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Peritoneal sperm recovery can be consistently demonstrated in women with unexplained infertility
FERTILITY AND STERILITY
Vol. 53, No. 6, June 1990
Copyright 0 1990 The American Fertility Society
Printed on acid-free paper in U.S.A.
Peritoneal sperm recovery can be consistently demonstrated in women with unexplained infertility
SamuelS. Ramsewak, M.R.C.O.G.*t Christopher L. R. Barratt, Ph.D.:j: Tin-Chiu Li, Ph.D., M.R.C.O.G.
Hilary Gooch Ian D. Cooke, F.R.C.O.G.
Department of Obstetrics and Gynaecology, University of Sheffield, Jessop Hospital for Women, Sheffield, England
The technique of laparoscopic recovery of sperm from the peritoneal cavity has been previously used as an indicator of sperm transport. Templeton et al. 1 and Templeton and Mortimer 2 performed a succession of clinical studies concentrating on the unexplained infertile couple and showed that sperm recovery, performed 6 to 12 hours after insemination or coitus, showed no difference between either method, but a higher spontaneous pregnancy rate occurred in patients who had sperm recovered than those with negative results. The sperm recovery rate recorded was 63%,1 and when compared with 100% in salpingectomy specimens examined up to 24 hours by Settlage et al., 3 this raised the question of a defect of sperm function in this group of patients. The aim of this study was to assess sperm recovery rates in women with unexplained infertility under conditions of appropriate timing in the cycle, with the interval from insemination to laparoscopy constant at 18 to 20 hours and with meticulous laboratory analysis of the peritoneal aspirate. MATERIALS AND METHODS
The patients were recruited over a 6-month period from the Infertility Clinic at the Jessop Hospital for Women. The characteristics of the couples Received August 1, 1989; revised and accepted February 9, 1990. * Commonwealth Medical Fellow. t Present address: Samuel S. Ramsewak, M.R.C.O.G., Mt. Hope Women's Hospital, Trinidad, West Indies. :j: Reprint requests: Christopher L. R. Barratt, Ph.D., Jessop Hospital for Women, Sheffield S3 7RE, England.
1106
Ramsewak et al.
Communications-in-brief
recruited were: (1) normal history and physical examination of both partners, (2) ages between 18 and 35 years, (3) cycle lengths of 28 ± 2 days, (4) bilateral tubal patency on hysterosalpingogram, (5) evidence of ovulation from previous biphasic temperature charts and plasma progesterone estimations, and (6) normal semen analysis. 4 All patients had norethindrone tablets in the luteal phase of the previous cycle to induce a withdrawal bleed, and ward admission was timed on day 14 ± 2 depending on the usual cycle length. The couple was asked to abstain from sexual intercourse for 3 days before laparoscopy. On the day of admission, the male partner was asked to provide a semen sample by masturbation at between 1 P.M. to 2 P.M. The semen was allowed to liquefy and after setting aside 0.5 to 1.0 mL for insemination, the rest was used for laboratory analysis (see below). One hour after production, the patient was seen on the ward, examined using a bivalve speculum, and a sample of cervical mucus obtained for assessment by Insler scoring and insemination performed by gentle instillation of the semen at the external os. The following morning, laparoscopy was performed in the operating theater so that the time elapsed was 18 to 20 hours. Routine laparoscopy was performed and aspiration of fluid from the Pouch of Douglas performed using a chorionic villous biopsy needle (Rocket, London, England) which has a blunted end and a side-entry point. Each fimbria! end was washed with Ham's F-10 solution (Gibco, Grand Island, NY) and the flushing aspirated. The samples were taken immediately to the laboratory for analysis. After liquefaction of the semen sample, it was Fertility and Sterility
analyzed4 for sperm density, motility, morphology, presence of antisperm antibodies, penetration into bovine mucus, and the hypo-osmotic swelling test. A drop each of peritoneal fluid (PF) and aspirate was placed on a slide and examined to confirm the presence of sperm, but no assessment of density, motility, or morphology was performed. The remainder of the samples were then centrifuged at 700 X g for 10 minutes, resuspended in Ham's F-10 medium and red cells lysed with Zap-o-Globin (Coulter Electronics Ltd., Luton, England), and centrifuged again. The supernatant was discarded and the pellet resuspended in 1 mL distilled water, mixed, and centrifuged again. The deposit was resuspended in 15 ~L distilled water and slides prepared using 5 ~L drops, fixed with absolute ethanol, and allowed to dry in air. They were then systematically and thoroughly examined using phase contrast microscopy at 400X magnification and sperm were photographed to provide a permanent record. The average time spent for preparation and examination of each patient's specimen was 8 hours.
