Semen Preparation

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Tohoku J. Exp. Med., 1992, 168, 583-590

Methods of Semen Preparation forIntrauterine Insemination and SubsequentPregnancy Rates

PANAYIOTISM. ZAVOSand GRACE M. CENTOLAtUniversity of Kentucky, Andrology Institute of Lexingtonand Central Baptist Hospital, Lexington, KY 40502, USA;and t Department of Obstetrics and Gynecology, Universityof Rochester Medical Center, Rochester, NY 14642, USA

ZAVOS, .M. and CENTOLA, .M. Methods of Semen Preparation for Intra-uterine Insemination and Subsequent Pregnancy Rates. Tohoku J. Exp. Med.,1992, 168 (4), 583-590 Semen for insemination, either intrauterine or in vitro,must be prepared to remove seminal plasma products and/or select the healthierpopulation of sperm prior to use. Traditionally, a double wash technique isperformed, with or without subsequent swim-up to isolate the motile fraction ifnecessary. More recently, the use of the SpermPrep filtration method has gainedacceptance, with the benefits of removal of leukocytes and seminal debris from thespecimen as well as enhancement of overall sperm quality. In the current studywe compared the traditional double wash method without the swim-up to Sperm-Prep filtration. Intrauterine inseminations (IUI's) were performed in 307 cycleson 148 infertile couples at two different infertility centers in the USA. Aftercomplete diagnostic evaluation the couples were offered JUT before proceeding toany other form of assisted reproductive technologies. Semen samples were prepar-ed in human tubal fluid media supplemented with 5% human serum albumin(HSA ; location 1) or in Ham's F-10 media supplemented with 3% HSA (location2), either with the SpermPrep filtration method (ZBL, Inc., Lexington, KY 40523,USA) or the double sperm wash (SW) procedure. Similar sperm numbers wereused for the JUT procedure in both treatment groups and locations. The Sperm-Prep method resulted in significantly higher pregnancy rates (PR) than the SWprocedure, independent of location. The clinical pregnancy rates per cycle werestatistically lower (p


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584 P.M. Zavos and G.M. Centolaartificial reproductive techniques. SpermPrep ; spermatozoa ; spermwash ; semen fitration ; IUI ; pregnancy rates

Improvements in rates of conception could be realized if spermatozoa areselected on the basis of their motility, progressive motility, and morphologicalcharacteristics. Such selection of spermatozoa could be properly applied at thetime of intrauterine insemination (JUT) or other forms of assisted reproductivetechniques (ART) because the seminal plasma and other background materialsand debris should be removed from the spermatozoa before these procedures areperformed.A number of manipulative techniques for fresh semen are currently availableto remove the undesirable spermatozoa, debris and other factors and to increasesperm quality. These various techniques include the most popular ones, simplesperm wash, swim-up or sperm rise methods (Centola and Zavos 1991), andswimdown or sedimentation type methods (Dmowski et al. 1979). Less popularmethods include Ficoll centrifugation (Free et al. 1991), Percoll density gradient(Kaneko et al. 1980; Horvath et al. 1989), and Sephadex or glass wool fiberfiltration (Katayama et al. 1989; McClure et al. 1989). It should be emphasized,however, that with many of these manipulative techniques the increase in spermquality is often achieved at the expense of numbers of recovered spematozoa thatmay not be especially advantageous for patients with various seminal deficienciessuch as oligozoospermia. Also, equally important, the relatively lengthy timeperiod required to perform these procedures is additionally disadvantageousbecause the life expectancy of low quality spermatozoa may be limited.

Recently, a new technique, SpermPrep (ZBL, Inc., P.O. Box 23777, Lexin-gton, KY 40523, USA), has been introduced that yields higher levels of spermrecovery and is rapid and reproducible (Moslein-Rossmeissl and Taubert 1989;Pickering et al. 1989; Pretorius and Kruger 1990). Because of these advantages,the SpermPrep technique could have significant effects in the manner that speci-mens are prepared and improved before their use in the various artificial (non-coital) reproduction procedures. The present study was designed to compare thetraditional double sperm wash method to the new SpermPrep filtration techniquewhen processing semen for JUT and their possible effects on pregnancy rates.

