Andrology: An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen

The efficiency of spermatozoa prepared by swim-up or by Percollcentrifugation was assessed in an in-vitro fertilization programmeon 71 semen samples of a well-defined quality [total numberof type A (WHO criteria) motile spermatozoa]: category I (n= 21) with > 100 x 10⁶, II (n = 31) with 15–100 x 10⁶,III (n = 11) with 5–15 x 10⁶ and IV (n = 8) with <5 x 10⁶ type A motile spermatozoa. Oocytes were inseminated4 h after oocyte retrieval, alternately with spermatozoa derivedfrom swim-up and Percoll preparation. Both selection proceduresresulted in a significantly higher (P < 0.001) percentagemotility as compared to fresh semen. For low-quality samples(III and IV), however, swim-up was more effective in selectinghighly motile (P = 0.004) and morphologically normal spermatozoa(P < 0.05). For high-quality samples, this difference mighthave been masked by introducing a swim-up step to remove Percollparticles. Regardless of the initial sperm quality, the meanfertilization rate was significantly higher (P = 0.003) whenPercoll-treated spermatozoa were used for insemination (51.3versus 37.8%). For semen of groups I and II, no difference infertilization capacity was observed according to the sperm preparationmethod. Despite the lower percentage motility and normal morphologyfor the Percoll compared to the swim-up treatment in groupsIII and IV, fertilizing capacity was significantly (P < 0.001)in favour of this selection method (65.3 versus 26.5% in groupIII, 47.6 versus 11.6% in group IV). Based on these results,it may be concluded that a subgroup of patients exhibiting poorsemen quality can benefit from Percoll semen preparation interms of improved fertilizing capacity.     

Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples.

Two methods of density gradient centrifugation, Percoll (P) and mini-Percoll (MP), were compared with the swim-up technique for preparing spermatozoa from each of 40 abnormal semen samples. P and MP produced similar results with a mean recovery of spermatozoa with progressive motility which was significantly higher (18-19%) than that achieved with swim-up (5%). However, the swim-up method resulted in the recovery of spermatozoa with a higher mean motility (89 versus 58%), velocity (69 versus 56 microns/s), percentage with normal morphology (22 versus 16%) and intact acrosomes (61 versus 36%) than P and MP. The mean amplitude of lateral head displacement was the only characteristic of spermatozoa in semen which correlated with the recoveries of motile spermatozoa. Combining MP and swim-up methods for 10 samples produced a higher recovery (11 versus 6.9%) of spermatozoa with significantly better mean motility (94 versus 87%) than did swim-up alone. Although P and MP resulted in greater yields of motile spermatozoa than the swim-up preparation, the latter procedure selected higher proportions of spermatozoa with improved characteristics (velocity, intact acrosomes and normal morphology) which correlate with fertilization rates in vitro. It is concluded that P and MP are not superior to swim-up. However, sequential MP and swim-up preparation improves yields of high quality spermatozoa from some abnormal semen samples and therefore has potential for improving fertilization rates.     

Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures

Many groups currently use two methods for the separation of motile spermatozoa, swim-up (S-up) and centrifugation on discontinuous Percoll gradient (PGC), and comparison of results indicates that PGC is superior. In this study we have attempted to identify the factors explaining this difference. This laboratory has long-standing expertise in seminology, thus the parameters of sperm morphology were the obvious first choice for detailed study. First, the respective effects of S-up and PGC on sperm morphology were analysed in different types of ejaculates: 62 semen samples with normal parameters and 41 with poor parameters. Both separation techniques resulted in improved morphology in the final preparation but only the increase of morphologically normal spermatozoa in the final Percoll suspension was significant. Second, application of these techniques in our in-vitro fertilization (IVF) programme revealed that, together with the improvement of sperm morphology, a higher pregnancy rate was obtained after PGC. The ongoing pregnancy rates per oocyte retrieval were 21.1% for the S-up technique and 33.3% for the PGC technique. These data show that spermatozoa selected by PGC present an improved morphology which we believe to be linked to improvement of the quality of the in-vitro fertilized embryos and ultimately the percentage of successful IVF results.     

