Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane-coated silica particles at different g forces

C.M. Nicholson1, L. Abramsson2, S.E. Holm3 and E. Bjurulf1,4

1 Department of Obstetrics and Gynecology, 2 Department of Urology and Andrology and 3 Department of Clinical Bacteriology, Umeå University Hospital, S-90185 Umeå, Sweden


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of density gradient centrifugation through silane-coated silica particles (PureSperm®) using 100, 200, 300 and 500 g on bacterial contamination of sperm samples and recovery of motile spermatozoa from sperm samples were investigated with conventional culturing techniques and microscopic visual assessment. The recovery of motile spermatozoa was variable and was not improved using 500 g compared to the recommended 300 g. The bacterial contamination was highly decreased by gradient centrifugation through PureSperm® and was almost abolished when strict aseptic techniques were used, with changes to sterile Pasteur pipettes and tubes prior to washing procedures.

Key words: assisted reproduction/bacteria/density gradient centrifugation/semen preparation/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Density gradient centrifugation of liquefied ejaculate through polyvinylpyrrolidone (PVP)-coated silica particles (Percoll; Pharmacia, Uppsala, Sweden) was used for sperm preparation (Bolton and Braude, 1984Go) until 1996. Percoll has since been withdrawn from use in clinically applied assisted reproduction and has been replaced by other products. Percoll is known selectively to clean sperm samples from bacterial contamination (Bolton et al., 1986Go), but the efficiency of new compounds, e.g. silane-coated silica particle solution (PureSperm®; Nidacon International AB, Gothenburg, Sweden), for removing bacterial contamination has not been proven.

The clinical importance of a procedure that eliminates bacteria from the semen is evident as bacteriospermia is common (Willén et al., 1996Go; Cottell et al., 1997Go). Moreover there is a tendency of some bacteria to adhere to spermatozoa (Friberg and Fullan, 1983Go; Wolff et al., 1993Go; Diemer et al., 1996Go), and to impair both motility (Kaur et al., 1986Go) and the inducibility of the acrosome reaction (el Mulla et al., 1996Go). The clinical importance is further strengthened by another finding (Huyser et al., 1991Go) that, in bacterial infested cultures, the oocytes are degenerated. However, it is somewhat surprising that the presence of bacteria in the original semen sample has been reported to have no effect on fertilization, cleavage or pregnancy rates in in-vitro fertilization (IVF) treatments (Riedel et al., 1984Go; Forman et al., 1987Go; Stovall et al., 1993Go; Liversedge et al., 1996Go; Bussen et al., 1997Go). It is not always clearly stated whether or not antibiotics were added to the culture media used in these studies. The only micro-organism reported to decrease pregnancy rates is Ureaplasma urealyticum (Montagut et al., 1991Go), where the authors speculate on an endometrial effect.

The benefit of adding antibiotics to semen preparation solutions and IVF media has been questioned both by media producers and IVF clinics. The most widely used antibiotics in IVF are a combination of penicillin and streptomycin. If these antibiotics are excluded, the embryos cleave faster (Magli et al., 1996Go). Penicillin G is degraded to 50% within 24 h (Neftel et al., 1983Go), while streptomycin is stable (Kassem et al., 1983Go) at 37°C. A prerequisite for the reduction or exclusion of antibiotics from IVF media is the use of laboratory methods which eliminate the bacterial contamination originating from the semen sample.

In cases where the male has low sperm counts and intracytoplasmic sperm injection (ICSI) is necessary, it is important to have a sperm preparation method with high and reliable recovery. When performing other forms of assisted reproduction, such as conventional IVF and intrauterine inseminations, a sperm preparation with good recovery could be crucial. Silane-coated silica particles have been reported both to provide equal sperm recovery compared to PVP-coated particles (Claassens et al., 1998Go; Centola et al., 1998Go) and to have a lower recovery (Chen and Bongso, 1999Go).

The purpose of this study was to evaluate the effect of various centrifugation forces and the usage of aseptic techniques on bacterial contamination, recovery of spermatozoa and sperm motility under antibiotic-free culturing conditions.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, one sperm sample from each of 30 patients undergoing fertility investigation or IVF treatment were examined. The patients were between 23 and 47 years old (mean 35.9). The infertility diagnosis of the couples was of a female origin in 21 cases and was idiopathic in nine cases. No cases of diagnosed male infertility were included. For participation in the study a cut-off value of 80x106 motile spermatozoa in the ejaculate was set. No patients were excluded because of previous infections.