MATERIALS ND METHODSEjaculate collection

Ejaculates were collectedfrom all the males who participated in the current study.Ejaculates were collectedwith 2-4 days of abstinenceeach time. Patients collected heirejaculateseither by using the seminal collectiondevice(SCD) at intercourse Quinlivanetal. 1982; Rogerset al. 1991)or via masturbation.Semen evaluation

After semensampleswere producedand completely iquefied within 15-30min), each


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Semen Preparation for Intrauterine Insemination 585specimen was evaluated according to standard procedures recommended by the WorldHealth Organization (WHO) using a phasecontrast microscope (Russell and Rogers 1987).Semen measures included volume, sperm count per milliliter, percentage sperm motility,grade of sperm motility (Sofikitis et al. 1992), and sperm morphologic features. All seminalparameters were evaluated by the same technicians as per location. Spermatozoa wereprepared either via the SpermPrep method or the double sperm wash method, evaluated andthen used for IUI.SpermPrep filtration

The SpermPrep was used according to the manufacturer's specifications and instructions(ZBL, Inc.) and also according to previously described methodology (Zavos 1985, 1991,1992a; World Health Organization 1987). It should be emphasized that proper standardlaboratory techniques were employed in our laboratory during the filtration process andwere applied similarly during the sperm wash. Those techniques included complete steril-ity and maintenance of all semen diluents, the SpermPrep filter, and all other materialswithin a temperature range of 30 to 35C. Filtration was begun by placing the properlyresuspended spermatozoa in the filter. At the end of filtration (10 to 15 min), the filtratewas centrifuged and resuspended in Ham's F-10 or human tubal fluid (HTF), assessed andused for IUI purposes.Double sperm wash

The semen was diluted 1:1 with the buffer of choice (HTF or Ham's F-10), mixed andcentrifuged at 300 x g for 10 min. This process was repeated and the generated pellet wasgently resuspended in the buffer of (HTF or Ham's F-10) choice, evaluated as previouslydescribed (Russell and Rogers 1987) and used for IUI purposes. Adequate numbers ofmotile spermatozoa were obtained from the double wash-resuspended aliquot for IUI tomatch the numbers of total motile sperm used in the SpermPrep reconstituted aliquot forJUT purposes.Patient group

One hundred-forty eight couples participated in this study at two different locations inthe USA (Table 1). The mean female age was 28.1 years (range 24 to 37), and the meanduration of the infertility was 3.8 years (range 1 to 9 years). Each couple underwent anexamination that included a medical history, physical examination, semen analysis, evalua-tion of basal body temperature, serum progesterone determinations in the luteal phase of themenstrual cycle, postcoital tests, hysterosalphingogram, and laparoscopy. Couples witheither tubal damages, subnormal semen samples according to the WHO (Russell and Rogers1987), or immunological infertility were not included in the study.Direct JUT insemination with controlled ovarian stimulation was mostly offered tocouples with unexplained infertility, minimal endometriosis or with cervical factor beforeproceeding to any other form of artificial reproductive technology.Study design

The couples were randomized before the first treatment cycle into two groups, one groupbeing treated with sperm prepared by the traditional double wash method and the otherwith sperm prepared via the SpermPrep filtration method. In subsequent treatment cycles,the sperm preparation method was not alternated. The sperm parameters before and afterpreparation and the post-preparation parameters were compared. The pregnancy rates afterdirect JUT using either the double wash or SpermPrep preparations were also compared. Incomparison of sperm parameters, the Student's t-test was used. Biochemical pregnancieswere only included in the calculations of sperm parameters.


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586 P.M. Zavos and G.M. Centola

Performance of IUIIntrauterine insemination (JUT) was performed following proper ovulation predictionsand the luteinizing hormone (LH) was determined in serum or urine (Ovusticks ; Mono-

clonal Antibodies, Mountain View, CA, USA). In ovulation induction patients, at leasttwo follicles of a minimum 15 mm were present. Ovulation was then induced by aninjection of 10,000 IU human chorionic gonadotropin (hCG, Profasi ; Serono, USA) and theinseminations were performed 22 to 42 hr (mean 38 hr) after the hCG injections. In cyclesin which an endogenous LH surge was detected (Ovusticks), it was supported by an hCGinjection (10,000IU), and the insemination was performed the day after. Clinical preg-nancies were always verified by ultrasound scanning in the 7th week of gestation. If thepregnancy could not be verified by ultrasound, it was regarded as biochemical.Insemination

Patients undergoing JUT were instructed not to have intercourse for 2-3 days before theday of semen collection and insemination. A mean volume of 0.4 ml (range 0.3 to 0.5 ml)of washed sperm sample was aspirated into an 11.5 cm Tomcat catheter (Sherwood Medical,St. Louis, MO, USA) which had been fashioned to the natural curvature of the uterinecavity. The cervix was exposed with a bivalve speculum and the tip of the catheter waspassed into the uterus until it lay about 0.5 cm from the top of the uterine cavity in thefundal region. The sperm fraction was then gently expelled and the catheter was gentlywithdrawn. The patients rested in a supine position for 20-30 min after the inseminationwas performed.

RESULTSClinical data of the patient population that participated in the current study

is shown in Table 1. There were no differences in the ages between the two sementreatments of the husbands or wives, neither within each location nor between thetwo locations (p >0.05). Similarly, no differences were noted in the time intervalthat the couples were trying to conceive between the two treatments, either withinlocation or between locations (p >0.05).