Andrology: Percoll semen preparation enhances human oocyte fertilization in male-factor infertility as shown by a randomized cross-over study

The aim of this study was to compare two methods of semen preparation:multiple tube swim-up and Percoll separation, using a randomizedcross-over clinical study, in which sperm parameters, oocytefertilization rates, embryo quality and cell stage were analysed.Overall, there was no difference between the two preparationmethods in the normozoospermic cycles. In the male-factor cycles,Percoll extracted a higher total number of spermatozoa (P =0.02), increased the concentration of motile spermatozoa (P= 0.02), increased the total number of motile spermatozoa persample (P = 0.02), and enhanced the recovery rate of motilespermatozoa (P = 0.04) compared to swim-up. There was a significantimprovement in fertilization rates (P = 0.0006), in the percentageof embryos over 2-cell stage on day of transfer (P = 0.004),and in the number of replaced embryos per transfer (P = 0.01)in the Percoll as compared to swim-up cycles. There was no significantdifference in embryo quality. We conclude, therefore, that inadvanced reproductive procedures where sperm dysfunction exists,semen preparation with Percoll should replace the swim-up technique.     

Effect of swim-up, Percoll and Sephadex sperm separation methods on the hypo-osmotic swelling test.

The separation of spermatozoa from the seminal plasma is required to prepare the spermatozoa for intrauterine insemination, in-vitro fertilization and sex selection. This study evaluated the effects of sperm preparation techniques on the functional integrity of the sperm membrane, as measured by the hypo-osmotic swelling test (HOS). Thirty-four semen specimens obtained from the male partner of infertile couples were evaluated. A semen analysis and HOS test were performed on each specimen. The remainder of the specimen was divided into three equal aliquots, the first prepared using Percoll, the second using a swim-up method and the third using a Sephadex column. After the preparation, a semen analysis and HOS test was performed on each aliquot. The mean and standard deviation for the HOS test was 72.9 +/- 8.5% initially, 71.2 +/- 13.1 after Percoll, 75.2 +/- 15.1 after swim-up and 62.4 +/- 14.5 after Sephadex. Analysis of variance showed that the mean HOS score was the same after Percoll and swim-up as it was initially but significantly lower after preparation with Sephadex. There was also a higher proportion of abnormal semen specimens (HOS less than 50%) after preparation with Sephadex than after the other preparation methods. We recommend the use of the HOS test as part of a screening panel for sperm separation.     

The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization

Silane-coated silica particles (PureSperm) were evaluated as an alternative to Percoll for gradient separation of spermatozoa, for use in assisted reproduction. Recovery of motile and morphologically normal spermatozoa after using a four-layer Percoll and a two- and four-layer PureSperm gradient respectively was recorded. In-vitro fertilization (IVF) results after using PureSperm for the sperm preparation were also evaluated. No difference in sperm recovery or sperm motility was found when comparing the use of Percoll and the four-layer gradient of PureSperm. When using a two-layer PureSperm gradient, motility was significantly decreased (P < 0.05) compared to Percoll. Normal sperm morphology increased from 8-17.2% after using Percoll and to 12.7% and 11.4% after using a four-layer and a two-layer PureSperm gradient respectively. All gradient preparations showed a significant decrease in the teratozoospermia index compared to the ejaculate (P < 0.01). No significant differences in IVF results regarding fertilization and pregnancy rates were found when PureSperm or the swim-up technique were used for the sperm preparation. PureSperm seems to be an acceptable alternative to Percoll but although the percentage of sperm recovery was higher after PureSperm we still recommend the swim-up technique to be the first choice, as a higher percentage of progressive motile spermatozoa is obtained without using other chemicals than IVF culture medium.     

Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization

Human and bovine cervical mucus penetration tests (n = 57) wereperformed preceding IVF to test their prognostic value as spermfunction tests for IVF. This evaluation also induded resultsfrom conventional semen analysis and from a computerized spermanalysis system. The bovine cervical mucus penetration testwas shown to be at least as valuable as the human cervical mucuspenetration test in evaluating sperm function. The migrationdistance of the vanguard sperm (P < 0.001) and the spermdensity at a fixed migration distance in the mucus column (P< 0.05) correlated most closely with the IVF results. A clearparallelism with the out come of the ‘swim up’ techniquewas also found. Of the sperm parameters examined, only spermmotility In the ejaculate (P < 0.05) correlated significantlywith the results of IVF. It is concluded that the outcome ofa bovine cervical mucus penetration test depends on the samesperm functions as re quired for IVF. Therefore, this test maybe of predictive value in an IVF programme.     

A follow-up study of sperm preparation for IVF by swim-up in a solution of hyaluronate

Spermatozoa prepared for in-vitro fertilization (IVF) by swim-up in a balanced salt solution containing hyaluronate gave rates of fertilization, cleavage and pregnancy which were not significantly different from those obtained with sperm prepared by swim-up in standard IVF medium followed by centrifugation. However, the content of prostaglandin F2alpha in the final sperm suspension was higher using hyaluronate but this seemed to be of no consequence for IVF. Thus, preparation of normal sperm samples for IVF may be simplified by performing swim-up in a balanced salt solution containing hyaluronate.     