The ejaculate was collected by masturbation into a sterile container (Falcon No. 2070; Becton Dickinson Co., Franklin Lakes, NJ, USA) and allowed to liquefy for 30 min on a rocking table. After semen analysis and microbiological examination, the remaining semen sample was split into four aliquots and carefully placed on top of PureSperm® gradients. Each gradient comprised two 1.0 ml layers of 90 and 45% (v/v) respectively, diluted in Gamete-100 medium (Scandinavian IVF Science, Gothenburg, Sweden) prepared in sterile conical tubes (Falcon No. 2095; Becton Dickinson Co.). The PureSperm® gradients were run at 100, 200, 300 and 500 g for 20 min at room temperature and without braking. The supernatants were removed using sterile Pasteur pipettes, starting at the surface using a circular motion around the inside of the tubes until approximately 100 µl were left. The sperm pellets were carefully mixed with the remaining supernatants and resuspended in 2.5 ml Gamete-100. This was performed in two different ways, either in the same tubes or after transfer of the pellets, with clean Pasteur pipettes, to clean tubes where the resuspensions were performed. The tubes were allowed to stand for 5 min, after which they were centrifuged at 200 g for 10 min. The supernatants were removed except for 200 µl in which the sperm pellets were resuspended. This washing procedure was repeated once. Samples were taken from the sperm suspensions for microbiological cultivation and sperm analysis.

Semen and sperm suspensions were analysed by microscopic visual assessment using a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) for the number of motile and immotile spermatozoa.

Microbiological examinations of the ejaculates and sperm suspensions were performed. The ejaculates and sperm suspensions were plated with a standard 10 µl plastic loop onto blood agar (Columbia II agar base, BBL; Becton Dickinson Co.), and saboraud agar (Lab M Ltd, Manchester, Lancashire, UK). A total of 50 µl samples were also inoculated in 1 ml of Todd Hewitt bouillon (BBL; Becton Dickinson Co.). This was performed within 1 h from the end of the liquidation or washing procedure. These bacteriological cultures were all incubated at 37°C. The blood agar plates were cultured for 24 h and an additional 24 h if the initial reading was negative. The Todd Hewitt bouillons were incubated for 48 h, then plated on blood agar dishes and incubated as previously mentioned. The fungal cultures were incubated at 30°C for 7 days. After the incubations, the numbers of colonies were counted and the isolates identified using routine laboratory techniques.

A control assay was performed to evaluate a possible bacteriostatic effect of PureSperm®. Isolates of three bacterial species were investigated: Escherichia coli, Staphylococcus epidermidis and Streptomycin sanguis. Four wells/strain were cut in blood agar dishes each filled with 1 ml PureSperm®. A suspension of 105 bacteria/ml was prepared of each isolate. The long edges of several sterile object glasses were dipped in respective bacterial suspensions and stamped over the wells containing PureSperm® on the blood agar dishes. After 24 h of incubation at 37°C, bacterial growth was noted in the vicinity of the PureSperm® wells.

Values are presented as mean ± SEM. Group comparisons were made using two-tailed Student's paired t-test, when comparing different centrifugation forces. When comparing other groups of quantitative data, two-tailed Student's unpaired t-test was used. When comparing non-quantitative data, two-sided Fisher's exact test was used. Correlations were tested using Pearson's test. Differences were considered significant for P values of 0.05 or less.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There was a small but significant increase of both motile and total number of spermatozoa with increased centrifugal force. The recovery of motile spermatozoa was 18.7 ± 2.1% (range: 5.7–47.1) when centrifugation was performed at 100 g and 23.1 ± 3.3% (range: 4.3–80.3), 22.9 ± 2.7% (range: 5.7–56.7) and 25.3 ± 3.2% (range: 1.6–71.1) (P < 0.05 compared to 100 g) respectively when centrifugations were performed at 200, 300 and 500 g. The percentage of motile spermatozoa in the original specimen was 58.9 ± 2.5%. Centrifugation through PureSperm® increased the percentage of motile spermatozoa significantly compared to semen. Even though the recovery of motile spermatozoa was increasing using higher g force, the percentage of motile spermatozoa to total spermatozoa was significantly decreased (P < 0.05) from 87.5 ± 1.7% (100 g) to 80.6 ± 2.8 (200 g) and 77.5 ± 2.6 (300 g) and further decreased to 71.6 ± 3.8% when centrifuged using 500 g.

