Intrauterine insemination outperforms intracervical insemination in a randomized, controlled study with frozen, donor semen

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1 FERTILITY AND STERILITY Copyright 1992 The American Fertility Society Printed on acid-free paper in U.S.A. Intrauterine insemination outperforms intracervical insemination in a randomized, controlled study with frozen, donor semen Phillip E. Patton, M.D.*t Kenneth A. Burry, M.D.* Amy Thurmond, M.D.t Miles J. Novy, M.D.* Don P. Wolf, Ph.D.* Oregon Health Sciences University, Portland, Oregon Objective: To assess the efficacy of intrauterine insemination (lui) in a donor insemination program. Design: Prospective randomized clinical trial. Setting: Donor insemination program. Patients, Participants: Women undergoing insemination were randomly assigned to receive either lui or intracervical insemination for a maximum of six cycles. Interventions: None. Main Outcome Measure(s): Cycle fecundity rates between the two routes were compared. Results: The monthly fecundity rate for intracervical insemination was 5.1 % compared with 23% by lui. By life table analysis, pregnancy rates for lui were significantly higher than intracervical insemination (P = 0.02). Conclusions: Intrauterine insemination with quarantined donor sperm is superior to intracervical insemination. Fertil Steril 1992;57: Key Words: Route of insemination, intrauterine insemination with frozen donor semen Therapeutic insemination with donor sperm is a common treatment alternative for infertility attributable to a male factor or for single women who desire pregnancy. Until a few years ago, fresh semen was used for this procedure; however, because ofthe potential for infectious disease transmission, only cryopreserved semen quarantined for a minimum of 6 months is now recommended for use in therapeutic insemination by donor (1-3). Unfortunately, cryopreservation is associated with decreased post-thaw recovery of motile sperm, and those sperm that do survive are fragile (4). Insemination by a conven- Received July 27, 1991; revised and accepted November 27, * Department of Obstetrics and Gynecology. t Reprint requests: Phillip E. Patton, M.D., Department of Obstetrics and Gynecology L-466, 3181 Southwest Sam Jackson Park Road, Portland, Oregon :j: Department of Radiology. tional intracervical route with quarantined, cryopreserved sperm results in a decreased cycle fecundity when compared with fresh semen, and despite ongoing improvements in cryotechnology, cycle fecundity rates using cryopreserved donor semen have remained relatively stable. In view of the mandated, exclusive use of quarantined, cryopreserved semen and the recognition that thousands of women seek pregnancy through donor insemination, strategies that improve cycle fecundity are highly desirable. Because pregnancy initiation is undoubtedly dependent on the number and quality of sperm at the site of fertilization, the assessment of the optimal method of insemination would be beneficial. In the present study, we conducted a controlled, prospective, randomized clinical trial comparing conventional intracervical insemination and intrauterine insemination (lui) in women undergoing therapeutic insemination with frozen donor semen. Patton et al. lui using donor semen 559

2 Patients MATERIALS AND METHODS Candidates for the study were women desiring therapeutic insemination by the Fertility Clinic at the Oregon Health Sciences Center between 1988 and The protocol and consent form was approved by the internal review board. The primary indication for insemination was azoospermia (28), severe oligospermia (11), or the patient was single (11). Patients were placed into one oftwo treatment groups by random assignment for a maximum of six cycles of insemination after enrollment. Preinsemination screening tests included an analysis of ovulatory function by basal body temperature (BBT) records and urinary luteinizing hormone (LH) assays. Tubal patency was evaluated by hysterosalpingography (HSG), which was performed in women with historical risk factors or at the completion of the study in women failing to become pregnant. A total of 4 women were excluded from the study analysis secondary to an abnormal hysterosalpingogram (3 in lui and 1 in intracervical insemination group). Nineteen women did not have HSG. Ofthese, 13 conceived and 6 dropped out before study completion. Each subject received either a single lui or intracervical insemination on the day after detection of the LH surge by conventional urine kits (OvuQuick; Monoclonal Antibodies, Inc., Mountain View, CA); if the surge was detected on Friday, a single insemination was conducted on the same day. Testing was initiated approximately 4 days before the predicted time of ovulation, based on review of previous BBT records. Cycle adequacy was evaluated retrospectively after each insemination by assessing follicular and luteal phase lengths as estimated by BBT. An optimum cycle was defined as a follicular phase length of at least 10 days and a luteal phase length of 12 or more days. Abnormalities in ovulatory function were treated with clomiphene citrate (CC); 50 to 150 mg/d for 5 days). Sperm donors were recruited from the medical community and screened by established guidelines (1,3). Semen samples were obtained by masturbation