A prospective study to evaluate the efficacy of modified swim-up preparation for male sex selection

A previously published study reporting the use of a modifiedswim-up technique for sperm preparation prior to inseminationwhich resulted in a high percentage of male births has beencriticized for its lack of controls. The present prospectivestudy was initiated to investigate further the efficacy of modifiedswim-up preparation for male sex-selection when applied in aproperly defined control group. Our results showed that theproportion of males born in singleton pregnancies was 50% inthe group inseminated following sperm preparation with Percolland in the control group with no sperm preparation comparedwith 88.5% in the group treated with the modified method ofswim-up sperm preparation prior to insemination. This high rateof males in the group treated with modified swim-up was alsoobserved in singleton pregnancies of women taking ovulationinducing drugs (primarily clomiphene citrate). This contrastswith previous publications in which a higher rate of femaleswas found in clomiphene citrate patients using the albumin separationtechnique. How the mechanism of the swim-up procedure may resultin a high male birth rate remains unclear. A high percentageof y enriched semen was found using fluorochrome quinacrinemustard staining but this procedure may falsely stain autosomalchromosomes. If analysis using sensitive DNA probes fails toconfirm the y enrichment of the spermatozoa, one must hypothesizethat the modified swim-up procedure damages the x-spermatozoa.     

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.     

Male factor as determinant of in-vitro fertilization outcome.

The effect of different semen parameters was evaluated in 200 consecutive couples in an in-vitro fertilization (IVF) programme. All semen analyses were performed on the native aliquot of semen which was subsequently prepared and used for in-vitro insemination. Morphology evaluation using strict criteria (kappa 0.46 and r = 0.565) was compared with progressive motile sperm density (kappa 0.37 and r = 0.333) and the conventional World Health Organisation (WHO) evaluation of morphology (kappa 0.31 and r = 0.378). Results show that morphology evaluation using strict criteria is the best predictor of IVF and density of progressively motile spermatozoa can be an optional method. The combined results of strict morphology and motile concentration progressively showed that if both parameters were below the cut-off points of 5% and 3 x 10(6)/ml respectively, the fertilization rate per oocyte was very low (18%). No pregnancies were achieved in this group. When both parameters were above the cut-off points, the fertilization rate per oocyte was high (72%) (P less than 0.005) and the pregnancy rate per embryo transfer was 27%. Predictive values indicate that morphology evaluation using strict criteria and the number of progressive motile spermatozoa can be used as patient selection criteria for infertility clinics.     

Improvement of sperm quality in abnormal semen samples using a modified swim-up procedure

A modified swim-up procedure for the selection of motile andmorphologically normal spermatozoa is described. Applied toabnormal (astheno and asthenoterato) semen samples, the recoveryfigures are comparable to those obtained by other authors innormal samples. The elimination of seminal plasma from the beginningof the procedure avoids contact of spermatozoa with decapacitatingfactors.     

Andrology: Effectiveness of various sperm processing methods in removing seminal plasma from insemination media

The effectiveness of the wash and swim-up (1 and 2 wash cycle),two layer Percoll gradient, SpermPrep and underlay sperm preparationmethods in removing seminal plasma from insemination suspensionswas investigated. The number of wash cycles needed to rid spermsuspensions of seminal plasma (n = 15) was also determined.All sperm preparation methods were compared to the control washand swim-up method, performed using Earle's buffered salt solution(EBSS), without serum supplementation so as not to mask seminalplasma concentrations. Control processing comprised 1 ml semensubjected to two wash cycles in EBSS followed by a swim-up periodof 60 min under 5% CO₂ in air. The Percoll method comprised1 ml semen layered on a discontinuous 36 and 81% Percoll gradient(n = 14). In the SpermPrep method, 1 ml semen was run througha SpermPrep II column (n = 10), and underlay samples (n = 12)were processed by two wash cycles, after which sperm pelletswere resuspended in the remaining medium, layered under 1.2ml EBSS and allowed to swim up under 5% CO₂ in air for 1 h.Seminal plasma and supernatant fractions obtained after eachprocessing phase were stored at -20°C and protein concentrationswere determined by spectrophotometry. The wash and swim-up methodwas the most effective in removing seminal plasma from spermsuspensions, followed by the two-layer Percoll gradient, underlayand finally the SpermPrep II processing methods.     