RESULTS
Fifteen patients were originally recruited but 1 was excluded because the laparoscopy was performed on day 7 of the cycle because of an error of organization. This patient and 1 other had Stage 2 endometriosis noted at the time of laparoscopy. The age of the patients was 29.1 ± 1.2 years (mean ± SEM) and the duration of infertility ranged from 3 to 5 years. On the day of admission, semen analyses performed on the samples produced for insemination showed a mean sperm density of 120 ± 24.5 X 106 /mL, combined grade I and II motility 51%± 2.2%, percentage ideal forms 40% ± 2.8%. Penetration into bovine mucus was uniformly good with vanguard readings 35.3 ± 5.4 mm; no sample had antisperm antibodies and the hypoosmotic swelling test showed the mean number of coiled forms to be 78.3% ± 3.4%. These results are in keeping with normal ranges for fertile men in our laboratory. 4 Table 1 shows details of the day of insemination in the cycle, the interval to laparoscopy, and the outcome at laparoscopy. Apart from the two women with endometriosis who have started treatment with danazol tablets (Danol; Winthrop, Surrey, England), follow-up of patients 5 months after laparoscopy has revealed that no woman has as yet achieved pregnancy. Vol. 53, No.6, June 1990
Table 1 Results of Laparoscopic Sperm Recovery in Relation to Day of Cycle and Interval From Insemination
Patient no.
Cycle day of insemination
Interval to laparoscopy
14 15 16 14 15 14 12 15 14 14 12 16 14
19.5 18.5 18.5 18.5 18.5 19.0 19.0 18.5 18.0 19.5 18.5 18.0 19.0 18.5 19.0
Sperm recovery
h
1 2 3 4 5 6 7
8 9 10 11 12 13 14" 15" a
7
14
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes
Comment: endometriosis.
DISCUSSION
The possibility of failure of sperm transport along the female genital tract has been proposed by Templeton and colleagues 1 •2 who observed a 63% and 55% laparoscopic sperm recovery rate among patients with unexplained infertility. This compares with 100% from salpingectomy specimens examined up to 24 hours after insemination reported by Settlage et al. 3 in 1974. However, the findings in this study that sperm could be consistently recovered in the PF of women with unexplained infertility suggests that abnormal sperm transport in the female genital tract may not be an important cause of infertility in this group of women. The discrepancy in the better sperm recovery rate reported by Templeton and Mortimer2 (63%) and us (100%) is unlikely to be a result of bias in the population, as the women included in both studies had unexplained infertility defined in a similar manner. It is unclear whether the insemination recovery interval may have any bearing on sperm recovery rates. Templeton and Mortimer 2 recovered sperm 6 to 12 hours after insemination, whereas we recovered sperm 18 to 20 hours after insemination. It was of interest to note that very high sperm recovery rates were also reported by Asch 5 when the interval ranged from 23 to 30 hours. It is arguable that the observation of sperm in the peritoneal cavity does not imply that sperm are present at the tubal site of fertilization, although it is clear that they would have had to pass this point. Ramsewak et al.
Communications-in-brief
1107
Work in sheep by Hunter and Nichol, 6 in which oviduct ligation was performed after mating and subsequent tubal inspection 2 to 3 days later suggests that viable sperm are sequestered in the caudal isthmus for as long as 17 to 18 hours from where they are subsequently transferred up the tract. A similar situation may exist in the human and further study is needed to ascertain this.
SUMMARY
Diligent analysis of PF 10 to 20 hours after midcycle intracervical insemination with husband's semen in couples with unexplained infertility showed that sperm are consistently able to transverse the reproductive tract in this group of patients. However, this finding does not necessarily imply that the sperm were retained at the site of fertilization or that they were competent to achieve oocyte fertilization. Therefore, further experiments obtain-
1108
Ramsewak et al.
Communications-in-brief
ing sperm from the tubal isthmus to assess the effects of their sequestration there on their ability to fertilize human oocytes are needed. REFERENCES 1. Templeton A, Aitken J, Mortimer D, Best F: Sperm nmction in patients with unexplained infertility. Br J Obstet Gynaecol 89:550, 1982 2. Templeton AA, Mortimer D: The development of a clinical test of sperm migration to the site of fertilization. Fertil Steril37:410, 1982 3. 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. Fertil Steril24:655, 1973 4. Barratt CLR, Dunphy BC, Thomas EJ, Cooke ID: Semen characteristics of 49 fertile males. Andrologia 20:264, 1988 5. Asch RH: Laparoscopic recovery of sperm from peritoneal fluid in patients with negative or poor Sims-Huhner test. Fertil Steril27:1111, 1976 6. Hunter RHF, Nichol R: Transport of spermatozoa in the sheep oviduct. Preovulatory sequestering of cells in the caudal isthmus. J Exp Zool228:121, 1983
Fertility and Sterility
Vol. 53, No. 6, June 1990
Copyright 0 1990 The American Fertility Society
Printed on acid-free paper in U.S.A.
