The development of a clinical test of sperm migration to the site of fertilizrition *

Transcription

1 FERTILITY AND STERILITY Copyright c 1982 The American Fertility Society Vol. 37, No.3, March 1982 Printed in U.s A. The development of a clinical test of sperm migration to the site of fertilizrition * A. Allan Templeton, M.B., Ch.B., M.R.C.O.G.t David Mortimer, B.Sc., Ph.D.:j: Department of Obstetrics and Gynaecology, University of Edinburgh, Edinburgh, Scotland In those patients whose infertility is unexplained after routine investigations, it is likely that there are defects of gamete transport, fertilization, or implantation. This paper describes the development of a test of sperm migration to the site of fertilization. A method of laparoscopic sperm recovery from the peritoneal cavity and fimbrial rinsings following insemination at midcycle is described. In all, 47 patients were studied, and spermatozoa were recovered in 55%. There was no difference in the success rate following artificial insemination or coital insemination. Successful sperm recovery could not be attributed to better semen quality or better timing of the procedure in the menstrual cycle. The spontaneous pregnancy rate in the positive sperm recovery group was significantly higher than in the negative group. A comparison was made between the results of the postcoital test and the results of laparoscopic sperm recovery. There was a poor correlation between the two tests. It is suggested that laparoscopic sperm recovery is useful in the investigation of and the further management of that group of patients whose infertility is unexplained. Fertil Steril 37:410, 1982 In a recent article in this journal entitled "The frustrations of being 'normal' yet 'infertile'," Wallach drew attention to the continuing clinical problem posed by those patients in whom there is no apparent cause for infertility.1 In a recent review of the literature, we found an overall incidence of unexplained infertility of 15%, although, in our own practice, the incidence is 24%.2 The cause of this phenomenon is unknown, but it is likely that in some patients there is a failure of gamete transport, fertilization, or implantation. These are 'all areas that cannot be investigated Received June 21, 1981; revised and accepted November 5, I *This work was supported by the Wellcome Trust. treprint requests: Dr. A. Allan Templeton, Lecturer, Department of Obstetrics and Gynaecology, University of Edinburgh, 37 Chalmers Street, Edinburgh, EH3 9EW, Scotland. *Present address: Department of Obstetrics and Gynaecology, University of Birmingham, Birmingham, B15 2TG, England. satisfactorily by current clinical techniques. We have attempted to develop a clinical test that would be helpful in investigation of one of these areas, that is, sperm transport to the site offertilization. Currently, tests of sperm behavior in the female genital tract are confined to the in vivo or in vitro assessment of the interaction between spermatozoa and cervical mucus. Although it is likely that in a small proportion of patients, estimated at around 5%,3, 4 problems do arise in this area, there are often uncertainties in interpreting results. The postcoital test is a widely used and useful method of assessing the cervical barrier, although some workers have expressed doubts about its value. 5-9 Matthews et al. have shown that where the preovulatory period is defined by serial measurements of plasma luteinizing hormone (LH), the incidence of poor sperm penetration in an in vitro system (Kremer's test) is exceedingly small.10 Furthermore, the significance of the postcoital test in predicting the ability of spermatozoa to reach the site of fertilization has 410 Templeton and Mortimer Clinical test of sperm migration Fertility and Sterility

2 been called into question by Asch, who demonstrated spermatozoa at the fimbriated end of the fallopian tubes in patients who had consistently poor or negative postcoital tests. 11, 12 This study describes an attempt to develop a clinical test of sperm migration using laparoscopic sperm recovery from the pelvis. The assumption made is that sperm recovered from the peritoneal cavity have demonstrated their ability to reach the site of fertilization in the fallopian tube. PATIENTS MATERIALS AND METHODS All patients were seen at the Infertility Clinic, Royal Infirmary, Edinburgh. Couples were recruited into the study from January 1978 until March Thus, the recruitment period stretched over 27 months. The purpose of the study was explained to each couple, and each partner's consent was given. All patients were managed clinically by one of us (A. T.), and all laparoscopies were also carried out by him. Couples were selected if each partner had a normal medical history. Particular regard was paid to the gynecologic history, so that only those in whom it was anticipated that the genital tract might be normal were recruited. Similarly, only those women with regular