Comparison between the quality and function of sperm after semen processing with two different methods

 M.E. Hammadeh¹,  P.M. Zavos^2,3, P. Rosenbaum¹,   W. Schmidt¹

¹Dept. of Obs/Gyn, University of Saarland,  66421 Homburg/Saar, Germany
²Andrology Institute of America and ³Kentucky Center for Reproductive Medicine & IVF, Lexington, KY, USA

Asian J Androl  2001 Jun; 3:  125-130

Keywords: sperm; semen processing;  spermPrep^TM;  percoll gradient centrifugation;  IVF

Abstract

Aim: To compare the recovery rate of morphologically normal and chromatin condensed spermatozoa from native semen samples using the SpermPrep^TM filtration columns and Percoll gradient centrifugation and to determine the influence of the two processing techniques on fertilization and pregnancy rates in an IVF-ET program. Methods: Sixteen semen samples obtained from patient's husband were included in this study. Each was divided into two aliquots. The first aliquot was processed with SpermPrep^TM filtration columns and the second, Percoll gradient centrifugation. Smears were made before and after semen processing with both methods for the evaluation of chromatin condensation (chromomycine CMA3) as well as morphology (strict criteria) of spermatozoa. One hundred and seventy oocytes were retrieved from the patients and the oocytes from each patient were subdivided in to two sets: one set was inseminated using spermatozoa processed with SpermPrep^TM and the other inseminated after semen processing with Percoll gradient centrifugation. Results: The Percoll method yielded a significantly higher percentage of chromatin condensed (90.86.5% vs 82.38.8%, P=0.017) and morphologically normal spermatozoa (12.97.4% vs 6.94.8%, P=0.001) in comparison to SpermPrep^TM. Whereas, sperm count recovery rate was significantly higher after the use of SpermPrep^TM than after the Percoll gradient centrifugation. The fertilization rate was similar between the two methods. Conclusion: Semen processing with Percoll should be recommended for intracytoplasmic sperm injection as the natural selection is bypassed and the SpermPrep^TM technique could be recommended for IVF and IUI programs as the sperm concentration plays a more significant role in these procedures.

1 Introduction

The ideal sperm preparation method should select morphologically normal, motile sperm from the ejaculate. It should also minimize contamination and iatrogenic damage to sperm during processing^[1]. Several techniques^[2-4] have been developed to remove the undesired sperm, debris, and increase the overall sperm quality.

It is well known that certain seminal parameters such as sperm morphology^[5], some aspect of sperm motility^[6], and the ability of spermatozoa to undergo acrosome reaction^[7] are strongly correlated with fertilization rate in vitro. A significantly different fertilization rate has been reported for patients with similar semen parameters, suggesting that a more sensitive test is needed to identify the inherited defects which render certain spermatozoa unable to fertilize^[8]. Many studies have demonstrated that male factor infertility patients possess hidden anomalies in the composition of their sperm nuclei, displaying a higher level of loosely packaged chromatin and damaged DNA^[9,10].

Furthermore, it has been suggested that a low DNA content in spermatozoa of subfertile men may be responsible for inducing alterations in sperm shape^[11]. The chromatin of spermatozoa first undergoes dramatic changes during spermatogenesis. The histones which bind  to DNA in somatic cells and in germinal cells  through the spermatocyte  stage become replaced, by protamines in spermatid during spermiogenesis^[12]. The protamine, small basic  proteins containing much  arginine, bind more tightly to the minor groove of DNA than do histones and this results in compaction of chromatin in the sperm nucleus, a process which is termed sperm chromatin condensation. Therefore, chromatin condensation is disturbed when lysine-rich somatic histone are not sufficiently substituted by arginine-and cysteine-rich protamines during spermatogenesis^[13].

A great deal of evidence suggest that these processes are vital for male fertility, and that complete chromatin packaging is essential  for normal sperm functioning^[14]. Thus, it has been shown that incomplete replacement of histones by protamines^[15], aberrant ratios of protamine 1 to proptamine 2^[16] are associated with male subfertility

Furthermore, a correlation  between  abnormal sperm chromatin packaging and the presence of DNA strand breaks has also been  shown to exist^[17].

Several  fluorochromes and dyes have been used to assess chromatin packaging quality in spermatozoa. Chromomycin A₃ (CMA₃) has been  shown to bind as a Mg²⁺ -coordinated dimmer at the minor groove of GC rich DNA helix, a positional preference that can make it a competitor to protamines, and induces a conformational perturbation in the DNA helix resulting in a wider and shallower minor groove at its binding site^[18].