Peritoneal sperm recovery can be consistently demonstrated in women with unexplained infertility
SamuelS. Ramsewak, M.R.C.O.G.*t Christopher L. R. Barratt, Ph.D.:j: Tin-Chiu Li, Ph.D., M.R.C.O.G.
Hilary Gooch Ian D. Cooke, F.R.C.O.G.
Department of Obstetrics and Gynaecology, University of Sheffield, Jessop Hospital for Women, Sheffield, England
The technique of laparoscopic recovery of sperm from the peritoneal cavity has been previously used as an indicator of sperm transport. Templeton et al. 1 and Templeton and Mortimer 2 performed a succession of clinical studies concentrating on the unexplained infertile couple and showed that sperm recovery, performed 6 to 12 hours after insemination or coitus, showed no difference between either method, but a higher spontaneous pregnancy rate occurred in patients who had sperm recovered than those with negative results. The sperm recovery rate recorded was 63%,1 and when compared with 100% in salpingectomy specimens examined up to 24 hours by Settlage et al., 3 this raised the question of a defect of sperm function in this group of patients. The aim of this study was to assess sperm recovery rates in women with unexplained infertility under conditions of appropriate timing in the cycle, with the interval from insemination to laparoscopy constant at 18 to 20 hours and with meticulous laboratory analysis of the peritoneal aspirate. MATERIALS AND METHODS
The patients were recruited over a 6-month period from the Infertility Clinic at the Jessop Hospital for Women. The characteristics of the couples Received August 1, 1989; revised and accepted February 9, 1990. * Commonwealth Medical Fellow. t Present address: Samuel S. Ramsewak, M.R.C.O.G., Mt. Hope Women's Hospital, Trinidad, West Indies. :j: Reprint requests: Christopher L. R. Barratt, Ph.D., Jessop Hospital for Women, Sheffield S3 7RE, England.
1106
Ramsewak et al.
Communications-in-brief
recruited were: (1) normal history and physical examination of both partners, (2) ages between 18 and 35 years, (3) cycle lengths of 28 ± 2 days, (4) bilateral tubal patency on hysterosalpingogram, (5) evidence of ovulation from previous biphasic temperature charts and plasma progesterone estimations, and (6) normal semen analysis. 4 All patients had norethindrone tablets in the luteal phase of the previous cycle to induce a withdrawal bleed, and ward admission was timed on day 14 ± 2 depending on the usual cycle length. The couple was asked to abstain from sexual intercourse for 3 days before laparoscopy. On the day of admission, the male partner was asked to provide a semen sample by masturbation at between 1 P.M. to 2 P.M. The semen was allowed to liquefy and after setting aside 0.5 to 1.0 mL for insemination, the rest was used for laboratory analysis (see below). One hour after production, the patient was seen on the ward, examined using a bivalve speculum, and a sample of cervical mucus obtained for assessment by Insler scoring and insemination performed by gentle instillation of the semen at the external os. The following morning, laparoscopy was performed in the operating theater so that the time elapsed was 18 to 20 hours. Routine laparoscopy was performed and aspiration of fluid from the Pouch of Douglas performed using a chorionic villous biopsy needle (Rocket, London, England) which has a blunted end and a side-entry point. Each fimbria! end was washed with Ham's F-10 solution (Gibco, Grand Island, NY) and the flushing aspirated. The samples were taken immediately to the laboratory for analysis. After liquefaction of the semen sample, it was Fertility and Sterility
analyzed4 for sperm density, motility, morphology, presence of antisperm antibodies, penetration into bovine mucus, and the hypo-osmotic swelling test. A drop each of peritoneal fluid (PF) and aspirate was placed on a slide and examined to confirm the presence of sperm, but no assessment of density, motility, or morphology was performed. The remainder of the samples were then centrifuged at 700 X g for 10 minutes, resuspended in Ham's F-10 medium and red cells lysed with Zap-o-Globin (Coulter Electronics Ltd., Luton, England), and centrifuged again. The supernatant was discarded and the pellet resuspended in 1 mL distilled water, mixed, and centrifuged again. The deposit was resuspended in 15 ~L distilled water and slides prepared using 5 ~L drops, fixed with absolute ethanol, and allowed to dry in air. They were then systematically and thoroughly examined using phase contrast microscopy at 400X magnification and sperm were photographed to provide a permanent record. The average time spent for preparation and examination of each patient's specimen was 8 hours.
