Preliminary experiences with gamete intrafallopian transfer (GIFT)*

Transcription

1 FERTILITY AND STERILITY Copyright 198 The American Fertility Society Vol. 5, No.3, March 198 Printed in U.SA. Preliminary experiences with gamete intrafallopian transfer (GIFT)* Ricardo H. Asch, M.D. t Jose P. Balmaceda, M.D. Linda R. Ellsworth, M.D., Ph.D. Peng C. Wong, M.R.C.O.G.:j: Division of Human Reproduction, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas I! I j i, l,! This article describes the first series of patients to undergo gamete intrafallopian transfer (GIFT) as a treatment for infertility. Ten patients with the diagnosis of either unexplained infertility or male factor were treated with human menopausal gonadotropin and human chorionic gonadotropin before surgery by laparoscopy or minilaparotomy. Semen was collected 2.5 hours before oocyte pickup at surgery and treated by the technique of wash and swim-up. After gamete evaluation, one or two oocytes and 0,000 actively motile sperm were loaded into a catheter and introduced through the fimbria. The contents of the catheter were gently emptied at a site approximately 1.5 cm inside each fallopian tube. Patients received progesterone in oil, 12.5 mg/day, from day after GIFT until up to 8 weeks of gestation. Four patients became pregnant: two pregnancies aborted; the other two pregnancies proceeded to the delivery of viable infants. GIFT may be considered as an alternative to in vitro fertilization in infertility cases in which at least one fallopian tube is patent. Fertil Steril 5:'3, 198 Since the introduction of in vitro fertilization (IVF) and embryo transfer, 1 many techniques have been developed to manipulate fertilized and unfertilized human gametes for the treatment of Received September 2, 1985; revised and accepted December 2, *Recipient of the 1985 Squibb Award. Presented at the Forty-First Annual Meeting of The. American Fertility Society, September 28 to October 2, 1985, Chicago, Illinois. treprint requests: Ricardo H. Asch, M.D., Division of Human Reproduction, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas :j:postdoctoral Fellow. Present address: Department of Obstetrics and Gynaecology, National University of Singapore, Kandang Kerbau Hospital, Singapore, Republic of Singapore. infertility. Procedures such as intrauterine insemination,2 ovum transfer,3 and transfer of sperm and oocytes into the uterine cavity have been documented in the literature. We have recently reported the successful development of a new technique, gamete intrafallopian transfer (GIFT),5, an approach that involves the djrect transfer of preovulatory oocytes and washed sperm into the fallopian tubes. In an attempt to mimic the early physiologic processes that lead to gestation in the human, this technique results in the placement of both gametes at the normal site of fertilization. We present here the complete details of the first ten patients to undergo the GIFT technique in our institution and report the resulting successful birth of one set of twins and one singleton. 3 Asch et al. Preliminary experiences with GIFT Fertility and Sterility