Treatment of human sperm sample with protamines eliminates CMA₃ positively and prevent the identification of endogenous DNA nicks by in situ nick translation, a technique that reveals damaged DNA. Therefore, Chromoycin A₃, is a useful tool for the rapid screening of subfertility in man, as it seems to allow an indirect visualization of protamine-deficient, nicked and partially denatured DNA^[19].

Monaco and Rasch^[20] suggested that the decline in mithramycin and CMA₃ staining intensely observed in maturing spermatozoa of fish, frogs and rabbits reflected changes in protein composition and in DNA packaging ratios.

2 Materials and methods

2.1 Samples

Semen samples (n=16) obtained from patients undergoing IVF treatment were included in this study. The samples were assessed according to WHO standards^[21], except for morphology which was assessed according to strict criteria. Each semen sample was divided into two aliquots, the first was processed with the SpermPrep^TM filtration columns and the second with Percoll gradient centrifugation. Smears were prepared before and after semen processing with both methods for morphological examination and chromatin condensation analysis, which was visualized after staining with chromomycine CMA₃.

2.2 Assessment of sperm morphology

Morphology was evaluated from the native and post selection samples by one observer, according to strict critera^[22] and taking into consideration the specific effects e.g., size alteration of a specific staining method^[23]. Slide preparation was in accordance with the method established by Kruger et al^[22]. A total of 5 L of semen were pipetted onto a slide to make thin smears. The slide was air-dried for 3 min, then fixed for 15 s in methyl alcohol. It was stained with Papanicolaou staining. Bright field illumination with a magnification of 100 under an oil immersion objective was used for the evaluation. For each semen sample 200 spermatozoa were evaluated.

The following criteria for normal spermatozoa were used: (i) smooth, oval head, 5-6 mm in length and 2.5-3.5 mm in diameter; (ii) well defined acrosome, 40-70% of the sperm head; (iii) mid-piece without defect, slender, axially attached with <1 m; (iv) tailpiece uniform, free from kinks, uncoiled, width thinner than mid-piece, length-45 m; and (v) cytoplasmic droplets (remnants), which compromise less than half the head area, are acceptable but must be retained in the mid-piece region only. Multiple defects in individual cells were classified  as amorphous unless there were only multiple tail defects. The semen samples were either classified as normal (14% morphologically normal spermatozoa) or abnormal (<14% morphologically normal spermatozoa).

2.3 Hypo-osmotic-swilling  test

The HOS-test was performed, before and after semen processing, according to the method described by Jeyendran et al^[24]. In a word, it was performed by mixing 100 L of  sperm suspension with 1 mL of hypo-osmotic solution (equal parts of 150 mOsmol fructose and 150 mOsmol sodium citrate solutions), followed by 30 min incubation at  37. After incubation, one drop was observed under the light microscope.

Evaluation was accomplished using bright field microscopy (400). A minimum of 200 spermatozoa were examined per slide and the percentage of spermatozoa that showed typical tail abnormalities indicative of swelling was calculated.

2.4 Eosin-nigrosin test (viability assessment)

The Eosin-nigrosin test in the present study was used to evaluate the viability of the spermatozoa before and after semen processing in both techniques according to the method described by Eliasson and Treich^[25]. Briefly, one drop of semen was mixed on a slide with 1 drop of the 0.5% aqueous yellowish eosin solution and one drop of Nigrosin (10% in distilled water) and covered with a cover slip. After 1-2 min, the spermatozoa stained red (dead spermatozoa) can be distinguished from the unstained spermatozoa (alive). Nigrosin was used as a counter-stain to facilitate visualization of the unstained live cells^[41].

2.5  Assessment of chromatin condensation chromomycin (CMA₃)

CMA₃ was performed as described by Bianchi et al^[19]. Briefly, semen aliquot before and after preparation were washed in Dulbecco's Ca-Mg free phosphate buffer saline (PBS) and centrifuged at 2500 rpm for 10 min. The spermatozoa were washed again, fixed in methanol/glacial acetic acid (3:1) at 4 for 5 min and then spreaded on clean slides. Each slide was treated for 20 min with 100 L of CMA₃ solution (Sigma)  (0.25 mg/mL in Mc Ivaine buffer, pH 7.0, containing 10 mM MgCl₂). The slides were then rinsed in buffer, mounted with buffered glycerol, and fluorochrome positivity was examined using a Zeiss photomicroscope III employing a combination of exciter: dichroic: barrier filters of BP 436/10: FT 580: LP 470. A total of 200 spermatozoa were counted on each slide.