ovulatory cycles, within the limits of28 ± 4 days, were recruited for the study. Ovulation was confirmed by plasma progesterone estimation in the luteal phase of at least two consecutive cycles. Each man had a series (usually three) of seminal analyses, and only men with sperm quality in the accepted fertile range were studied. In each case, the mean semen parameters met the following requirements: semen volume, 1.5 ml; sperm count, > 20 x 106/ml; sperm motility, > 40%; and normal sperm morphology, > 40%. In our routine practice, the next step in investigations is diagnostic laparoscopy.13 This is usually carried out in the luteal phase, but in each of the patients studied here, it was scheduled for the estimated time of ovulation, based on the previous menstrual history. Subsequent confirmation of the time in the cycle that laparoscopy was performed was obtained by integration of the following data: (1) dates of previous and next menstrual period; (2) plasma LH, estradiol, and progesterone assay on the day before, day of, and day after surgery; and (3) histologic assessment of an endometrial biopsy.14 All couples were asked to abstain from intercourse for at least 3 days prior to the investigation, and all subsequently gave assurance that they had complied. Two modes of insemination were used in the study. The first group of patients underwent artificial insemination with the husband's semen (AIH). In this group, a semen specimen obtained by masturbation was delivered to the ward early on the morning of the day of surgery and was analyzed and utilized for insemination within 2 hours of ejaculation. The standard clinical technique for intracervical insemination was used, whereby a plastic catheter (Quill, Everett Medical Products Limited, Mitcham, Surrey, England, U. K.) was inserted into the external cervical os and the semen gently expelled. Minimal pressure was exerted and, in each case, semen was seen to spill from the cervix around the catheter back into the vagina. Care was taken in the positioning and removal of the speculum to ensure that the vaginal pool was left undisturbed. In the second group of patients, insemination was by coitus; and, depending on the timing of surgery, these patients were asked to have intercourse either late in the evening before admission to the hospital or early on the morning of admission. All patients in the coital group were seen the day prior to surgery so that we could be sure that they were fit for anesthesia, so that they could have blood taken, and so that we could be sure that they understood the instructions. The occurrence of intravaginal ejaculation was checked by our questioning the patients and also by our verifying the existence of spermatozoa in the cervix. LAPAROSCOPY AND SPERM RECOVERY Diagnostic laparoscopy was carried out by the standard techniquep In each case a careful pelvic inspection of the uterus, tubes, and ovaries was carried out. The mobility of each of the pelvic organs was carefully assessed, and the pelvic floor and uterosacral ligaments were examined closely for the presence of endometriosis. The laparoscopy was performed either 6 or 12 hours following insemination. However, the rate of successful sperm recovery between the two timing groups does not differ,15 and, for the purpose of this study, these two groups are considered together. In addition to the routine laparoscopic assessment, the fluid present in the cul-de-sac or the pouch of Douglas (POD) was aspirated with a blunt-ended needle (2 mm in diameter). Following aspiration, the fimbriated end of each fallopi- Vol. 37, No.3, March 1982 Templeton and Mortimer Clinical test of sperm migration 411

3 f Table 1. Number of Patients Recruited into the Study and Reasons for Exclusions AlH Coitus Total No. of patients recruited Excluded for pelvic problems Affecting sperm transport Affecting fertility Excluded for technical reasons Excluded because of poor 3 3 semen quality on day of insemination Actual study group an tube was separately rinsed with 10 ml of sterile medium (medium 199 with Earle's salts and HEPES buffer, Flow Labs, Irvine, Scotland, U. K.), and the rinsings were subsequently aspirated from the pouch of Douglas. Finally, an endometrial biopsy was taken with a Sharman curette. The procedure usually lasted 20 to 30 minutes. In each case, the patient was discharged from the hospital the following day, having been told the result of the laparoscopic inspection. PROCESSING OF MATERIAL FROM THE FEMALE TRACT The volumes of all flushings were measured with the use of 10-ml "blow-out" glass pipettes, and from each a known fraction (usually 1I10th) was taken for subsequent processing and scoring of sperm numbers and appearance. During the course of the study, several improvements in the processing methods were made to facilitate the scoring of spermatozoa in the material recovered from the female tract. Each improvement was fully evaluated against its predecessor on