after 24 to 48 hours of sexual abstinence. All donors had normal semen parameters according to World Health Organization Guidelines (5) before freezing and evidence of recovery of at least 50% of the initial motile sperm after freezing. Preinsemination and postinsemination semen parameters were measured by conventional light microscopic techniques and/or by computer-assisted video image analysis (CellSoft; Cryoresources, Ltd., New York, NY). Liquified semen was cryopreserved by first diluting 1:2 or after November 1989, 3:4 with a TES Tris-egg yolk mixture to a final glycerol concentration of 6% or 7.5%, respectively (Irving Scientific, Santa Ana, CA). The mixture was then ali quoted into 1.2-mL Nunc cryovials. After a 90-minute hold at 4 DC, freezing was accomplished by exposure to liquid nitrogen vapors. The sample was then plunged into liquid nitrogen and stored therein for a minimum of 6 months when the donor retested negative for human immunodeficiency virus before release. Thawing of one or two vials per procedure was conducted in room air or in a 37DC water bath. Both lui and intracervical insemination were performed by personnel in the Division of Reproductive Endocrinology. Semen samples for lui and intracervical insemination were derived from similar donor groups. Semen for intracervical insemination was thawed and used directly without cryoprotectant removal. Semen samples for lui were washed two times with three volumes of Ham's F-10 medium (GIBCO, Grand Island, NY) or human tubal fluid medium (6) supplemented with 2 mg/ml of human serum albumin. The supernatant (300 X g for 7 minutes) was removed and discarded, and a fresh aliquot of buffer (0.5 ml) was added. After incubation for 1 hour in 5% CO 2, 37DC, the supernatant (swim-up) was removed, and an aliquot was analyzed for sperm density and motility. Alternatively, the sperm pellet in its entirety was resuspended in 0.25 to 0.50 ml of medium and used directly. At least 20 X 10 6 motile cells were required before insemination by either route. Intracervical insemination (1.5 to 2.5 ml) was performed by placing a speculum into the vagina to expose the cervix. The ectocervix was wiped with a cotton swab to remove vaginal secretions and excess cervical mucus. Semen was then delivered into the cervical canal with a syringe-cannula system (17 g; Intracath-Deseret, Sandy, UT) and allowed to pool against the cervix for 20 minutes. After vaginal-cervical preparation, lui (0.25 to 0.3 ml) was performed with the same syringe cannula system by gently inserting the flexible cannula through the cervical os into the upper fundal area of the uterine cavity. The suspension of washed sperm was injected over 30 to 60 seconds, and the patient remained recumbent for 10 to 15 minutes. All pregnancies were confirmed by standard serum assays of human cho- 560 Patton et al. lui using donor semen Fertility and Sterility

3 Table 1 Number of Pregnancies in Women Undergoing lui or Intracervical Insemination as a Function of Cycle Number Route of insemination Cycle no. Intracervical Intrauterine Totals 1 2/22 8/28 10/50 (20)* 2 1/19 8/20 9/39 (23) 3 0/16 1/12 1/28 (04) 4 1/13 1/9 2/22 (09) 5 0/6 0/7 0/13 (0) 6 0/3 1/6 1/9 (11) Totals 4/79 19/82 23/161 * Values in parentheses are percents. rionic gonadotropin and ultrasound evidence of a gestational sac. Statistical Analysis Life table analysis was used to compare fecundity between intracervical insemination and lui cycles. Logistic regression was used to examine the effect of covariances such as age, parity, and cycle quality. Analyses were conducted with the SPSS (SPSS, Inc., Chicago, IL) statistical package for IBM-PCs. Comparison of variables between intracervical insemination and lui were performed by application ofthe Student's t-test or X2 analyses with a significance level set at RESULTS There were 69 patients enrolled in the study. Nineteen women (9 intracervical insemination and 10 lui) were eliminated for the following reasons: dropped out of the study before the first insemination, more than a single route of insemination (4), more than one insemination per cycle (2), uterine structural anomalies (4), and gonadotropin use (2). Fifty patients underwent at least one therapeutic insemination cycle, 22 by intracervical insemination and 28 by lui. There were a total of 79 treatment cycles by intracervical insemination and 82 by lui used in data analysis. Ofthe 22 patients randomized to intracervical insemination, 4 became pregnant (pregnancy rate [PR], 18.2%) in 79 cycles of insemination (monthly fecundity, 5.1 %). Three ofthe four pregnancies occurred in the first two cycles, and only one pregnancy was conceived after more than two cycles of insemination (Table 1). In women randomized to lui (n = 28), 19 became pregnant (67.9%) in a total of 82 insemination cycles for a monthly fecundity of 23%. Sixteen of the 19 pregnancies occurred in the first two insemination cycles (Table 1). Using cycle number as a measure of time, a life table was constructed, and the results, as cumulative PRs, have been plotted in Figure l. A comparison of curves by insemination route gave a high probability that the two routes were different (Lee-Desu statistic, P = ). To explore the effects of confounding factors such as patient age, parity, and cycle quality on cycle outcome, logistic