The semen samples were prepared via the SpermPrep method and by thedouble sperm wash technique. The different sperm parameters before and afterpreparation are presented in Table 2. Before preparation the semen parameters

TABLE1. Clinical data of patient populations studied (means +s.E.)


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Semen Preparation for Intrauterine Insemination 587for the 123 semen samples prepared by the SpermPrep and the 184 semen samplesprepared via the double sperm wash differed insignificantly. After preparationwith the SpermPrep method, the mean sperm density was lower (p


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those inseminated with SpermPrep recovered sperm. This was necessary toovercome the lower motility ratio (51.4+6.8%) of the double washed spermaliquots. The percentage recovery of spermatozoa was higher with the doublewash (83.8%) than with the SpermPrep method (54.1%). The differences in therecorded recovery are shown in Table 2. The conception rates for sperm prepara-tions with SpermPrep and double sperm wash by location and within locationwere different (p


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intrauterine insemination. Androk gia, 21, 519-523.9) Pickering, S.J., Fleming, T.P., Braude, P.R., Bolton, V.N. & Gresham, GAG. (1989)Are human spermatozoa separated on a Percoll density gradient safe for therapeuticuse? Fertil. Steril., 51 1024-1029.

10) Pretorius, E. & Kruger, T. (1990) Fluorochrome acridine orange test. In :.HumanSpermatozoa in Assisted Reproduction, edited by A.A. Acosta, R.J. Swanson, S.B.Ackerman, T.F. Kruger, J.A, van Zyl & R. Menkveld, Williams and Wilkins, p. 233.11) Quinlivan, W.L.G., Preciado, K., Long, T.L. & Sullivan, H. (1982) Separation of

human X and Y spermatozoa by albumin gradients and Sephadex chromatography.Fertil. Stern., 37, 104-107.12) Rogers, B;J., Wamil, B. & Zavos, P.M. (1991) Comparison of the fertilizing potentialof human spermatozoa processed by swim-up or Sephadex filtration columns. Pro-

ceedings of the Seventh World Congress on IVF and Assisted Procreation, 318.13) Russell, DL. & Rogers, B.J. (1987) Improvement in the quality and fertilization

potential of a human sperm population using the rise technique. J. Androl., 8, 25-33.14) Sofikitis, N., Miyagawa, I. & Zavos, P.M. (1992) The SpermPrepTM iltration methodselectively entraps single stranded DNA spermatozoa. Jpn. J. Fertil. Steril., 37,10-13.15) World Health Organization (1987) WHO Laboratory Manual for the Examination

of Human Semen and Semen-Cervical Mucus Interaction, 2nd edition. Cambridge,The Press Syndicate of the University of Cambridge, p. 62.16) Zavos, P.M. (1985) Characteristics of human ejaculates collected via masturbationand a new Silastic seminal fluid collection device (SCD). Fertil. Steril., 43, 491-492.17) Zavos, P.M. (1991a) Selection de spermatozoides viables a partit d'echantillons despermes humains congelesdecongeles : Comparaison de la methode du "swim-up" et

d'une nouvelle methode de filtration : le SpermPrepTM. Contracept. Fertil. Sex., 19,293-297.

18) Zavos, P.M. (1992b) Preparation of human frozen-thawed specimens using theSpemPrepTM filtration method : Improvements over the conventional swim-upmethod. Fertil. Steril., 57, 1326-1330.19) Zavos, P.M. (1992) Amelioration des caracteristiques qualitatives des spermat-ozoides humains cryoconserves apres recuperation par la methode de filtration Sperm-PrepTM I. Contracept. Fertil. Sex., 20, 541-545.

20) Zavos, P.M. & Goodpasture, J.C. (1989) Clinical improvements of specific seminaldeficiencies via intercourse with a seminal collection device versus masturbation.Fertil. Steril., 51, 190-193.21) Zavos, P.M. & Centola G.M. (1990) Qualitative and quantitative improvements inhuman spermatozoa recovered via the swim-up and a new semen filtration method.Infertility, 13, 25-34.

22) Zavos, P.M. & Cohen, MR. (1980) The pH of cervical mucus and the postcoital test.Fertil. Steril., 34, 234-238.23) Zavos, P.M., Sofikitis, N., Toda, T. & Miyagawa, I. (1991) Improvements in qualita-tive characteristics of cryopreserved human spermatozoa following recovery via theSpermPrepTM I filtration method. Tohoku J Exp. Med., 163, 283-290.24) Zavos, P.M., Sofikitis, N., Toda, T. & Miyagawa, I. (1992) Selection and preparationof human spermatozoa for artificial insemination using the new and improved Sperm-PrepTM I filtration method. Jpn. J. Fertil. Steril., 37, 14-19.

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