2 Table 1. Clinical Profile of the First Ten Infertile Couples to Undergo GIFT" Case Age Infertility Dia~- Female Male nosls Type Duration UI 1 7 b MF 1 8 b UJ< 1 F,2 M UI 1 2b MF UI 2 F,1 M MF UI 2 5 b UI UI 1 18 auj, unexplained infertility; MF, male factor. bpregnancies. cyasectomy reversal. MATERIALS AND METHODS OVARIAN STIMULATION After exhaustive studies, patients undergoing ovarian stimulation were diagnosed (Table 1) as having either unexplained infertility7 or a male factor. All of them had previously undergone numerous therapeutic trials, which included induction of ovulation with clomiphene citrate and/or human menopausal gonadotropin (hmg) with and without intrauterine insemination. Patients received hmg, 2 ampules (150 IU offollicle-stimulating hormone and 150 IU of luteinizing hormone; Pergonal, Serono Laboratories, Inc., Randolph, MA) intramuscularly for ovarian stimulation beginning on day 3 of the menstrual cycle. Patients underwent pelvic ultrasound examination at the beginning of the cycle to rule out adnexal masses and then each day after day. Serum estradiol levels were measured on day 3 and daily from day by specific radioimmunoassay.8 Human chorionic gonadotropin (hcg; Profasi, Serono Laboratories, Inc.),,000 IU intramuscularly, was given when two or more follicles reached 1 mm in diameter on ultrasound and serum estradiol levels reached 300 to 500 pg/ml for each main follicle (> 1 mm in diameter). Thirty-six hours after hcg injection, follicles were aspirated via laparoscopy or minilaparotomy (a suprapubic 3- to -cm incision). SEMEN WASH AND SWIM-UP Semen specimens were obtained 2.5 hours before surgery and allowed to liquefy for 30 minutes Yol. 5, No.3, March 198 yrs before semen analysis was performed. The count, motility, morphology, and forward progression score (0, none; 1, poor; 2, good; 3, excellent) of sperm were recorded. Semen was gently mixed at a ratio of 1:3 (vol/vol) with Ham's F-1O medium (GIBCO, Grand Island, NY) with % fetal cord serum (1:3 vol/vol) containing streptomycin (000 U/0,000 ml) and penicillin (12,000 U/0 ml) and centrifuged for minutes at 00 x g. After the supernatant was removed, 0.5 to 1 ml of medium was layered onto the pellet and sperm were allowed to swim out of the pellet for 5 minutes. After removal of the medium above the pellet, sperm parameters were reassessed and the sperm concentration was adjusted to 0,000/25 j.ll whenever possible. The semen specimens for cases 2 and 5 were collected in 8 ml of medium because of high viscosity and asthenospermia. For case 2, which had severe oligospermia as well, the pellet was resuspended and allowed to settle for 5 minutes after the original swim-up was unproductive. After preparation, samples were incubated in 5% CO2 and air at 37 C until tran~fer. OOCYTE RECOVERY AND GAMETE INTRAFALLOPIAN TRANSFER After follicular aspiration, recovered oocytes were placed in individual culture dishes containing 0 IJJ of Ham's F-1O medium with 50% fetal cord serum under silicon oil. If necessary, the cumulus oophorus was spread slightly, with the use of sterile 27 -gauge needles, to facilitate identification of polar bodies. With the use of Nomarski optics, the oocytes were classified and photographed and then maintained in an incubator with 5% CO2 and air at 37 C until transfer. 00- cytes were classified as follows: grade 1, germinal vesicle; grade 2, thick cumulus and corona (C + C); grade 3, partially dispersed C + C; grade, well-dispersed C + C; grade 5, presence of polar body. Upon completion of oocyte evaluation, gametes were loaded into a catheter for transfer. A Deseret Intracath (#3132, The Deseret Company, Sandy, UT) catheter "with a 1-ml tuberculin syringe at one end was rinsed twice with medium containing 50% Ham's F- and 50% fetal cord serum. The catheter was loaded with 25 j.ll of the sperm preparation containing 0,000 sperm (which had been placed in a drop on an inverted culture dish lid), an air space of 5 j.ll, one or two of Asch et al. Preliminary experiences with GIFT 37

3 Table 2. Characteristics of the Individualized Induction of Follicular Development in the First Ten GIFT Cases a Case hmg No. ampules Serum estradiol at hcg in jection pglml al, laparoscopy; ML, minilaparotomy. bpregnancies. Operative route Lb MU ML MLb ML L L MU ML L the more mature oocytes in 25 fll of medium, another airspace of 5 fll and, finally, fll of medium. The prepared catheter was transported to the adjoining operating room under sterile conditions and inserted into the aspiration needle, which had been placed in the operating channel of the laparoscope. Under laparoscopic or direct (minilaparotomy) visualization, the catheter tip was guided into the fimbriated end of the fallopian tube, inserted a distance of approximately 1.5 cm, and the contents of the catheter gently discharged. The catheter was then returned to the laboratory and examined to assure that it had been completely emptied. The same procedure was then repeated for the second fallopian tube. A period of approximately 5 minutes to 1 hour was required to perform the entire operative procedure. POSTOPERATIVE CARE Patients were discharged on the day of the procedure or the following day, as appropriate for their recovery from general endotracheal anesthesia. All patients received progesterone in oil, 12.5 mg intramuscularly daily from day after transfer until up to 8 weeks of gestation. Serum (3-hCG levels were measured by radioimmunoassay from day 7 after transfer, and pelvic ultrasound scans were performed 3 to 5 weeks after GIFT. RESULTS Table 1 shows the clinical profile of the ten infertile couples who underwent the GIFT procedure. Table 2 characterizes the regimen for gonadotropin stimulation of follicular development in each one of the women. Table 3 shows the results of the pelvic ultrasound in terms of number and size (maximal diameter) of follicles per patient at the time of hcg injection. In addition, it shows the number and maturity of oocytes aspirated during pickup and of those transferred during GIFT. As can be noted, this scheme of ovarian stimulation generally produced a good cohort of preovulatory oocytes in most subjects. Table reveals the characteristics of the semen analysis before and after the wash and swim-up technique. As shown, most of the semen specimens presented marked improvement in both motility and progression.. The diagnosis of pregnancy was established when at least two consecutive sera obtained on and after day 1 after GIFT revealed concentra- II I Ii 'I Table 3. Detailed Description of the Follicular Diameter (by Ultrasound) and of the Maturity of Oocytes Aspirated and Transferred in the First Ten GIFT Cases a Case Follicular diameter (measured by ultrasound) Maturity of oocytes aspirated b Maturity of oocytes transferred b mm 1 20,18,1, ,1,1,15,15,12 3 1,1,15,1, 17,17,15,1,1 5 17,1,15,15,,,19,18, ,1,15,1,13, ,19,1,1 9 2,19,18,,, 22,17,1, ad, degenerated; CL, corpus luteum. bsee text for scoring criteria. CPregnancies. no. 5 5 no. no.,,5,5,,5,5 c 2,3,,5 2,3,,5 c 3,,, 3,,, 3, 2 3, 2 c,,,,,d,d,d 8,,, 3,, 3 3,, 3 1,5 2 1,5 2 CL,,5,D,D 5,,5 3 c 1,1,1,3,5,5,5,D 8 3,5,5,5,,,5,,,5 38 Asch et al. Preliminary experiences with GIFT Fertility and Sterility

