A trial of superovulation in ovum donors undergoing uterine lavage

FERTILITY AND STERILITY

Vol. 51, No.1, January 1989

Printed in U.S.A.

Copyright<> 1989 The American Fertility Society

A trial of superovulation in ovum donors undergoing uterine lavage

Mark V. Sauer, M.D.* Robert E. Anderson, M.D. RichardJ. Paulson, M.D. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Southern California School of Medicine, Los Angeles, California

In an attempt to increase the number of fertilized ova recoverable by uterine lavage, the authors superovulated ovum donors using a combination of clomiphene citrate and human menopausal gonadotropin. Six women underwent seven lavage cycles with uterine flushes performed 96, 120, 144, and 168 hours after ovulation and artificial insemination. Ova were recovered from three women between 96 and 144 hours after ovulation. However, no ova were recovered from the other three donors, two of whom were later noted to be pregnant. Retained pregnancies occurred despite postlavage administration of highdose contraceptive pills, endometrial aspiration, and, in one case, RU 486. None of the infertile women became pregnant as a result of the trial. The authors conclude that, although superovulation may increase ovum production, without reliable contragestion, such practice is unsafe for ovum donors undergoing uterine lavage. Fertil Steril 51: 131,1989

We previously reported the successful recovery of fertilized human ova from women undergoing uterine lavage for purposes of ovum donation, using a specially designed catheter. 1 Originally, all procedures were performed following spontaneous ovulation, with ova recovered in 44% of inseminated cycles. It has been demonstrated that ova retrieved 90 to 130 hours after conception are most likely to successfully implant when transferred to a recipient, if development has progressed to the blastocyst stage. 2 Following spontaneous ovulation, the likelihood of recovering a blastocyst from a fertile donor during a lavage sequence is at best 25%. 3 Pregnancy rates in women who receive transferred embryos obtained by uterine lavage are approximately 10%. 1•4 This statistic reflects the Received July 20, 1988; revised and accepted September 16, 1988. 'Reprint requests: Mark V. Sauer, M.D., Women's Hospital, 1240 North Mission Road, Room L-946, Los Angeles, California 90033. Vol. 51, No.1, January 1989

transfer efficiency of blastocysts (50%), found in 25% of lavage cycles. The purpose of this study was to enhance ovum yield, and subsequent blastocyst recovery, by superovulating donors before uterine lavage, thus increasing the probability for pregnancy in recipient couples.

MATERIALS AND METHODS

We performed 28 lavages and collected data prospectively between July 1987 and April1988. Study subjects consisted of six normal, presumably fertile women who underwent uterine lavage in order to donate ova to six infertile women. All participants were informed of the risks of the procedure and gave written consent for participation, in conformity with guidelines established by the California Medical Center Institutional Review Board, which had approved the study. Ovum recipients were married women between 27 and 40 years of age. All were previously diagnosed with _premature ovarian failure. Recipients Sauer et al.

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were free of any major medical or surgical illness, and were screened psychologically. They were placed on a regimen of oral micronized estradiol (E 2 ) and vaginal progesterone (P) prior to attempted embryo transfer. 5 Husbands exhibited normal semen analyses with a minimum of 20 million motile sperm per ml and normal sperm morphology. Semen cultures were free of pathogens, and all males were serologically negative for human immunodeficiency virus (HIV). Ovum donors were selected by the recipient couple prior to enrolling in the study. Donors ranged in age from 21 to 37 years, were regularly ovulatory, and were free of any medical, surgical, or psychological disorders. Three donors were sisters of the recipient women, and three were paid participants. Prior to study entry, all demonstrated normal pap smears, negative cultures for cervical pathogens, and were serologically negative for HIV. In this trial, the nonsurgical uterine lavage sequence involved the following steps: Synchronization

Recipients began pharmacologic replacement of hormones 2 to 3 days before the expected onset of the donor's menses. This compensated for the shortened follicular phase of the donor's stimulated cycle. Ovulation Induction