2.6 IVF  procedure

The female partners in all the groups underwent controlled ovarian hyperstimulation after pituitary desensitization using gonadotrophin-releasing hormone analogue (Gn-RHa) in the mid luteal phase. Thereafter, stimulation with human menopausal gonadotrophin (HMG, Menogon, Ferring, Kiel, Germany) or follicular stimulating hormone (FSH Gonal-F, Serono, Germany) was performed. When at least three follicles were > 18 mm in the diameter and the serum oestradiol concentration > 800 pg/mL, ovulation was induced by administration of 10,000 IU human chorionic gonadotrophin (hCG) Transvaginal follicle aspiration was carried out 36 h. after hCG injection. Retrieved oocytes were cultured in Hams F-10 medium supplemented with 15% patients serum at 37, in 5% CO₂. One hundred and seventy-nine oocytes were retrieved (10.53.9/patient). The oocytes from each patient were randomly divided into two groups. One group was inseminated with spermatozoa obtained with SpermPrep^TM filtration and the second group, with the Percoll gradient centrifugation technique.

Fertilization was recorded after 18-24 hours if the two pronuclei were detected. The percentage of improvement in spermatozoa with regard to sperm count, morphology and chromatin condensation and membrane integrity as well as the fertilization rates were compared between the two methods.

2.7 Statistical analysis

3 Results

Tab.1 summarises the results obtained in this study. The Percoll gradient centrifugation method  yielded a significantly higher percentage of morphologically normal sperm (12.97.4% vs 6.25.0%; P=0.001) and chromatin condensed spermatozoa (90.86.5% vs 82.28.8%; P=0.017) as compared to the SpermPrep^TM method. However, sperm count recovery with the SpermPrep^TM was significantly higher in comparison to the Percoll method (44.235.5 vs 33.330.7; P=0.02).

On the other hand, 170 oocytes were retrieved from the patients, the oocyte from each patient was randomly divided into two groups. The first group (n=85) was inseminated using spermatozoa processed with Sperm Prep method, whereas the second group (n=85), with Percoll gradient centrigugation. One hundred and twenty oocytes were fertilized (fertilization rate 70.6%): Fifty-two oocytes in the first group (fertilizaion rate 61.1%) and 68 oocyte in the second group (fertilization rate 80.0%). No significant difference was observed between the two groups with respect to the fertilization rate. 

Table 1. Comparison between semen parameters after processing either with Sperm prep filtration columns or percoll gradient centrifugation. ^aP>0.05, ^bP<0.05, ^cP<0.01 compared with Percoll.

4 Discussion

The separation of spermatozoa from seminal plasma to allow capacitation and expression of their intrinsic fertilising ability is a fundamental prerequisite of assisted reproduction technology.

Several studies have been published on the effect of individual methods of semen preparation on sperm morphology. Density gradient centrifugation separate spermatozoa according to their density and favours the isolation of the motile and normal morphology spermatozoa^[26].

Besides, 99% of Percoll-filtered sperm contained chromatin that was fully condensed, whereas 15% of swim-up sperm still possessed incompletely condensed chromatin^[27].

Sperm morphology is possibly the sperm variable most consistently related to in vitro fertilization success rate^[28]. A logistic regression model, including DNA status and sperm morphology, revealed that sperm morphology (strict criteria) and the concentration of progressive motile sperm were the principal predictors for in vitro fertilisation^[29].

The percentage of post-preparation morphologically normal forms, determined by the strict criteria(Kruger et al^[22]), is known to be strongly correlated with the likelihood of achieving pregnancy following IVF^[30].

Nevertheless, Miller et al^[31] have shown that fertilization rates were related to the initial semen parameters.

Parinaud et al^[32] demonstrated that sperm selection on a percoll gradient increased the percentage of morphologically normal spermatozoa. However, morphology of spermatozoa in the percoll fraction  had the same predictive value for IVF as did for the native semen. They suggest that impairment of spermiogenesis, which presumably induces sperm abnormalities, is more important than the actual characteristics of the spermatozoa. On the other hand, morphology as a good and simple predictor of fertilization is difficult to accurately set up a high inter-laboratory and inter-technician variability^[33].

The usefulness of assessment of the integrity of chromatin  structure has been highlighted by many studies^[9,34]. An assay that could give us information on molecular structures believed to be involved directly in fertilization may be more relevant and less subjective than sperm morphology.

Besides, poor chromatin packaging and possible DNA damage may contribute to failure of sperm decondensation after ICSI with and subsequent fertilization failure^[19,35].