Sperm chromatin packaging as an indicator of in-vitro fertilization rates

The development of a sequential diagnostic schedule for patients consulting for infertility disturbances would be an ideal method of approach for clinicians in the absence of an aetiological or pathophysiological diagnosis. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology as well as in-vitro fertilization (IVF) rates, with chromatin staining among fertile and subfertile men. Two semen smears were prepared from each specimen obtained from 72 men to record normal morphology and chromatin packaging as recorded by chromomycin A(3) (CMA(3)) staining. Following the semen analyses, the 72 men were divided into the two morphological groups, namely <4% and >4% normal forms. Significantly different percentages of CMA(3) staining (mean +/- SE) were recorded between the two morphological groups, namely 65.9% +/- 3.5 and 44.5% +/- 1.7 (P +/- 0.001). A highly negative significant correlation existed between percentage of normal morphology as recorded by strict criteria and CMA(3) staining. A highly significant and positive correlation was recorded for normal morphology and IVF rates (r +/- 0.45, P +/- 0.0001). A significant negative correlation (r +/- -0.51, P +/- 0.0001) existed between CMA(3) values and IVF rates. The discriminating power of nuclear maturity, as recorded by CMA(3) staining, to identify abnormal morphology values and poor IVF rates was calculated with receiver operator characteristic (ROC) analyses. The areas under the ROC curves were 0.86 for sperm morphology and 0. 74 for IVF rates. The calculated threshold values for CMA(3) staining to distinguish between morphology groups were 48 and 50% for IVF. Chromatin packaging assessment is a valuable addition to the sequential diagnostic programme in an assisted reproductive arena.     

A self-migration method for preparation of sperm for in-vitro fertilization

Human sperm samples (n = 211) were prepared for in-vitro fertilization(IVF) and embryo transfer by a self-migration procedure in Earle'smedium containing highly purified hyaluronic acid (Hya) (MW3 000 000) included to increase the viscosity of the medium.The method resulted in the recovery of a significantly higherpercentage of motile spermatozoa compared with the traditionalcentrifugation method, 87.5 ± 0.9% versus 76.1 ±1.3% (P < 0.001). When comparing media with and without Hyain the selfmigration method for preparation of normal spermsamples, the media containing Hya resulted in the recovery ofa significantly higher percentage of motile spermatozoa, 89.0± 0.8% versus 73.8 ± 2.0% (P < 0.001). In agroup of 80 consecutive couples entering our IVF programme,sperm samples from 44 of the men were allocated at random forthe self migration method in medium containing Hya and spermsamples from 36 men for preparation by centrifugation and swim-up.Significantly more pregnancies were achieved in the group preparedin medium containing Hya. It is concluded that self-migrationof sperm in a medium containing Hya is simple and rapid, andresults in a high recovery of motile spermatozoa which can beused for in-vitro insemination of human oocytes with favourableresults.     

Successful fertilization and pregnancy outcome in in-vitro fertilization using cryopreserved/thawed spermatozoa from patients with malignant diseases.

Cryopreservation of spermatozoa before treatment is the only proven effective method available to circumvent the sterilizing effect of therapy in some patients with malignant diseases. Because of impaired sperm quality after freezing and thawing in-vitro fertilization/embryo transfer (IVF/ET) was indicated in 10 patients (12 cycles) during 1986-1990. The patient's mean age was 33.4 +/- 1.6 years. The following diagnoses were made: seminoma (1), testicular carcinoma (3), leiomyosarcoma of the prostate (1), Wegener's granulomatosis (1), non-Hodgkin's (1) and Hodgkin's lymphoma (3). When motile spermatozoa could be recovered after thawing, the total fraction of motile spermatozoa after swim-up separation ranged from 0.2 to 4.2 x 10(6) spermatozoa/ml (eight patients, nine cycles). In all these cases, insemination was performed with multiple oocytes per dish. Fertilization was achieved when swim-up recovered a mean of 1.8 +/- 0.5 x 10(6) spermatozoa/ml and when insemination was performed with at least a calculated concentration of motile spermatozoa of 1 x 10(5) spermatozoa/oocyte. The fertilization rate of preovulatory oocytes was 60%. Four patients achieved a pregnancy: two of them delivered a single healthy baby, one delivered triplet healthy babies and one had a preclinical abortion. In two patients (three cycles), no motile spermatozoa were recovered after thawing, and micromanipulation of oocytes for assisted fertilization was performed. Although fertilized oocytes were transferred, those couples did not achieve a pregnancy. Patients with lymphopathies had the best results, whilst those with testicular neoplasms had the poorest outcome, thus suggesting a poor gametogenic function in the non-affected testis. These results give hope to some patients with malignant diseases to maintain their reproductive capacity through sperm banking and IVF/ET.     