either duplicate aliquots of tract specimens or on artificially produced "flushings." The final method, used in the majority of cases, is described in detail. The aliquot of each flushing was treated with twice its volume of 0.01 % (w/v) aqueous saponin solution (saponin powder, Coulter Electronics, Harpenden, Hertfordshire, U. K.) to lyse contaminating red blood cells, centrifuged at 500 x g for 10 minutes and the pellet resuspended in 1.0 ml of fresh saponin solution. After a second centrifugation under the same conditions, the pellet was resuspended in 15 ILl of distilled water, and the suspension was spotted onto a clean microscope slide as three equal (6 or 7 ILl) droplets. Slides were dried overnight in a 37 C incubator, fixed in absolute ethanol, and allowed to air-dry. Glycerine jelly (Raymond A. Lamb, London, U. K.) melted at 60 C was used to mount the preparations. For scoring, the entire area of each drop was scanned under phase-contrast optics at magnification ranging from x 250 to x 500 (depending upon the amount of residual background material and on the microscope used). The number of spermatozoa present were counted, and this figure was used to calculate the total number present in the whole flushing, although for the purposes of this study, sperm recovery was deemed positive if any spermatozoa were detected. Before laparoscopy, but under anesthesia, the cervical mucus from each patient was aspirated into a 1-ml tuberculin syringe. The cervix and cervical mucus were assessed with the Insler score,16 and the mucus was then examined under a high-power lens for the presence of spermatozoa. Spermatozoa were classified as highly progressively motile, progressively motile, and nonmotile; and the average number in each group was calculated after examination of several highpower fields. PATIENTS' REVIEW Six weeks after laparoscopy each couple was seen at the Infertility Clinic. Details of the menstrual history were recorded, and the laparoscopic findings were explained. Thereafter, the couples were reviewed periodically, but no treatment was instituted. All patients were reviewed for the last time in March Most patients who had not become pregnant were still attending the clinic, but those who were not were contacted by phone or by mail. The details of any pregnancies and the outcome were recorded. Table 2. Prognostic Value of Laparoscopic Sperm Recovery in AlB and Coital Patients 4 Sperm recovered at laparoscopy Pregnant Not pregnant Sperm not recovered at laparobcopy Pregnant Not pregnant AlHpatients (n = 25) Coital patients (n = 22) Total (n = 47) Tests of significance: AIH patients: "Sperm recovered, pregnancy," versus "sperm not recovered, pregnancy": X 2 = 3.89; P < Postcoital patients: "Sperm recovered, pregnancy" versus "sperm not recovered, pregnancy": X2 = 4.07; P < Total group (n = 47): "Sperm recovered, pregnancy" versus "sperm not recovered, pregnancy": X2 = 7.45; P < Templeton and Mortimer Clinical test of sperm migration Fertility and Sterility

4 Table 3. Insemination Data in Patients Who Had AlHa Volume (m}) 2.8 ± 0.5 Sperm count (x 106/ml) ± 70.0 % Progressively motile sperm 51.5 ± 4.1 % Morphologically normal 48.2 ± 2.2 sperm Cervical score (Insler 8.4 ± 0.7 et aj.16) Plasma estradiol (pmol/l) 789 ± 116 a All results mean ± standard error of the mean. Sperm recovered, be Sperm recovered, not Sperm not recovered, Sperm not reo came pregnant yet pregnant became pregnant covered 3.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 84 The laparoscopic sperm recoveries following AIH were carried out during the period February 1978 to November 1978; and thus the follow-up period ranged from 28 to 37 months (32.8 ± 3.0 months, mean ± standard deviation), while the laparoscopic sperm recoveries following coital insemination were carried out in the period February 1979 to May 1980, and thus the follow-up period ranged from 10 to 25 months (18.6 ± 4.9 months, mean ± standard deviation). RESULTS The study group and exclusions are summarized in Table 1. Twenty-five patients who had AIH and 22 patients who had coital insemination were included in the review. Successful sperm recovery was recorded in 14 (56%) of the 25 AIH patients and in 12 (55%) of the 22 coital patients. The pregnancy rates in both groups are recorded in Table 2. For the AIH patients where sperm recovery was successful, 8 of14 became pregnant, whereas when sperm recovery was not successful, only 2 of 11 became pregnant (X 2 = 3.89; P < 0.05). In the coital group, 4 of 12 where sperm recovery was successful became pregnant, whereas none of 10 where sperm recovery was not successful became pregnant (X2 = 4.07; P < 0.05). This meant that for all the patients (AIH and coitus) 12 of 26 patients with successful sperm recovery became pregnant, whereas only 2 of 21 patients where sperm recovery was unsuccessful became pregnant (X2 = 7.45; P < 0.01). The higher pregnancy rate in the AIH patients can presumably be attributed