regression was conducted. The mean age ± SD for women who received lui (30.7 ± 5) was not different when compared with the age of women who received intracervical insemination (32.0 ± 5). Slightly more nulliparous women received intracervical insemination compared with lui (P = 0.04). A significant variable was cycle quality. All women had a follicular phase length> 10 days. One hundred twenty-nine cycles were considered optimal with a BBT elevation ~ 12 days posturinary LH surge. Thirty-two cycles were not optimal, as demonstrated by BBT evidence of anovulation or a luteal phase length rise of <12 days. Thirteen women in the intracervical insemination group had 23 suboptimal cycles. Eight" of these women had optimal cycles when treated with CC, and one of these women conceived. Six women in the lui group had 9 suboptimal cycles. All were treated with CC, and two conceived. An additional woman was on CC before study entry and conceived during her first cycle of insemination. When CC-stimulated cycles were considered optimal by this study's criteria, the PR was similar to that of unstimulated optimal cycles. Cumulative Proportion Pregnant P = Cycle Number Figure 1 Life table analysis of pregnancy outcome as a function of the route of insemination. (lui, intrauterine insemination; ICI, intracervical insemination). Patton et al. lui using donor semen 561

4 Because of the potential relationship between sperm concentration and pregnancy outcome, the concentration of motile cells was analyzed in relation to the route of insemination. The number of motile cells per insemination used for intracervical insemination (49.2 X 10 6 ± 21.9, mean ± SD) was no different when compared with lui (43.7 X 10 6 ± 23). Pregnancy outcome as a function of the number of motile cells inseminated was also analyzed. When the number of motile cells inseminated during the first and second insemination cycle was compared between women who became pregnant and those who did not, no significant differences in cell counts were noted (data not shown). The number of motile cells inseminated in women who became pregnant (44 ± 15 X 10 6, mean ± SD) was not different than the average cell count for women who did not become pregnant (47 ± 16 X 10 6 ). After completion of the study (6 insemination cycles), a total of 9 women opted to receive the alternate route of insemination. An additional 4 women who did not complete the study were also moved to the alternate arm. In these 13 women, a total of 84 insemination cycles were reviewed. There were no pregnancies in women who received intracervical insemination after lui (0/61 cycles), but 5 of 23 (21.7%) cycles of lui were successful in women who originally received intracervical insemination. DISCUSSION Cycle fecundity estimates for donor insemination by intracervical insemination using cryopreserved semen are generally lower than those obtained with fresh semen. The mandated use of quarantined, cryopreserved semen in clinics practicing therapeutic insemination has stimulated the timely development of strategies to improve the performance of frozen sperm. The results of the present study indicate that route of insemination is a critical variable that can directly affect pregnancy outcome with cryopreserved semen. In this randomized prospective protocol, cycle fecundity after lui (23%) was higher when compared with intracervical insemination (5.1%). Furthermore, the cumulative PR, as calculated by life table analyses, for lui using cryopreserved samples approached rates established after intracervical insemination using fresh donor semen. Several studies now suggest that the route of insemination may be an important variable in ther- apeutic insemination (Table 2). However, the definitive demonstration that lui is superior to intracervical insemination remains uncertain primarily because of the potential impact of confounding variables (10). Factors that can directly or indirectly affect pregnancy outcome include the timing, number, and technique of insemination; the assessment and treatment of female fertility factors; recipient age and indication for insemination; the number and quality of sperm used; and the method of sperm preparation. We used a study protocol designed to reduce the impact of confounding factors on pregnancy outcome. Recipients were screened for ovulatory disorders and tubal or peritoneal disease. The mean age of the study population as well as the indication for donor insemination was similar for the two study groups. Insemination timing was based on an objective, identifiable marker (urinary LH surge) and only a single insemination was performed each cycle. Moreover, because ofthe well-recognized decrease in cycle fecundity over time, the study was limited to a maximum of six insemination cycles. We did observe that multiparity was a potential confounding factor. Whether the slight increase in nulliparous women noted in the intracervical insemination group is clinically significant remains to be determined. Our protocol was designed to minimize the impact of sperm density. Because variations in motile sperm concentration delivered at the time of intracervical insemination for either fresh or frozen donor sperm can directly affect PR (11), the study was structured to deliver a similar concentration of motile sperm per insemination for both intracervical insemination and lui. Although the optimal number of motile sperm for insemination is unknown, all samples used in the present study Table 2 A Summary of Therapeutic Insemination Studies With Quarantined Donor Semen: Effect of Route of Insemination Intracervical Cycle fecundity Intrauterine 9/229 (4)- 23/238 (10) 12/230 (5) 20/82 (24) 4/79 (5) 19/82 (23) - Values in parentheses are percents. t Byrd et al. (7). + Barratt et al. (8). Silva et al. (9). Reference Byrd et ai., 1990t Barratt et ai., Silva et ai., 1989 This study 562 Patton et al. lui using donor semen Fertility and Sterility

