods |
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As mentioned before, the indications for ICSI were in all cases severe male factor infertility. Couples were recruited only if the previous sperm count fulfilled at least two of the three criteria for severe OAT, i.e. concentration <5x106/ml, progressive motility [World Health Organization (WHO), 1992] <20%, normal morphology <10%. There were no exclusion criteria for OAT subsets.
For 60% of patients it was the first ICSI trial, for 25% the second, 6% of patients had the third trial and 9% had the fourth ICSI cycle. There were no significant differences between groups A and B.
Men were randomized according to their year of birth (even = A/uneven = B) in two groups: 55 men had semen collection into sterile dry pots (group A) and 59 of the men had samples collected into 20 ml HEPES buffered Ham's F-10 medium (Sigma Aldrich Chemie, Steinheim, Germany) with 10% human serum albumin (group B). All patients gave informed consent to this prospective randomized trial. The mean age of the men was 35 ± 4.8 years. All patients were clinically asymptomatic for male accessory gland infection and were not treated with any antibiotic therapy.
Semen analysis and sperm preparation
All native semen samples and samples after preparation were evaluated manually according to WHO guidelines (WHO, 1992) for volume, total sperm count, concentration, leukocytes, motility, vitality and morphology (expressed as percentage normal forms). Bacteria and detritus were scored under x1250 magnification and subdivided in four grades (03).
After sample collection into medium (group B), the ejaculates were incubated for 30 min at 37°C and mixed gently. Ham's F-10 medium supplemented with 10% human serum albumin (HSA) was used for all washing procedures. The spermatozoa were then washed by centrifugation and prepared by a swim-up technique with moderate centrifugation at 23 g for 10 min. The supernatants were removed and centrifuged again for 10 min at 93 g. The pellets were carefully resuspended and centrifuged again for 3 min at 145 g. Fresh medium (0.3 ml) was gently layered over the pellet, followed by incubation at 37°C for 3060 min for swim up. The motile spermatozoa were finally recovered, washed once by centrifugation at 145 g for 3 min and used for injection.
The ejaculates of group A were prepared by a conventional swim-up procedure. The whole ejaculate was centrifuged at 209 g in several Falcon tubes for 10 min and was then resuspended in 0.5 ml Ham's F-10 medium with 2% HSA. A second and a third centrifugation step was performed at 145 g for 2 min. The supernatants were then removed, leaving the pellet intact. The sperm preparation was incubated for 12 h at 37°C. The supernatant was removed and added to 5 µl of 10% polyvinylpyrrolidone (PVP) solution (ICSITM 100, Scandinavian IVF, Gothenburg).
Ovarian stimulation and ICSI procedure
Ovarian stimulation was induced by a mild step-down human menopausal gonadotrophin (HMG; Pergonal; Serono Pharma, Unterschleissheim, Germany) or FSH (Gonad F; Serono Pharma SPA, Bari, Italy or Puregon; NV Organon, Oss, Netherlands) protocol starting on day 3 of the cycle after pituitary down-regulation by 0.2 mg nafareline 24 times a day (Synarela®, Heumann, Nürnberg, Germany) 68 days prior to the expected period. Human chorionic gonadotrophin 10 000 IU (HCG; Pregnesin; Serono Pharma, Germany) was administered late in the evening of the day during which the mean diameter of the dominant follicle reached 18 mm and after 67 days of a steady rise in serum oestradiol concentrations. Oocyte recovery was performed by the vaginal route 3436 h after HCG administration.
IVF medium (Scandinavian IVF) was used for all oocyte cultures. Oocytes were cultured at 37°C in 5% CO2 at pH 7.4. At 4 h after recovery, the ICSI procedure was performed after enzymatic removal of the cumulus complex and mechanical dissection of the corona radiata. The maturity of the oocytes was determined and only metaphase II oocytes were used for ICSI procedure. The injection was performed on a heated stage at 37°C of a inverted microscope under oil in two microdroplets of medium containing the ova and two containing PVP with the spermatozoa. A single spermatozoon was then immobilized mechanically before drawing it into the microinjection pipette with the tail first. The injection pipette containing the spermatozoon was advanced towards the oocyte being held by another pipette. The injection needle was then advanced to penetrate the oolemma and the spermatozoon was injected. Prior to expelling the spermatozoon, rupture of the oolemma was confirmed by aspiration of a small volume of the oocyte cytoplasma into the injection pipette. After injection, oocytes were transferred to fresh IVF medium.
After incubation for 1618 h, the oocytes were checked for the presence of pronuclei as evidence of fertilization. Fertilization was defined as extrusion of the second polar body and presence of pronuclei. Fertilized oocytes in excess of three were cryopreserved or withdrawn. Fertilized oocytes were transferred to G1 medium (Scandinavian IVF), kept in culture for another 48 h and were then placed into G2 medium (Scandinavian IVF). A maximum of three embryos was transferred into the uterus 4 days after oocyte recovery. The culture duration was stepwise extended from 2 to 3 days in our clinic a few years ago. Since sequential media are available we stepwise extended the time of culture again from 4 to now 5 days routinely. In the time where the study has been undertaken, the culture duration was 4 days. The luteal phase was routinely supported by vaginal progesterone (Crinone 8%; Serono Pharma, Germany) suppositories (300 mg/day) and by at least one injection of 5000 IU HCG.
Pregnancy assessment
A single serum ß-HCG measurement was performed 14 days after embryo transfer. Clinical pregnancies were defined as presence of a gestational sac on ultrasound scan.