Improvement of survival and fertilizing capacity of human spermatozoa in an IVF programme by selection on discontinuous Percoll gradients

Two techniques for the separation of spermatozoa were compared: swim-up migration (SUM) and centrifugation on a discontinuous Percoll gradient (CPG). Their respective effects on sperm motility were analysed by computer-assisted videomicrography in either normal or asthenozoospermic groups. In both groups, there was no difference in any of the motion parameters between the two treatments after 1-h incubation. However, a clear difference was observed after 24 h when excellent motility was retained only in the CPG-treated group. A total of 350 ejaculates were produced by the husbands of women undergoing oocyte retrieval in an IVF programme. Spermatozoa were treated by CPG when the infertility was due to poor quality spermatozoa (n = 91), when there was a known previous history of semen infection (n = 73) or when frozen semen, originating from a donor, was used (n = 36). In all other cases (n = 150), spermatozoa were treated by SUM. The cleavage rates obtained were 32.2, 70.1, 60.9 and 68.6% respectively in the four categories. The clinical pregnancy rates per oocyte retrieval were 19.8, 31.5, 22.2 and 18.0% respectively. Forty-eight births occurred in the CPG group: 28 boys and 20 girls, all normal. We conclude that CPG is useful, both in cases of poor semen quality and in tubal infertility, in which the clinical pregnancy rate increased significantly from 18.0 to 31.5%.     

A new sperm collection method for in-vitro fertilization: collection at tail of epididymis.

We have developed a method for the collection of spermatozoa from the tail of the epididymis in patients with severe asthenozoospermia. The treatment was applied to 25 cycles, of which 16 cycles provided spermatozoa in good condition with concentration and motility greater than 50 x 10(4)/ml and greater than 50% respectively. Spermatozoa thus obtained were subjected to IVF in 11 cycles and to ZIFT in five cycles; five IVF cases reached cleavage and two of those cases resulted in pregnancy. Two of the five ZIFT cases also achieved a pregnancy. Despite limits on frequency, the sperm collection process is thought to be very useful for the treatment of severe asthenozoospermia.     

Epididymal sperm aspiration in conjunction with in-vitro fertilization and embryo transfer in cases of obstructive azoospermia.

Epididymal sperm aspiration is a new treatment for vasal agenesis. In previous reports, epididymal spermatozoa resulted in pregnancy by utilizing in-vitro fertilization (IVF) or gamete intra-Fallopian transfer (GIFT). We sought to investigate the efficacy of epididymal sperm aspiration in conjunction with IVF in patients with congenital absence of the vas deferens or with secondary extended obstruction of spermatic ducts. Fifty-eight attempts were performed in 23 patients (25-50 years). Eight patients (34.7%) had vasal agenesis and 14 (60.8%) presented with vasal secondary extended obstruction. The sperm count was adequate (greater than or equal 20 x 10(6)/ml) in 13.8% of sperm retrievals and sperm motility of 20% was obtained in 15.5% of sperm retrievals. Fourteen attempts at IVF were performed with epididymal sperm counts of 2-44 x 10(6)/ml and motilities of 0-45%. A mean of six mature oocytes (0-13) were inseminated in each case. Five embryo transfers were performed in five patients' wives (35.7%) and two couples had an early pregnancy loss (14.2%). Epididymal sperm aspiration is an advance in treating such patients, as an adequate number of mature spermatozoa can be obtained and used for IVF. However, spermatozoa directly aspirated from the proximal epididymis and with fertilizing capacity in vitro, gave a high rate of embryo degeneration (greater than 50%) after embryo transfer.     

Quality control in an in-vitro fertilization laboratory: use of human sperm survival studies

A bioassay procedure is described for quality control testingof various disposable items used in routine IVF procedures.This biosassay is performed over 4 days and uses the survivalof human sperm in vitro at room temperature to assess whichproducts are suitable for use. New products were tested forcytotoxicity using a general screening method and subsequenthatches of every suitable item tested to detect interbatch variation.Products were considered suitable or unsuitable for use dependingupon a calculated sperm survival index. Two main types of productwere found to be cytotoxic, namely certain brands of syringeand surgical gloves, the common feature of both being the presenceof rubber components. The bioassay was also used to investigatefurther the cytotoxic effect of the powdered and starch-freesurgical gloves. The cytotoxic substances from both types ofsurgical glove were readily transferred to an embryo replacementcatheter by touch, and washing of the gloves reduced this effectonly moderately. The bioassay has proved inexpensive and convenientbut more importantly it has been invaluable for detecting potentialsources of cytotoxicity before they are introduced into a standardIVF protocol.