to the longer follow-up period. There were no significant differences among the groups in the duration of infertility at the time of laparoscopic sperm recovery. Similarly, there were no significant differences among the groups in the ages of husbands and wives, respectively. In order to exclude the possibility that positive sperm recovery merely reflects better semen quality or better timing of the procedure in the menstrual cycle, the semen parameters, the cervical score, and the plasma estradiol levels-were compared in all four groups. The results are summarized in Table 3 for AIH patients and in Table 4 for postcoital patients. There were no significant differences among the groups, and thus the results cannot be explained in this way. A comparison was then made between the results of the postcoital test after artificial insemination and after coitus and the findings of spermatozoa in the POD or in fimbrial flushings. The results are summarized in Table 5 for AIH and in Table 6 for coital patients. There was no correlation between the finding of progressively motile sperm in the post-aih or postcoital cervical mucus specimen and positive laparoscopic sperm recovery. In fact, in 52% of patients with progressively motile sperm in the mucus, laparoscopic sperm recovery was negative, while in 31% of patients with nonmotile cervical sperm, laparoscopic sperm recovery was positive. Table 4. Insemination Data in Patients Who Had Coitusa Sperm re- Sperm re- Semen param- covered, be- covered, not Sperm not eters came preg- yet preg- recovered nant nant (n = 10) (n = 4) (n = 8) Volume (m}) 3.1 ± ± ± 0.3 Sperm count 87.2 ± ± ± 21.7 (x 10 6 /m}) % Progressively 52.5 ± ± ± 3.4 motile sperm % Morphologically 46.2 ± ± ± 1.8 normal sperm Cervical score 8.7 ± ± ± 0.8 (Insler et a1. 16) Plasma estradiol 1145 ± ± ± 65 (pmol/l) asemen data based on analysis nearest to procedure. All results mean ± standard error of the mean. Vol. 37, No.3, March 1982 Templeton and Mortimer Clinical test of sperm migration 413

5 Table 5. Comparison of Postcoital Test (After Artificial Insemination) and Laparoscopic Sperm Recovery from the Pelvis Postcoital test" Spenn re- Spenn not Total covered recovered (D = 25) (D = 14) (D = 11) Highly progressively 8 (57%) 7 (64%) 15 (60%) motile sperm seen Progressively motile 2 (14%) 3 (27%) 5 (20%) Only nonmotile 4 (29%) 1 (9%) 5 (20%) abased on assessment of several high-power fields. DISCUSSION The study described here was undertaken with the intention of developing a routine clinical test that would indicate when sperm transport to the site offertilization had been successful. In almost half the patients studied, spermatozoa were recovered from the peritoneal cavity, and there was no difference in the success rate after artificial or coital insemination. An early study had confirmed that the success rate was the same whether recovery was attempted 6 hours or 12 hours after insemination.15 There are no strictly comparable studies of laparoscopic sperm recovery in fertile women in the literatur~. However, if it is accepted that spermatozoa that reach the fallopian tubes will be found in the peritoneal cavity, and there is evidence to support this assumption,17 then the successful sperm recovery rate reported here in infertile patients is much lower than previously reported in groups of fertile patients undergoing salpingectomy (100% and 85%, respectively),18, 19 suggestive that failure of sperm migration to the site of fertilization may be the problem in some of those patients whose infertility was previously unexplained. Unfortunately, there is no way of assessing the reproducibility of laparoscopic sperm recovery, and it is possible that a negative finding in one cycle could be a positive finding in the next. One way around this problem might be the use of transvaginal aspiration of peritoneal fluid in successive cycles. This method has met with some success,20 although Kelly has pointed out the dangers of contamination with vaginal spermatozoa. 21 However, the fact that the pregnancy rate in the group of patients with successful sperm recovery is significantly higher than in the group with negative recovery suggests that these results are meaningful. The favorable prognosis of positive laparoscopic sperm recovery is in agreement with the work of Koch, who found that 30% of women with spermatozoa in the peritoneal fluid became pregnant, whereas only 8% with no spermatozoa became pregnant.20 Similarly, Asch reported a favorable outcome in the patients he studied by a similar method. 