In 13 of 120 (11%) samples the motile sperm counts were under 2x106 after preparation, irrespective of the centrifugal forces used (data not shown).

When culturing from the original semen sample, only one lacked detectable bacteria, while none had detectable fungus. The most common bacterial type was coagulase negative staphylococci, followed by {alpha}-haemolytic streptococci (Table IGo). Other bacterial species were found sporadically. Thirteen semen samples had two or more types of bacteria.


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Table I. Number of patients with different bacterial species in semen and sperm suspensions after centrifugation through PureSperm® at different g forces
 
As seen in Figure 1Go, 30.83 ± 7.91 colonies were formed/10 µl semen in the original specimen. This number decreased significantly to 3.23 ± 0.96 colonies after preparation by density gradient centrifugation. When a strict aseptic laboratory procedure was used, with change of pipettes and tubes before washing procedures, the number of colonies was further decreased to 0.13 ± 0.05 colonies. No significant differences in bacterial numbers were obtained with different centrifugation forces (Table IIGo). Even though the bacterial parameters in both Figure 1Go and Table IIGo were measurements of concentration, the general picture and significant differences found were the same when the numbers of bacteria loaded on the gradients and in the prepared samples were calculated and compared. The number of samples without bacteria was significantly greater after preparation with strict aseptic techniques (40 of 68 cases) both compared to original samples (one of 30 cases) and after preparation without changing pipettes and tubes prior to washing procedures (eight of 52 cases). More samples were found to be bacteria-free when using lower g forces, but this difference was not significant.



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Figure 1. Number of bacterial colonies formed/10 µl semen and sperm suspensions after centrifugation through PureSperm® with or without changing pipettes and tubes before washing procedures. Bars with different superscripts differ significantly (P < 0.05) using Student's unpaired t-test.

 

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Table II. Number of bacterial colonies formed/10 µl semen and sperm suspensions after centrifugation through PureSperm® at different g forces with or without changing pipettes and tubes before washing procedures
 
In 46 of the total 150 samples, one or more additional bacterial types were isolated after seeding material from the overnight bouillon culture and, in 40 of these samples, it was the only way to isolate bacteria at all. In eight of the 120 samples after preparation there were bacterial species not found in the original sample. PureSperm® caused no growth inhibition of three test bacterial isolates on blood agar plates.

Staphylococci were recovered significantly more often than {alpha}-haemolytic streptococci when centrifugations were followed by changes of pipettes and tubes before washing procedures (Table IIIGo).


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Table III. Number of samples without staphylococci (CNS) or {alpha}-haemolytic streptococci ({alpha}-Strept) in sperm suspensions after preparation with change of Pasteur pipettes and tubes, when these bacteria were present in the original samples
 
The motility of spermatozoa did not relate to concentration of staphylococci, {alpha}-haemolytic streptococci or total bacteria in either the semen or the prepared spermatozoa suspensions.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic-free IVF media are commercially available. In order to reduce or exclude antibiotics in IVF media, it is necessary to use semen preparation methods which minimize, or rather remove, the microbiological contamination originating from the semen sample. In this respect the virulence in vivo of the microbe is of less importance than altered culture conditions caused by a microbe that divides rapidly in the medium. If the purpose is to evaluate the possibilities to culture gametes and embryos in an assisted reproductive programme with antibiotic-free media, antibiotic-free gradients and media should be used, as is the case in this study. Centrifugation through PureSperm® efficiently reduces the bacterial contamination. Changing of pipettes and tubes prior to the washing procedures is an important step which is not stated in the manuals either for PureSperm® (Nidacon) or for IxaPrep® (Medicult a/s, Jyllinge, Denmark). The fact that PureSperm® lacked an intrinsic bacteriostatic effect points toward a mechanical separation of bacteria rather than a pharmacological effect in density gradient centrifugation. Swim-up has also been reported to eliminate bacterial contamination (Wong et al., 1986Go) but, if antibiotics are excluded from the media, the bacteria remain in higher amounts after preparation (Kuzan et al., 1987Go). Further studies to compare the two methods should be performed.

