ARTIFICIAL INSEMINATION HOMOLOGOUS WITH OLIGOSPERMIC SEMEN SEPARATED ON ALBUMIN COLUMNS*

Transcription

1 FERTILITY AND STERILITY Copyright 1979 The American Fertility SOciety Vol. 31, No. I, January 1979 Printed in V.SA. ARTIFICIAL INSEMINATION HOMOLOGOUS WITH OLIGOSPERMIC SEMEN SEPARATED ON ALBUMIN COLUMNS* W. PAUL DMOWSKI, M.D., PH.D.t LILIANA GAYNOR, M.D. MARY LAWRENCE, B.S. RAMAA RAO, M.D. ANTONIO SCOMMEGNA, M.D. Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center, University of Chicago Pritzker School of Medicine, Chicago, Illinois 6616 Semen samples obtained from 27 infertile men were separated on human serum albumin (HSA) columns prior to artificial homologous insemination (AIH). The columns contained either a single 7.5% HSA layer or two 17.5% and 7.5% HSA layers. Separated specimens were free of seminal debris, had significantly improved motility and progressive drive, and had a decreased percentage of abnormal forms. The total sperm count was also significantly decreased after separation, especially with the two-layer technique. No conceptions occurred in 21 couples when two-layer separation was used, but four pregnancies in 12 couples resulted from AIH with semen separated on the single-layer column. The mean total count of motile sperm obtained for AIH was 5 million with the two-layer and 31 million with the one-layer technique. Fertil SteriI31:58, 1979 Male factor is a significant cause of infertility in about 4% of childless couples. The severity of the problem is reflected in the semen analysis, which may indicate absolute sterility as in patients with azoospermia or a relatively minor problem as in patients with mild oligospermia. In the majority of infertile males morphologically normal, motile spermatozoa are intermixed in the semen with morphologically abnormal and nonmotile forms, epithelial cells, white blood cells, amorphous material, and other cellular and noncellular debris. It can be assumed therefore, that separation of motile, morphologically normal spermatozoa from the abnormal forms and debris prior to homologous artificial insemination (AIH) should improve chances for conception in such cases. Received July 27,1978; accepted September 11, *Presented in part at the First International Symposium on Artificial Insemination Homologous and Male Subfertility, Bordeaux, France, May 6 and 7, treprint requests: W. Paul Dmowski, M.D., Ph.D., Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center, 29th Street and Ellis Avenue, Chicago, Ill A variety of techniques for sperm separation prior to AIH have been reported claiming varying rates of success in improving semen quality and facilitating conception. Most of these techniques, however, have not been widely accepted because of lack of reproducibility. A simple, natural form of sperm separation through collection of different fractions of the ejaculate, the so-called split ejaculate, has been widely used in clinical practice with reasonably good results. With this method seminal plasma is separated more or less efficiently from the sperm. The technique has been considered of value specifically in patients with inflammatory processes in the accessory sex organs. With this technique, a concentrated semen specimen, relatively free of seminal plasma, becomes available for AIH. However, no change in the percentage of abnormal or nonmotile spermatozoa and little change in the amount of sperm debris have been noted. A technique recently reported by Ericsson et al. 1 allows separation of progressively motile sperm from nonmotile forms and debris. Other investigators 2-4 using the technique of Ericsson et al. 1 have 58