11, 12 On the other hand, Ahlgren could find no correlation between sperm recovery and subsequent fertilityp The finding that the postcoital test did not correlate well with the success of sperm recovery emphasizes the limitations of this long-accepted method of investigation. In many of the cases studied here, where the postcoital test was good or excellent by accepted criteria, sperm transport to the site of fertilization could not be confirmed. Also, in many cases where only nonmotile sperm were seen in the cervical mucus, successful sperm transport seems to have occurred. This finding is in agreement with the only other similar study in the literature, where successful sperm recovery was found in 9 of 11 patients' with poor or negative results in the postcoital test. 11 Thus, if an in vivo or in vitro assessment of the interaction between spermatozoa and cervical mucus is to be made, it should be related to the success of sperm migration, as assessed by the finding of spermatozoa in the peritoneal cavity. In summary, this study has shown that laparoscopic sperm recovery is a useful method of investigation in the further management of the infertile couple. Although there may be logistical problems in establishing a routine, the test will provide more information in the group of patients whose infertility is unexplained, by assessing an area that current clinical tests cannot evaluate. The procedure adds only minutes to a routine laparoscopy, and it can be carried out after either AIH or coitus. Problems in scoring the material have been resolved, and the test should now be incorporated in the diagnostic workup of appropriate infertile couples. Table 6. Comparison of Postcoital Test and Laparoscopic Sperm Recovery from the Pelvis Postcoital test" Spenn re- Spenn not Total covered recovered (D = 22) (D = 12) (D = 10) Highly progressively 5 (42%) 4 (40%) 9 (41%) motile sperm seen Progressively motile 3 (25%) 1 (10%) 4 (18%) Only nonmotile 4 (33%) 5 (50%) 9 (41%) abased on assessment of several high-power fields. 414 Templeton and Mortimer Clinical test of sperm migration Fertility and Sterility

6 Acknowledgments. Our thanks to Eleanor Leslie, Clare Reid and Ken Donachie for excellent technical assistance and to the staff of the Infertility Clinic, Royal Infirmary, Edinburgh, for invaluable help. REFERENCES 1. Wallach EE: The frustrations of being "normal" yet "infertile." Fertil Steril 34:405, TempletonAA, Penney GC: The incidence, characteristics, and prognosis of patients whose infertility is unexplained. Fertil SteriI37:175, Katayama KP, Ju KS, Manuel M, Jones GS, Jones HW Jr: Computer analysis of etiology and pregnancy rate in 636 cases of primary infertility. Am J Obstet Gynecol 135:207, Sperotf.L, Glass RH, Kase NG: Investigation of the infertile couple. In Clinical Gynecologic Endocrinology and Infertility, Second Edition. Baltimore, Williams & Wilkins Co., 1978, p Jette NT, Glass RH: Prognostic value of the postcoital test. Fertil Steril 23:29, Giner J, Merino G, Luna J, Azuar R: Evaluation of the Sims-Huhner postcoital test in fertile couples. Fertil SteriI25:145, Moran J, Davajan V, Nakamura R: Comparison of the fractional post-coital test with the Sims-Hubner post-coital test. Int J Fertil19:93, Tredway DR, Settlage DSF, Nakamura RM, Motoshima N, Umezaki CV, Mishell DR: Significance of timing for the post-coital evaluation of cervical mucus. Am J Obstet GynecoI121:387, Templeton AA, Mortimer D: Laparoscopic sperm recovery in infertile women. Br J Obstet Gynaecol 87:1128, Matthews CD, Makin AF, Cox LW: Experience with in vitro sperm penetration testing in infertile and fertile couples. Fertil Steril 33:187, Ascb RH: Laparoscopic recovery of sperm from peritoneal fluid in patients with negative or poor Sims-Hubner test. Fertil Steril 27:1111, Asch RH: Sperm recovery in peritoneal aspirate after negative Sims-Hubner test. Int J Fertil23:57, Templeton AA, Kerr MG: An assessment of laparoscopy as the primary investigation in the subfertile female. Br J Obstet Gynaecol 84:760, Noyes RW, Hertig A, Rock J: Dating the endometrial biopsy. Fertil Steril 1:3, Templeton AA: Sperm function in patients with unexplained infertility. M.D. thesis, submitted to the University of Aberdeen 16. Insler V, Melmed H, Eichenbrenner I, Serr DM, Lunenfeld B: The cervical score: a simple semiquantitative method for monitoring of the menstrual cycle. Int J Gynaecol Obstet 10:223, Ahlgren M: Migration of spermatozoa to the fallopian tubes and the abdominal cavity in women including some immunological aspects. M.D. thesis, University of Lund, Sweden, Settlage DSF, Motoshima M, Tredway DR: Sperm transport from the external cervical os to the fallopian tubes in women: a time and quantitation study. Sperm transport, survival and fertilizing ability. INSERM 26:201, Croxatto HB, Faundes A, Medel M, Avendano S, Croxatto HD, Vera C, Anselmo J, Pastene L: Studies on sperm migration in the human female genital tract. Sperm transport, survival and fertilizing ability. INSERM 26:162, Koch VJ, Hammerstein J, Zielske F: Clinical meaning of spermatozoa found in the peritoneal fluid after vaginal and intrauterine insemination.. Fertil SteriI28:311, Kelly JV: Myometrial participation in human sperm transport: a dilemma. Fertil Steril 13:84, 1962 Vol. 37, No.3, March 1982 Te~pleton and Mortimer Clinical test of sperm migration 415