The chromosomal constitution of human sperm selected for motility*

FERTILITY AND STERILITY Copyright " 1986 The American Fertility Society

Vol. 46, No.4, October 1986 Printed in U.SA.

The chromosomal constitution of human sperm selected for motility*

Brigitte F. Brandriff, Ph.D.t Laurie A. Gordon, B.S. Suzanne Haendel, B.S. Linda K. Ashworth, B.S. Anthony V. Carrano, Ph.D. Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California, Livermore, California

The chromosome constitutions of sperm selected for motility according to the swimup technique were compared cytogenetically with those of sperm remaining in the semen with the use of the human sperm/hamster egg system, in which human sperm are fused with hamster eggs to give analyzable haploid chromosome complements. Three semen samples from one donor resulted in 153 chromosome complements from selected, highly motile sperm and 110 unselected, control complements. Four samples were donated by another man, from which 181 selected and 186 control complements were obtained. The frequencies of chromosomal aberrations recovered from the population selected for high motility and the unselected population were not statistically different from one another. Fertil Steril 46:686, 1986

Sperm motility is an important variable for judging the quality of a given semen sample. In vivo, only motile sperm are able to leave the seminal fluid and enter the cervical mucus. l Clinically, motility measurements have been correlated to other semen parameters, particularly morphologic features, concentration, total sperm number, and the hamster egg penetration test. 2 , 3 We studied the correlation between sperm motility and sperm chromosomal constitution by examining a population of sperm selected for high-

quality movement characteristics by the swim-up method. 4 Chromosomes were analyzed with the human spermihamster egg system introduced by Rudak et a1. 5 In this system, human sperm are fused with hamster eggs, the eggs are cultured for 14 hours to the first mitotic division and fixed, and chromosomes are analyzed by standard cytogenetic techniques. We found that highly motile sperm did not differ in types and frequencies of chromosomal abnormalities from those in the whole, unselected semen. MATERIALS AND METHODS

Received April 14, 1986; revised and accepted June 6, 1986. *Performed under the auspices of the Office of Health and Environmental Research, United States Department of Energy, by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48. tReprint requests: Brigitte F. Brandriff, Ph.D., Biomedical Sciences Division, L-452, Lawrence Livermore National Laboratory, University of California, P.O. Box 5507, Livermore, California 94550. 686

Brandriff et al. Chromosomes of motile human sperm

Semen samples were obtained from two donors whose sperm had been assessed previously and found to carry relatively high frequencies of structural chromosomal abnormalities (donor 16 ,7 and donor R7). These donors were chosen because fewer haploid chromosome complements would be needed to detect a difference in their high frequencies as a result of the swim-up proceFertility and Sterility

Table 1. Summary of Sperm Chromosome Aberrations for Donor I in Swim-up Versus Whole Semen Fractions Sperm pre/haration me od Swim-up Whole Total

n

Cells with structural aberrations

Hyperhaploid cells

Hypohaploid cells

Total abnormal cells

a

153 110 263

No.

%

No.

%

No.

9 10 19

5.9 9.1 7.2

2 0 2

1.3 0.0 0.8

1 1 2

%

No.

%

0.7 0.9 0.8

12 11 23

7.8 10.0 8.7

"Total number of sperm chromosome complements analyzed.

of their sperm cells (donors I and R, respectively7) provided a new series of semen samples to study the effect of selection for increased sperm motility on sperm chromosome abnormalities. Both donors had unremarkable personal and medical histories, except donor R had a vasectomy reversal 4 years before sampling. 7 Three semen samples from donor I were obtained over a period of 13 months, yielding 153 haploid chromosome complements from sperm that had been allowed to swim up into medium and 110 complements from sperm that had not migrated out of the whole semen (Table 1). Three samples obtained over a 2-month period from donor R yielded information on 181 swim-up and 107 whole sperm (Table 2). An additional 79 sperm chromosome complements from donor R were obtained subsequently from sperm capacitated in TEST-egg yolk buffer lO as a control on the whole-sperm results, because the historical controls for this donor were capacitated in the TEST buffer rather than in the standard high HSA medium. Historical control data for both of these men are summarized for comparison (Table 3). No statistically significant differences were found for either donor in the frequencies of abnormal chromosome complements obtained from sperm that had been isolated by the swim-up technique, compared with sperm remaining in the whole semen (z-test for independent proportions). Although in both individuals there was a slight tendency to decreased structural abnormalities in the swim-up sperm, this trend was further