2 Vol. 31, No.1 AIH WITH OLIGOSPERMIC SEMEN 59 confirmed the isolation of a clean semen fraction free of debris and containing up to 9% of motile sperm with good forward progression. Encouraged by these reports using two modifications of the technique of Ericsson et all we have performed sperm separation prior to AIH in 27 couples with male factor. MATERIALS AND METHODS Twenty-seven couples with a significant male factor of infertility as evidenced by oligospermia and/or low motility as well as a high percentage of abnormal forms on repeated semen analysis are the subject of this report. The mean age for both wife and husband was 31 and the average duration of marriage was 6.2 years (Table 1). All couples complained oflong-standing infertility and all had been treated previously with various regimens including AIH with split semen specimens. In 5 of the 27 couples no other causes for their infertility were identifiable; in the remaining 22 couples various infertility factors were present in the wife. Ovulatory dysfunction in the form of anovulation or sporadic ovulation was found in 17 wives; relative tubal factor in the form of tubal phimosis or peritubal adhesions but with hysterosalpingographic evidence of tubal patency was observed in 4; mild endometriosis in 4; and a short luteal phase in 2. Each wife with female factors contributory to infertility was treated simultaneously for her disorder. Ovulation timing, utilizing basal body temperature (BBT) records, cervical mucus changes, and vaginal cytology, was conducted during all cycles of insemination. Treatment with clomiphene citrate, human chorionic gonadotropin, or both, was used in patients with ovulatory dysfunction and short luteal phase. Hydropertubations were performed in patients with the relative tubal factor. Insemination At the time of expected ovulation and after at least 3 to 4 days of sexual abstinence, the husband TABLE 1. Characteristics of Twenty-Seven Couples Undergoing Sperm Separation for Male Factor Mean wife's age (range) Mean husband's age (range) Mean duration of marriage (yr) Mean duration of infertility (yr) Couples with male factor alone Couples with additional female factors Anovulation Relative tubal factor Endometriosis Short luteal phase 31 (25-38) 31 (26-39) was instructed to produce a semen sample by masturbation. Semen analysis as well as the sperm separation procedure were then carried out as outlined below. Insemination (AIH) was performed with the separated semen specimen in a final volume ranging between.5 and 1 ml which was injected into the cervical canal at the level of the internal os. If ovulation was unpredictable, AIH was repeated one to three times during the cycle until ovulation occurred. In some patients, when ovulation occurred during the weekend when the separation procedure was not available, AIH with split_ semen was carried out. Separation Technique The sperm separation procedure was performed as soon as liquefaction of the semen was complete, and all manipulations were conducted at room temperature. Two modifications of the technique of Ericsson et au were used. Two-Layer Technique. The liquefied semen sample was diluted 1:1 with Tyrode's solution and then centrifuged at 36 rpm (16 x g) for 15 minutes. The supernatant was discarded and the spermatozoa were resuspended in Tyrode's solution to contain approximately 6 x 1 6 of spermatozoa in each m1. Aliquots of.5 ml of sperm suspension were then layered onto isolation columns, the number of columns being determined by the total number of spermatozoa in the sample. Each isolation column contained two discrete layers of human serum albumin (HSA). The lower layer consisted of.5 ml of 17.5% HSA and the upper layer consisted of 1 ml of 7.5% HSA. After 1 hour the top layer containing the sperm suspension was removed and the remainder of the column was left for another 3 minutes. At that time the 7.5% and 17.5% HSA layers were collected separately and centrifuged at 36 rpm (16 x g) for 15 minutes. The supernatants were removed and the spermatozoa were resuspended in Tyrode's solution. The sperm suspension obtained from the 17.5% HSA layer was used for insemination in a total volume ranging between.5 and 1 m1. The time between production of the semen sample and insemination was 3 to 4 hours. At each step in the procedure the total number of spermatozoa and the percentage of motile sperm were determined in a hemocytometer. The mean drive (that is, the speed of progressive motion across a distance of.5 mm) was measured with a stopwatch. The percentage of abnormal forms was determined subsequently on slides stained for spermatozoa. Also at each step in the procedure, smears were