5 were derived from the same donor pool and prepared such that at least 20 X 10 6 motile sperm per inseminate were delivered. Furthermore, only a single insemination was employed each cycle because recent observations suggest that multiple inseminations with cryopreserved semen are superior to a single insemination (12). Cycle quality was an internal variable within each subject. This study revealed that the cycle quality after each insemination and the frequency of cycles not meeting optimal quality were similar in both the lui and intracervical insemination groups. Therefore, anovulation or luteal phase duration did not contribute to the difference in cycle fecundity between the two groups. The basis for improved cycle fecundity after lui with cryopreserved sperm remains unexplained. In a previous study, we found no benefit of lui over intracervical insemination using fresh donor semen (13). Unquestionably, human sperm incur significant cryodamage when frozen by current protocols including those that employ controlled rates of cooling. Such damage is readily apparent in post-thaw decreases in sperm motility, velocity, and survival when compared with nonfrozen controls. Moreover, alterations in acrosomal status, cellular metabolism, and sperm penetration of cervical mucus or of heterologous eggs in vitro demonstrate that both structural and functional changes occur upon cryopreservation (14-17). These alterations undoubtedly adversely affect the fertilizing capacity of cryopreserved sperm. Theoretically, lui should be advantageous because the filtering effects of cervical mucus are avoided, thereby increasing the effective concentration of sperm at the fertilization site. Intracervical insemination using donor samples with high concentration of motile cells, multiple inseminations, or fresh semen may achieve the same goal (11, 12). The total cost of lui is more expensive than intracervical insemination because of laboratory processing charges and the additional costs of thawed semen necessary to meet the criteria of sample adequacy used in this study. At our institution, the cost of lui per cycle is roughly 1.5 to 2 times greater than intracervical insemination. Given the anticipated threefold to fivefold increase in cycle fecundity, the method would appear cost-effective. The present results confirm and extend observations by others that fecundity rates are superior when lui is used in preference to intracervical insemination in a carefully screened donor and recipient population when strict criteria of sample adequacy are used (7-9). This study was designed as a carefully controlled clinical trial comparing lui with intracervical insemination, and fecundity rates in a larger and more diverse recipient population remain to be determined. Nevertheless, our preliminary results are encouraging. In an unselected population of donor sperm recipients over the time frame of 1988 to 1991, a fecundity rate of 13% (60 pregnancies/462 insemination cycles) was achieved using a single lui timed on the basis ofthe LH surge. Our results also suggest that lui is a reasonable option in women who fail to conceive after conventional donor intracervical insemination. The possibility, of course, exists for improving fecundity rates as reported in this study. Parameters that deserve attention include but are not limited to multiple inseminations, increased motile sperm density, chemical stimulation of motility, and tubal insemination. Acknowledgments. We thank Kathy Burry, R.N., for her advice and assistance in data collection, the secretarial support of Ms. Elizabeth Cook, and the statistical analyses of the data by Jonathan Fields, M.S., research associate. REFERENCES 1. The American Fertility Society. Revised new guidelines for the use of semen-donor insemination. Fertil Steril 1988;49: Center for Disease Control. Semen banking, organ and tissue transplantation, and HIV antibody testing. MMWR 1988; 37:1. 3. The American Fertility Society. New guidelines for the use of semen-donor insemination: Fertil Steril 1990;53(1 Suppl):IS Graczykowski JW, Siegel MS. Motile sperm recovery from fresh and frozen-thawed ejaculates using a swim-up procedure. Fertil Steril 1991;55: World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. New York: Cambridge University Press, Kuzan FB, Quinn P. Cryqpreservation of mammalian embryos. In: Wolf DP, editor. In vitro fertilization and embryo transfer: a manual of basic techniques. New York: Plenum Press, 1988: Byrd W, Bradshaw K, Carr B, Edman C, Odom J, Ackerman G. A prospective randomized study of pregnancy rates following intrauterine and intracervical insemination using frozen donor sperm. 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