Therefore, alteration in sperm chromatin might result in defective decondensation and DNA activation during fertilization, leading to a delay in the formation of the male pronucleus and /or the first division events. One consequence of this might be early embryonic wastage or poor embryonic development^[36].

The data presented in this study indicated that both sperm processing methods yield a significant improvement in the assessed characteristics when compared to the native specimens. The percentage of sperm vitality and membrane integrity were higher in the sperm prepared either with Sperm Prep (85.515.2% and 71.39.5%, respectively) or with Percoll gradient centrifugation (80.210.9% and 82.56.5%) in comparison to the native semen (40.717.6% and 63.814.2%) (Table 1). The mean percentage of morphologically normal spermatozoa, however, was reduced compared to the initial sperm. Moreover, there was a statistically significant difference between the two methods with respect to the recovery rate of morphologically normal  spermatozoa (P=0.001), chromatin condensation (P=0.017) and membrane integrity (P=0.040). The mean percentage of morphologically normal, chromatin condensed, and HOS-test positive spermatozoa in the proportion processed with Percoll gradient centrifugation was 12.77.8%, 90.76.5% and 82.56.5%, respectively, and the corresponding value in the semen prepared with SpermPrep^TM were 6.25.0%, 82.28.8% and 71.39.5%, respectively. Whereas, the sperm count in the aliquot processed by Percoll gradient centrifugation was significantly lower (P=0.020) as compared to SpermPrep^TM column filtration (33.330.7 vs 44.235.5 mill/mL, P=0.020) (Table 1). This is in agreement with results shown by Sofikitis et al^[37] who demonstrated  an increase of sperm motility, percentage of normal spermatozoa, and mean percentage of double-stranded DNA recovered after filtration with SpermPrep. Lopetz et al^[38] compared the efficacy of a disposable, pre-packed PD-10 Sephadex columns to select motile spermatozoa with Sperm Prep filtration column and centrifugation through Percoll gradients. They found that the number of motile cells and the proportion of total spermatozoa selected was similar for all methods. The straight line velocity of motile cells was lower in samples processed by SpermPrep^TM as compared to both methods. Besides, Percoll centrifugation improved the percentage of morphologically normal spermatozoa more than the other methods.

Yamamoto et al^[39] showed that sperm populations recovered via the SpermPrep^TMII filtration method possessed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyper-activated spermatozoa than sperm recovered by the swim-up method. However, sperm recovered via the SpermPrepII method did not show significantly different values for these parameters and for most of sperm morphometric parameters as compared to Percoll density gradient method. They pointed out that SpermPrep^TMII filtration and Percoll density gradient method were equally efficient in isolating sperm subpopulations with better functional parameters than the swim-up method.

In a study comparing the various parameters of matched normal and asthenozoospermic specimens prepared by SpermPrep and mini Percoll gradient centrifugation method, Joshi et al^[40] reported significantly better sperm motion parameters, recovery of motile fraction, morphology, hypo-osmotic swelling and nuclear stability as assessed by sodium dodecyl sulphate. On the other hand, by using  the prepared spermatozoa for oocyte insemination in the IVF program, a higher fertilization rate has been shown by using spermatozoa obtained by Percoll gradient centrifugation than SpermPrep^TM filtration column (80.0% vs. 61.1%). However, no significant difference was shown between the two groups.

Our finding that the percentage of morphologically normal sperm in a given semen sample appears to influence the ability of that sample to achieve fertilization  and pregnancy  is in agreement with a number of other studies^[41,6,42].

Therefore, SpermPrep column filtration may be used to prepare semen in the laboratory as a practical alternative to Percoll gradient centrifugation on an individual basis, depending on the initial semen sample.

5Conclusion

Semen processing via Percoll gradient centrifugation yielded a significantly higher percentage of morphologically normal and chromatin condensed spermatozoa as compared to sperm prepared by the SpermPrep^TM filtration method. How ever, sperm count was significantly higher in semen processed by SpermPrep^TM filtration columns compared to Percoll gradient centrifugation. No significant difference was shown between the groups with regard to the fertilization rate. Therefore, the Percoll gradient centrifugation method may be appropriate when ICSI technique is prescribed where the natural selection of sperm failed, whereas the SpermPrep^TM filtration columns technique may be more appropriate for IVF as the sperm recovery rate will play a more significant role.

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Correspondence to: Dr. M.E. Hammadeh, Department of Obstetrics/Gynecology,  University of Saarland, 66421 Homburg/Saar, Germany.
Tel: +49-6841-16-8117   Fax: +49-6841-16-8061
E-mail: frmham@med-rz.uni-sb.de
Received 2000-03-08     Accepted 2001-04-25