3 6 DMOWSKI ET AL. January 1979 prepared for fluorescent Y -body counts. For that S tl purpose the slides were air-dried and fixed in 95% ' > ethanol. Subsequently they were stained with quinacrine dihydrochloride, and the percentage of > Y sperm was detemined by fluorescence micros- ''; tl copy..2 'g One-Layer Technique. This technique was es- <!: sentially similar to the one described above with ;;j s' = se tl the exception that, instead of two layers of HSA, ti';.ece < «C'l one layer of7.5% HSA was used in the column. All other steps in the procedure were identical. :g :::; tl RESULTS t- The total sperm count, sperm motility, percent- ] tl 8 x age of abnormal forms, progressive drive, and :ll centage of Y spermatozoa were the parameters r- < \:: Ei evaluated at each step in the separation procedure. tl «' These data for the initial semen sample and for the <I' ' : 7.5% and 17.5% HSA fractions are shown in Table ;, 2. '. tl tl The total sperm count and the percentage of < C'! motile sperm were lower in both oligospermic.,., ;::, groups as compared with normal males, whereas <!: < tb ;;j Sa; the percentage of abnormal forms was higher and. = s E ti tl.e the forward progression of the sperm was more ti'; >., «<I' C'l ;,,..: sluggish. There was a progressive increase in the E-< &. S percentage of motile spermatozoa in the 7.5% and tl tl.s :::; <I' 17.5% HSA fractions along with an increase in the tl > t- i::i speed offorward progression and a decrease in the > C'l <I' percentage of abnormal forms.. ] tl tl e 2'8 x Both fractions, but specifically the 17.5% HSA t-,...; tl ';. e- fraction, were markedly freer of sperm debris, (J.) > Ei epithelial and white blood cells, and amorphous E: V V V tl.,... Q..,Q..,Q.., ' elements. A significant decrease in the total sperm <I' ' count was observed in both fractions, but was more C'i C'l C'l s:: s:: s:: marked in the higher concentration of HSA. El El El...:l... tl tl!xi Motile sperm recovery was 21% in the 7.5% HSA... -< cq E-< C'! ' '., fraction and only 1% in the 17.5% HSA fraction. t;-;a; s:: s:: s:: The mean total count of motile sperm used for AIH C'l., r- 'Sc'Sc 'Sc Ei.,.,-1. in couples subjected to the two-layer technique 1;] Ifr-S Or- tl tl S S S was just over 5 million, while in those in whom the.a ee < r- «<I' <I' <os o:s..s::..s::...,..., one-layer technique was used the mean count was III C'l > El El El 31 million. tl tl cbcl:: :::;...,...,..., Altogether 122 AIH procedures with separated > <I' <I' s:: s:: s:: as as as semen were performed in 9 cycles of 27 patients, t;:l - ;::<... resulting in 4 pregnancies and a pregnancy rate of,> oot E <1' ot- OJ OJ., 14.8% (Table 3). There was 1 conception in 5 2'8 x,..!. C'lti >,>,>, --- couples with male factor alone and there were 3 in taa;a; 22 couples with both male and female factors. :o:a OJ., OJ ::: There were no pregnancies in 21 couples in whom s:: d'...,...,..., as as as tl ti rn.,., the two-layer technique was used. Q) +:J M'' --- Q) 1-l ' -.. E!C'lS:: ::l ::l ::l In the four patients who conceived, two concepas as s:: 7o.lIasO 7o.lI@O tat;; ElU) tions occurred in the first cycle of AIH, one in the ElU)»>.c c E-< second, and one in the third (Table 4). Three con-

4 Vol. 31, No.1 AIH WITH OLIGOSPERMIC SEMEN 61 It TABLE 3. Results of AlH with Separated Semen in Twenty- Seven Couples with Male Factor AIH Total Concep- Preg- No. Cycles AIH tions nancy rate % Couples with male factor alone Couples with additional female factor Total Two-layer technique One-layer technique ceptions occurred in a cycle in which one AIH separation procedure was performed, while one patient conceived with three AIH procedures performed in the same cycle. In two patients an AIH split procedure was also performed in addition to AIH with separated semen in the conception cycle. The total count of motile sperm in the AIH specimen exceeded 1 million in three patients who conceived and was just over 5 million in one. All pregnancies terminated in the delivery of healthy infants-two males and two females. DISCUSSION The literature abounds with reports on techniques designed to separate normal from abnormal spermatozoa in subfertile husbands prior to AIH. Several physical separation methods such as filtration, centrifugation, washing and pooling, and chromatography have been reported with variable