dure. Samples were collected by masturbation into sterile plastic containers and delivered to the laboratory. Sperm differed from sample to sample in terms of initial quantity and quality of motility. Highly motile sperm were selected according to a swim-up technique similar to that of Gould et a1. 4 Two milliliters of Biggers, Whitten and Whittingham (BWW) mediums containing 0.3% human serum albumin (HSA, fraction V, Sigma Chemical Company, St. Louis, MO) was placed into sterile plastic centrifuge tubes, and an aliquot of 0.5 to 1 ml of semen was carefully expelled into the bottom of the tube from a sterile pipette. The tubes were tilted gently at an angle in a Petri dish to increase the surface area of semen in contact with the medium. After 1 hour at 37°C, the top layer of medium, containing sperm that had migrated out of the whole semen, was removed without disturbing the bottom semen layer. Aliquots of both the swim-up and whole semen were then washed and capacitated as described earlier. 6 An additional control population for donor R was obtained by capacitating sperm in TESTris (TEST) egg yolk buffer9 stored overnight at 4°C, as described previously.lO Egg preparation, culture, fixation, and analysis were conducted as described before. 6

RESULTS Two donors previously assessed and found to have structural abnormalities in 9.1% and 15.8%

Table 2. Summary of Chromosome Aberrations for Donor R in Swim-up, Whole Semen, and TEST-Buffer Fractions Sperm pre/haration me ad

na

Swim-up Whole TEST-buffer Total

181 107 79 367

Cells with structural aberrations

Hyperhaploid cells

Hypohaploid cells

Total abnormal cells

No.

%

No.

%

No.

%

No.

%

15 11 8 34

8.3 10.3 10.1 9.3

1 0 0 1

0.6 0.0 0.0 0.3

3 1 2 6

1.7 0.9 2.5 1.6

18 b 12 10 40

9.9 11.2 12.7 10.9

"Total number of sperm chromosome complements analyzed. bOne cell contained both a structural and a numeric aberration. Vol. 46, No.4, October 1986

Brandriff et a1. Chromosomes of motile human sperm

687

Table 3. Summary of Historical Data for Two Donors

Donor

n

I R

340 279

Cells with structural aberrations No. %

31 44

9.1 15.8

Hy~rhap-

100d cells No. %

1 1

0.3 0.4

Hypohaploid cells No. %

4 5

1.2 1.8

Data from Brandriff et aU

minimized when all abnormal complements (structural and numerical) were considered together. There were also no differences in donor R from sperm capacitated in high-albumin BWW and those capacitated in the TEST-yolk buffer. However, although the combined data from donor I were not different from results obtained 20 and 36 months earlier6, 7 donor R showed a statistically significant decrease in the frequency of sperm with structural abnormalities, compared with samples obtained 6 months earlier (44/279, or 15.8% versus 34/367, or 9.3%; P < 0.02, z-test for independent proportions). Although there was an unexplained decrease in the overall frequency of structural aberrations, all classes of chromosome abnormalities seen previously were seen again, including a low frequency of chromosome exchanges (dicentrics and translocations) seldom seen in sperm from normal men. DISCUSSION

One of the principle characteristics of "good" sperm necessary for fertilization is high motility. It has been shown in vivo that a vanguard population of highly motile sperm migrate out of the semen into the cervical mucus and are followed by a somewhat slower, albeit still motile, group.ll Transport to the site of fertilization depends on sperm motility, because immotile sperm are unable to penetrate the cervical mucus. 1 The small population of sperm that actually reaches the oviduct is motile and presumably has more morphologically normal sperm 12 than the initial seminal population. Although some researchers assert that this subpopulation is actively selected by the female reproductive tract in women (e.g., by cervical mucus l3 ) as well as in other species (e.g., uterotubal junction in the mouse I4 ), others maintain that the sperm effect their own selection by being physiologically advantaged, particularly by the quality of motility they exhibit. 12 Further, nonmotile sperm 15 or sperm with lowerquality motilityll tend to be abnormally shaped. 688