It should be noted that, in many cases, the bacteria were found only after seeding from the bouillon culture, indicating a low number of bacteria in the original preparations. In other studies this method has not been used.

It is notable that only one person produced a semen sample without bacteria. This is in concordance with earlier studies (Bolton et al., 1986Go; Hillier et al., 1990Go; Merino et al., 1995Go), while another study (Forman et al., 1987Go) found a lower number of contaminated semen samples. The bacteria found were mainly those present in the normal skin flora. They could have originated either from the distal part of the urethra, the preputium, or the hands, contaminating the semen during masturbation. A thorough washing procedure seems to decrease the bacterial contamination (Forman et al., 1987Go; Boucher et al., 1995Go).

The probability for staphylococci to contaminate the sperm suspensions was greater than for {alpha}-haemolytic streptococci. This could be explained by their cluster-like growth. An alternative explanation is their attachment to spermatozoa, but this explanation is less likely since, to the best of our knowledge, it has not been reported that staphylococci attach to spermatozoa.

We did not find the relationship reported earlier between sperm motility and bacterial burden (Gopalkrishnan et al., 1988Go; Diemer et al., 1996Go), possibly due to the attachment of bacteria to spermatozoa (Diemer et al., 1996Go). In this study we had only a few samples with the bacteria known to attach to spermatozoa (Friberg and Fullan, 1983Go; Wolff et al., 1993Go; Diemer et al., 1996Go).

Just as for bacteria, an absolute prerequisite to eliminate virus particles by sperm washing procedures is that the viruses do not enter the spermatozoa. There have been conflicting reports on whether human immunodeficiency virus (HIV) has the ability to infect spermatozoa (for reference, see Kim et al., 1999). In a promising case report (Marina et al., 1998bGo), it has been shown that PureSperm® can be used on an HIV-infected male in combination with ICSI without seroconversion of the mother or the child. HIV has been found in about 5% of the post-preparational sperm solutions when using Percoll (Marina et al., 1998aGo), while in a smaller material a complete loss of HIV particles was seen when using Percoll followed by swim-up (Kim et al., 1999Go). It is possible to help great numbers of HIV discordant couples to reproduce with negligible risk of seroconversion of mothers or children (Semprini et al., 1997Go). The authors do not state what sperm preparation they used, but the beauty of this treatment is a reliable detection of viral particles in the sperm solution after preparation rather than the sperm preparation itself.

The morphology and motility parameters used in ordinary semen analysis only partly mirror the ability of spermatozoa to fertilize the oocyte and form a viable embryo. When less than 2x106 motile spermatozoa are obtained after semen preparation, the ICSI procedure should be considered for at least some of the oocytes, regardless of what was originally planned (Stovall et al., 1994Go). This occurred in 11% of the cases we examined and we would have to re-evaluate the laboratory technique used if the samples were to be used in clinically assisted reproduction, even though at least 20x106 spermatozoa were put on top of the gradients. Just as in this study, a recent report also showed great variance in recovery when using silane-coated silica particles (Chen and Bongso, 1999Go). The recovery of motile spermatozoa was not improved using higher centrifugal force than the recommended 300 g.

In conclusion, at least in our hands, the recovery of motile spermatozoa was highly variable using density gradient centrifugation. Centrifugation through PureSperm® efficiently diminished the bacterial contamination if strict aseptic techniques were also used with changes to sterile Pasteur pipettes and tubes prior to the washing procedures.


    Acknowledgments
 
We express our thanks to Ms Carin Olofsson for expert technical assistance. Financial support was provided by the Swedish Society of Medicine and the Swedish Medical Research Council no. 13144. PureSperm® and Gamete-100 were kind gifts of Nidacon International AB and Scandinavian IVF Science respectively.


    Notes
 
4 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 14, 1999; accepted on November 1, 1999.