Clomiphene citrate (CC), 100 mg/day and 1 ampule human menopausal gonadotropin (hMG) were given together for 5 days starting on the third day of the donor's menstrual cycle. Human menopausal gonadotropin was continued at 1 ampule per day from day 8 until the mean diameters of the lead follicles were 18 to 20 mm. At that time, 10,000 units human chorionic gonadotropin (hCG) was administered (8 P.M.) to effect ovulation. Insemination

Intrauterine insemination of the donor was performed 36 hours after hCG injection, using a washed, swim-up, specimen. Cervical mucus was examined microscopically prior to insemination to ensure the absence of sperm. Ultrasound Monitoring

Paily transvaginal ultrasound examinations with a 5.0 MHz transducer (General Electric 3000, General Electric Medical Systems, Milwaukee, 132

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Superovulation and uterine lavage

WI) were used for monitoring follicular development. Ovaries were imaged immediately before insemination and again 6 hours later in order to document ovulation. Additional ultrasound scans were performed 24 and 96 hours after ovulation to note the presence or absence of luteinized unruptured follicles. Lavage

Recovery of ova by uterine lavage was performed transcervically using a Louw catheter (Fertility and Genetics Research, Inc. Los Angeles, CA) without anesthesia at 96, 120, 144, and 168 hours after presumed ovulation. Ovulation was presumed to occur 36 hours after hCG injection. Sixty milliliters of phosphate-buffered saline containing 1% serum albumin was used as the medium for lavage. Recovered fluid was scanned immediately for the presence of ova, as recently described. 1 Ovum Transfer

A single ovum transfer was delivered to a recipient using 20 ~l Hepes-buffered Ham's F-10 solution (Gibco Laboratories, Grand Island, NY) supplemented with 20% heat-inactivated filtered pa. tient serum. Implantation Blockade

Immediately after the final uterine lavage, all donors underwent an endometrial curettage with a Vabra aspirator (Berkeley Medevices, Berkeley, CA). Ovral (ethinyl estradiol 0.05 mg, and norgestrel 0.5 mg, Wyeth Inc, Philadelphia, PA) was then administered, 4 tablets in 24 hours, as a contragestive.6 RESULTS

Twenty-eight lavages were performed on six donors during seven insemination cycles. Table 1 outlines our experience. No donor experienced a premature luteinizing hormone (LH) surge. In all cases, E 2 levels continued to rise after the administration of hCG. Persistent, unruptured follicles were noted in six of seven cycles studied, representing 13 of 35 (37%) preovulatory follicles tracked. The median volume of lavage fluid recovered was 92%, with a range of 67 to 98%; this is consistent with the reported past performance of the catheter.1 The catheter was inserted easily in all donors, and anesthesia was not required. Fertility and Sterility

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Table 1 Summary of Seven Stimulated Cycles In Six Women Undergoing Uterine Lavage Donor

Peak E 2 a

Ruptured follicles/follicles b

pg(ml

p

Ova recovered

ng(ml'

age in hours

Retained 1 cell (96) 1 cell (144) 0 Morula (96)

1 2 3 4 5 (cycle a) (cycle b)

3303 1199 1182 2392 1294

8/12 2/5 2/2 4/6 2/3

64.2 43.7 31.5 45.2 54.3

951

2/3

53.8

1 cell (96)

6

1750

2/4

39.0

Retained

Preovulatory value. 96 hours after hCG. c 144 hours after hCG. a

b

Three of four recovered ova were undifferentiated (Fig. 1). The single developed ovum, a morula obtained 96 hours after ovulation, was transferred but failed to implant (Fig. 2). Two donors, both of whom were sisters of therecipient, retained pregnancies despite Vabra aspiration and the administration of Ovral. A single 600 mg dose ofRU 486 was administered to one woman 14 days after ovulation, when the {1-hCG level was 96 miU /ml. This failed to terminate the pregnancy, requiring office curettage at 6 weeks gestation. The other pregnant donor elected not to terminate the pregnancy and is serving as a surrogate mother for her sister. DISCUSSION