RESULTS
Fifteen patients were originally recruited but 1 was excluded because the laparoscopy was performed on day 7 of the cycle because of an error of organization. This patient and 1 other had Stage 2 endometriosis noted at the time of laparoscopy. The age of the patients was 29.1 ± 1.2 years (mean ± SEM) and the duration of infertility ranged from 3 to 5 years. On the day of admission, semen analyses performed on the samples produced for insemination showed a mean sperm density of 120 ± 24.5 X 106 /mL, combined grade I and II motility 51%± 2.2%, percentage ideal forms 40% ± 2.8%. Penetration into bovine mucus was uniformly good with vanguard readings 35.3 ± 5.4 mm; no sample had antisperm antibodies and the hypoosmotic swelling test showed the mean number of coiled forms to be 78.3% ± 3.4%. These results are in keeping with normal ranges for fertile men in our laboratory. 4 Table 1 shows details of the day of insemination in the cycle, the interval to laparoscopy, and the outcome at laparoscopy. Apart from the two women with endometriosis who have started treatment with danazol tablets (Danol; Winthrop, Surrey, England), follow-up of patients 5 months after laparoscopy has revealed that no woman has as yet achieved pregnancy. Vol. 53, No.6, June 1990
Table 1 Results of Laparoscopic Sperm Recovery in Relation to Day of Cycle and Interval From Insemination
Patient no.
Cycle day of insemination
Interval to laparoscopy
14 15 16 14 15 14 12 15 14 14 12 16 14
19.5 18.5 18.5 18.5 18.5 19.0 19.0 18.5 18.0 19.5 18.5 18.0 19.0 18.5 19.0
Sperm recovery
h
1 2 3 4 5 6 7
8 9 10 11 12 13 14" 15" a
7
14
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes
Comment: endometriosis.
DISCUSSION
The possibility of failure of sperm transport along the female genital tract has been proposed by Templeton and colleagues 1 •2 who observed a 63% and 55% laparoscopic sperm recovery rate among patients with unexplained infertility. This compares with 100% from salpingectomy specimens examined up to 24 hours after insemination reported by Settlage et al. 3 in 1974. However, the findings in this study that sperm could be consistently recovered in the PF of women with unexplained infertility suggests that abnormal sperm transport in the female genital tract may not be an important cause of infertility in this group of women. The discrepancy in the better sperm recovery rate reported by Templeton and Mortimer2 (63%) and us (100%) is unlikely to be a result of bias in the population, as the women included in both studies had unexplained infertility defined in a similar manner. It is unclear whether the insemination recovery interval may have any bearing on sperm recovery rates. Templeton and Mortimer 2 recovered sperm 6 to 12 hours after insemination, whereas we recovered sperm 18 to 20 hours after insemination. It was of interest to note that very high sperm recovery rates were also reported by Asch 5 when the interval ranged from 23 to 30 hours. It is arguable that the observation of sperm in the peritoneal cavity does not imply that sperm are present at the tubal site of fertilization, although it is clear that they would have had to pass this point. Ramsewak et al.
Communications-in-brief
1107
Work in sheep by Hunter and Nichol, 6 in which oviduct ligation was performed after mating and subsequent tubal inspection 2 to 3 days later suggests that viable sperm are sequestered in the caudal isthmus for as long as 17 to 18 hours from where they are subsequently transferred up the tract. A similar situation may exist in the human and further study is needed to ascertain this.
SUMMARY
Diligent analysis of PF 10 to 20 hours after midcycle intracervical insemination with husband's semen in couples with unexplained infertility showed that sperm are consistently able to transverse the reproductive tract in this group of patients. However, this finding does not necessarily imply that the sperm were retained at the site of fertilization or that they were competent to achieve oocyte fertilization. Therefore, further experiments obtain-
1108
Ramsewak et al.
Communications-in-brief
ing sperm from the tubal isthmus to assess the effects of their sequestration there on their ability to fertilize human oocytes are needed. REFERENCES 1. Templeton A, Aitken J, Mortimer D, Best F: Sperm nmction in patients with unexplained infertility. Br J Obstet Gynaecol 89:550, 1982 2. Templeton AA, Mortimer D: The development of a clinical test of sperm migration to the site of fertilization. Fertil Steril37:410, 1982 3. 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. Fertil Steril24:655, 1973 4. Barratt CLR, Dunphy BC, Thomas EJ, Cooke ID: Semen characteristics of 49 fertile males. Andrologia 20:264, 1988 5. Asch RH: Laparoscopic recovery of sperm from peritoneal fluid in patients with negative or poor Sims-Huhner test. Fertil Steril27:1111, 1976 6. Hunter RHF, Nichol R: Transport of spermatozoa in the sheep oviduct. Preovulatory sequestering of cells in the caudal isthmus. J Exp Zool228:121, 1983
Fertility and Sterility