Brandriff et aI. Chromosomes of motile human sperm

An important unanswered question is whether sperm that are superior phenotypically, as expressed by motility characteristics, are superior genetically as well. We used the human sperml hamster egg system to compare chromosome complements from populations of motile sperm selected by the swim-up method with the unselected sperm from the whole semen. The swim-up technique is somewhat analogous to the in vivo selection process for motility taking place in the cervix, although it is, of course, unknown whether the same population of sperm swims into the BWW medium as swims into the cervical mucus from the semen. We found that the population of sperm selected on the basis of motility had the same frequency of chromosomally abnormal sperm as the population of sperm "left behind" in the whole semen. Sperm separated by an albumin density gradient procedure, an alternative procedure that greatly enriches the population of highly motile sperm, also did not show a statistically significant difference in sperm chormosome aberrations between processed, highly motile populations and the sperm from unprocessed, whole semen. 16 In vitro studies with the human spermlhamster egg system are limited because it is not known what characterizes the individual human sperm that interact with the hamster eggs. If only the most motile sperm in whole semen ultimately fuse with the eggs, it would not be surprising to find no detectable difference in the chromosome constitution of the two populations. However, we do not know what role motility plays with this method, because transport through the female tract is not a factor. It is known that motility is not strictly necessary for sperm to function in this system. Immotile sperm with a variety of sperm tail abnormalities have been shown to bind to hamster eggs, which is a necessary first step toward fusion. 17 Sperm from a patient with Kartagener's syndrome in which there is a complete absence of motility due to ciliary dysfunction underwent capacitation and the acrosome reaction and ultimately fused with hamster eggs. 18 In our studies, sperm recovered from the TEST-yolk buffer generally had poor motility yet showed no diminished frequencies of fusion and subsequent production of chromosomes. Sperm prepared in this manner interact with hamster eggs as well as, if not better than, sperm capacitated by the standard high HSA procedure,10 suggesting that physiologic factors other than motility are more Fertility and Sterility

important in determining which sperm will fuse with hamster eggs to produce sperm chromosomes. Animal data provide supporting evidence that chromosomally abnormal sperm can participate in fertilization. With the use of a low sperm to egg ratio in vitro, Thadani 19 showed that most mouse sperm are competent to fertilize, provided they have access to the egg surface. Foldesy et al. 20 compared naturally fertilized rabbit eggs and eggs inseminated by sperm placed directly into the oviduct and found no difference in the survival of these eggs; they concluded that the sperm selected by the reproductive tract were not genetically elite. With the use of mice heterozygous for Robertsonian translocations, Redi et al. 21 showed that although the female reproductive tract selected against abnormally shaped sperm, morphologically normal sperm carrying gross genome imbalances were able to reach the fallopian tubes. On the basis of our data, we cannot definitively conclude that there are no genetic differences between motile and nonmotile sperm, but it seems obvious that motile sperm can carry chromosomally abnormal complements. The question of the genetic quality of motile populations of sperm is especially important inhuman in vitro fertilization programs (IVF). For IVF, it is important to use highly motile sperm,22 and some variation of the swim-up procedure often is used to select promising sperm populations for such programs. 23 Chromosomal abnormalities are one possible reason for the low success rate of implantation after IVF. 24 Direct chromosome analysis of human-human embryos have been limited to polypronuclear enibryos, which are clearly unusable for embryo transfer. These studies are difficult to do, and the available data base is still small. Both Angell et al. 24 and Rudak et al. 25 found high frequencies of aneuploidy in karyotyped IVF embryos. Although we found more structural than numerical abnormalities, our data provide evidence that highly motile sperm can carry chromosomal abnormalities and still function physiologically in the in vitro system. Similar defects might reduce the chances for implantation after IVF, because embryos with such defects probably would be arrested at an early stage of development. Acknowledgments. We thank Andrew J. Wyrobek, Ph.D., for help in obtaining samples; Barton L. Gledhill, V.M.D., Ph.D., R. Lowry Dobson, M.D., Ph.D., and Daniel Pinkel, Vol. 46, No.4, October 1986

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