Superovulation has been successfully employed in virtually all 15 mammalian species to undergo

Figure 1 Single cell, undifferentiated ovum recovered at 144 hours after ovulation. Vol. 51, No.1, January 1989

Figure 2 Morula recovered concommittently with a one-cell undifferentiated ovum 96 hours after ovulation. This transferred ovum did not result in pregnancy.

donor transfer. 7 In cattle, the introduction of superovulation was the principal advancement that established uterine lavage as an economically viable method for breeding. 7 Recovery of multiple, viable ova in these animals resulted in 60 to 80% live young per transfer customarily experienced on cattle ranches. Furthermore, there are no longterm detrimental effects to animal donors. Following repeated superovulatory cycles, no permanent detrimental effects have been described. It was our hope that increasing ovum yield in human donors would result in a similar increase in pregnancy rates for recipients. Compared with successful in vitro fertilization (IVF) programs, the per cycle pregnancy rate of nonsurgical donor ovum transfer is less efficient. 8 Yet, initially, when IVF was performed in unstimulated cycles, the pregnancy rate was equally poor, approximately 1 in 60. 9 Similarly, controlled ovarian hyperstimulation and artificial insemination in women with longstanding unexplained infertility has demonstrated enhanced pregnancy rates. 10 Retained pregnancy in the ovum donor is an inherent risk of the uterine lavage procedure. However, in designing this study, we believed the risk to be acceptably small. Pope et al. 11 reported a lower than expected rate of ovum retention (3%) in baboon ovum donors undergoing uterine lavage. This was attributed to a possible deleterious local effect on the endometrium created by flushing the cavity. In our original series involving 90 retrievals, ova were retrieved in only 44% of cycles, but retained pregnancies occurred in only 2% of cycles. 1 The efficiency of the catheter in recovering blastocysts was calculated to be between 67% and 100%. Sauer et al.

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In superovulated donors, high levels of endogenous E 2 , pharmacologic levels of sex steroids delivered exogenously after lavage, combined with the mechanical disruption of the endometrium by suction curettage should have further decreased the probability for successful implantation. Furthermore, in the event of ovum retention, administration of the contragestive RU 486 provided yet another potential safeguard against unwanted pregnancy. Remarkably, the inability to prevent implantation despite these measures makes superovulating donors a high-risk procedure. There are several explanations for why donor pregnancies may have occurred. First, as mentioned, the recovery efficiency of the Louw catheter is <100%. Thus, an increased number of ova would increase the risk that the donor will retain conceptuses. Second, multiple ovulations accompanied by delayed ovum pick-up after hCG administration would retard arrival of the ovum to the uterine cavity. A previous report has documented that ovulation occurs 22 to 47 hours after hCG administration in CC cyclesP Finally, elevated levels ofP, resulting from the development of multiple corpora lutea, could adversely affect tubal motility and retard ovum transport timeY Our data support a delay in ovum arrival. In spontaneous cycles, when or during which ova were recovered during the first lavage, the mean age of ova recovered was 108 hours, and only rarely (1% of cycles) were ova recovered on subsequent lavage dates. 2 However, following ovarian hyperstimulation, we noted recovery over a range of lavage times, as early as 96 hours in one case, and as late as 144 hours in another. Thus, it seems most likely that the retained pregnancies resulted from the arrival of the conceptuses in the uterus after the lavage and endometrial curettage were performed. In summary, superovulation of fertile women undergoing uterine lavage may well increase donor fecundity. However, the lavage schedule needs to be altered and normative data for ovum recovery reestablished. With the current protocol, hyper-

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stimulation adds the unacceptable risk of increasing the incidence of donor pregnancies. Current methods of contragestion in the face of hyperstimulation are not reliable in preventing pregnancies in donors. Despite the more invasive nature of in vitro fertilization, oocyte donors undergoing hyperstimulation and aspiration appear to face less risk from complications at the present time. REFERENCES 1. Sauer MV, Bustillo M, Gorrill MJ, Louw JA, Marshall JR,

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