Statistical analysis
Multiple correlations between WHO ejaculate parameters and fertilization and pregnancy rates were calculated using Spearman's coefficient of correlation. MannWhitney tests were used to compare semen parameters in the medium and non-medium group. The
2 test was used to calculate the significant difference in the pregnancy rate between the medium and non-medium group. A P value < 0.05 was considered significant. P values were given without correction for multiple comparisons.
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Results |
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Discussion |
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A prospective randomized trial showed that collection of spermatozoa into medium significantly reduces the percentage of antibody-bound spermatozoa versus dry collection (Byrd et al., 1994). In that study, there was no significant difference between the motility of the spermatozoa following collection with or without medium, which was confirmed in this study. Surprisingly, in the current study, a slightly higher percentage of motile spermatozoa in the non-medium group was observed. It should be noted, however, that the sperm parameters in the study by Byrd et al. were normal (Byrd et al., 1994
), whereas only OAT ejaculates were included in this trial. Agglutinizing and immobilizing antibodies block spermoocyte interaction by inhibiting capacitation and acrosome reaction (Tasdemir et al., 1995
; Bathla and Sidhu, 1996
), but the blocking mechanisms of antisperm antibodies are bypassed by the injection of the spermatozoa into the ooplasma leading to normal fertilization rates (Clarke et al., 1997
).
In a retrospective analysis, it was shown that serum-diluted spermatozoa can successfully fertilize oocytes in vitro with improved fertilization and pregnancy rates (Elder et al., 1990). Scobey et al. found that media influence the sperm capacitation and acrosome reaction (Scobey et al., 1995
). Although it has been shown that treating spermatozoa with agents known to induce the acrosome reaction does not increase the fertilization rate after conventional IVF or ICSI in fresh human oocytes (Liu et al., 1994
), it is commonly agreed that induction of the acrosome reaction is related to fertilization rates (Liu and Baker, 1998
). Spermatozoa from infertile men with sperm antibodies in serum and seminal plasma undergo premature acrosome loss, thereby diminishing their fertilizing potential (Harrington et al., 1998
). Although the acrosome reaction was not measured in this trial, it is proposed that the observed increase in implantation rate after ejaculation in medium may be due to the prevention of premature acrosome loss in the spermatozoa used for ICSI.
The effect of dilution by medium in OAT ejaculates on implantation rate was shown in a previous retrospective analysis (Zollner et al., 1999). Bacteria and detritus are very common in OAT ejaculates. The presence of micro-organisms does not indicate infection in all cases; it can also be asymptomatic colonization (Bussen et al., 1997
). None of the patients in the current study had symptoms of genital tract infection or were treated with antibiotics. Cottell et al. found that semen preparation in an antibiotic rich culture medium effectively eliminates 95% of organisms (Cottell et al., 1997
). Huyser et al. even found that semen processing was more effective after removing microbial contaminants from 57.4% of semen samples than antibiotic treatment which decreased the incidence of pathogens by 16.3% (Huyser et al., 1991
).
In this study, we chose not to screen samples for antisperm antibodies, because it is well known that in OAT ejaculates, the incidence of sperm-reactive antibodies ranges at ~5%, which is comparable with that in normal fertile men (Höbarth et al., 1994).
A possible explanation for the beneficial effect of HEPES medium in oligoasthenoteratozoospermic samples could be the reduction of reactive oxygen species (ROS). Increased concentrations of ROS are often found in the seminal plasma of infertile patients. Generation of ROS can be induced by leukocyte contamination (Vicari, 1999), the sperm preparation procedure itself and the presence of abnormal spermatozoa. Ochsendorf found that ROS generated by leukocytes during male genital tract infection impair sperm function (Ochsendorf, 1998
). ROS have been shown to exert a detrimental effect on the fertilization potential because they induce breaks in DNA. Ermilov et al. showed that HEPES medium was the strongest DNA protector among many IVF media (Ermilov et al., 1999
). Kuribayashi and Gagnon also found that H2O2 produced in sperm suspensions before IVF reduces their potential to promote embryo development in mice. Incubation of spermatozoa with catalase and thioredoxin before IVF resulted in an increased blastulation rate (Kuribayashi and Gagnon, 1996
). Sperm motility parameters and fertilization rates were not affected by this treatment. These observations are in agreement with the results of this study, because it was not possible to detect significant differences in fertilization rate between the medium and non-medium groups, but there was a significant higher pregnancy rate in the medium group. The induction of DNA damage in human spermatozoa by ROS during preparation was also highlighted in another study, which suggested that seminal plasma itself had a protective effect (Twigg et al., 1998
).
The paternal effect on the embryo does not begin until the four-cell stage, because the first two cell cycles of the embryo are controlled by maternal genes (Oehninger et al., 1998), which could explain the higher implantation rates in group B than in group A although the fertilization rates were similar in the two groups.
In summary, the data show that the addition of HEPES buffered Ham's F-10 medium to sample collection pots significantly improves the implantation and clinical pregnancy rate after ICSI in patients with OAT. Two aspects to minimize the risk of generation of ROS are combined in this technique, i.e. mild centrifugation and incubation of the semen sample with seminal plasma and medium. No measurements of ROS were performed in this study, and further investigations are necessary to show whether this beneficial effect on implantation is in fact due to the decreased formation of ROS. Ejaculation in medium is an efficient and simple method that could finally be applied in all patients with severe male factor infertility undergoing reproductive technologies.
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Notes |
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References |
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Submitted on October 2, 2000; accepted on February 15, 2001.