claims of success but generally with little or no reproducibility. Most of the techniques are complicated, their results have been erratic in the hands of different investigators, and none has been generally accepted. Our initial experience with the technique of Ericsson et al. I as applied to normal semen specimens indicated that this procedure could be used to isolate a motile sperm fraction in subfertile males. Reports of other investigators on the effectiveness of this method in isolating a clean semen fraction, free of debris and rich in. highly motile sperm, confirmed the original report of Ericsson et al. l and have been in agreement with our experience. On the recommendation of Ericsson we decided to use in the isolation columns two layers ofhsa: 7.5% and 17.5%, concentrations somewhat lower than those used for Y sperm separation. The selection oflower concentrations ofhsa (7.5% and 17.5% versus 1% and 2%) was based on a presumption that the spermatozoa of oligospermic men have a decreased ability for progressive motion through denser media as compared with those of men with normal fertility. As shown in Table 2, our results indicate a significant increase in sperm motility and a decrease in the percentage of abnormal forms in the second HSA fraction. Simultaneously, an improvement in the speed of forward progression of the sperm was observed. However, this improvement was accompanied by a significant decrease in the total sperm count. We were able to isolate only a small percentage of the highly motile spermatozoa from the original samples. Since the initial motile sperm count was low, we were not able to recover after separation with the two-layer technique numbers of motile spermatozoa adequate to achieve fertilization. Not a single conception occurred with 94 procedures performed in 68 cycles of 21 wives. From analysis of sperm separation parameters (Table 2) it became apparent that a significant proportion of motile spermatozoa was still present in the lower HSA concentration at the completion of separation. We decided then to apply a one-layer separation only, using 7.5% HSA concentration. With the one-layer technique a significantly higher number of motile spermatozoa could be isolated and the separated specimens were free of debris, had acceptable motility, and a low percentage of abnormal forms. With the one-layer technique we were able to achieve 4 conceptions in 12 patients for a conception rate of 33%. Our observations regarding the usefulness of a single- TABLE 4. Analysis of Four Conceptions with Separated Semen (One-Layer Technique) in Couples with Male Factor Day ofbbt AIH Specimen Total % No. No. of Day of Dip Rise sperm % Abnormal Drive Pregnancy AIH cycles AIH count Motile forms outcome ,18a Female Male , Male ,14, Female aday of AIH split. x 1' sec

5 62 DMOWSKI ET AL. layer separation are in complete agreement with the data recently presented by Black et al,5 Contrary to the original report by Ericsson et al.,t we were not able to detect any significant changes in the percentage ofy spermatozoa before and after separation on 7.5% and 17.5% HSA columns. In conclusion, a one-layer HSA separation technique, utilizing a concentration of 7.5% HSA, can be used effectively to clean nonmotile and abnormal forms, other cellular and amorphous elements, and sperm debris from abnormal semen samples. This technique offers another chance for conception to a couple with a relative male factor. The two-layer technique does not appear to be of January 1979 value in such cases, probably because of a significant decrease in the total sperm count. REFERENCES 1. Ericsson &1, Langevin CN, Nishino M: Isolation of fractions rich in human Y sperm. Nature 246:421, Ross A, Robinson JA, Evans HJ: Failure to confirm separation of X- and Y-bearing human sperm using BSA gradients. Nature 253:354, Glaub JC, Mills RN, Katz DF: Improved motility recovery of human spermatozoa after freeze preservation via a new approach. Fertil Steril 27:1283, Ericsson &1: Isolation and storage of progressively motile human sperm. Andrologia 9:111, Black JB, Peduto JC, Servy EJ: Male factor infertility treated by isolation of progressively motile sperm (abstr). Fertil Steril 29:241,1978