4 Table. Comparison of the Characteristics of the Semen Before and After the Wash and Swim-up Technique in the First Ten GIFT Cases a Semen Case Prewash Postwash C M P M P b Ib b b ac, count (l0/ml); M, motility (%); P, progression (see text for scores). bpregnancies. tions of ~-hcg > 50 miulml. In addition to the four pregnancies diagnosed, patient 5 had a low, transient elevation of serum ~-hcg 15 days after hcg injection but no delayed menses. Patients 2 and miscarried during the first 2 months of gestation. Patient 1 had an uneventful twin pregnancy and delivered vaginally at 2 days after GIFT. Both newborns were normal, weighed 3 gm and 300 gm, and had karyotypes of,xx and,xy, respectively. Both infants had Apgar scores of 9/ (1 and 5 minutes). Patient 8 had an uneventful singleton pregnancy and was delivered of a normal female infant at 237 days after GIFT via cesarean section performed for premature rupture of membranes and arrest of labor. The newborn weighed 2835 gm, her karyotype was,xx, and her Apgar score was 9/ (1 and 5 minutes). DISCUSSION This report clearly demonstrates that GIFT is a successful technique for the treatment of infertility and documents the delivery of the first two GIFT pregnancies. The transfer of the gametes into the fallopian tubes can be easily accomplished by laparoscopy or minilaparotomy, and the postoperative course is similar for both procedures. The GIFT technique is simple; however, it requires some expertise in the laboratory handling of human gametes: sperm and oocytes. In order to place both gametes at the site of normal fertilization in the human, the fallopian tube, the gametes must be appropriately treated. The semen Vol. 5, No.3, March 198 specimens were treated by the technique of washing and swim-up, with a method similar to that originally described by Ericsson. 9 This procedure facilitates isolation of fractions of viable human sperm from the ejaculate. We observed a marked improvement in sperm motility and forward progression in the isolated fractions. In addition, we have demonstrated recently that the sperm washing technique removes microorganisms from human semen, as verified by electron microscopic studies and a significant decrease in the number of positive cultures. Thus, we believe that it is safe to introduce a sperm preparation handled in this fashion into the fallopian tubes with minimal risk of inducing pelvic inflammatory disease. After oocyte collection during surgery, the 00- cytes are transported to the adjacent laboratory for microscopic evaluation of their maturity. This assessment is essential in order to select the preovulatory oocytes in preference to those that are immature or degenerated. During the design of this study, we were uncertain about the number of gametes that would be needed to ensure pregnancy after GIFT. The number of sperm that actually reach the site of fertilization in the human is not clearly known, because of a paucity of research concerning sperm transport through the female genital tract ll or to the peritoneal fluid. 12 We decided to transfer approximately 0,000 active sperm per oviduct because this number has proven to be sufficient to fertilize oocytes in vitro.13 It is tempting to speculate that we may be able to reduce the number of sperm andlor oocytes transferred during GIFT, possibly varying the number according to the cause of the existing infertility. We are currently using the rhesus monkey (Macaca mulatta) as an animal model for GIFT and hope to determine the minimum number of gametes that must be transferred to ensure success.1 The possibility that the CO2-induced pneumoperitoneum during laparoscopy may significantly alter the intraabdominal ph, affecting gamete performance, is now under investigation. The pregnancies produced by GIFT may be due to improvement of several reproductive processes, according to the cause of infertility. In cases of unexplained infertility or endometriosis, the transport of the gametes to the fertilization site may be deficient.15, 1 It should be noted that, in highly fertile women, the efficiency of oocyte pickup by the tube at the moment of ovulation is no greater than 3%.17 It may be that, in women Asch et ai. Preliminary experiences with GIFT 39

5 for whom no cause for infertility can be demonstrated or in those having endometriosis, the efficiency of the oocyte pickup mechanism is extremely W.lB, 19 This factor should be further investigated. The luteinized unruptured follicle syndrome has been proposed as a cause ofinfertility in some couples.20 Oocyte entrapment in follicles during stimulated cycles has been documented, despite the presence of obvious ovarian ovulatory stigma identified at laparoscopy.2l In both of these events, the GIFT technique would be an appropriate method of correcting the mechanical deficiency responsible for oocyte retention, allowing both gametes to join at the normal site of fertilization. In cases of severe oligospermia, cervical factor, and/or immunologic factor, GIFT may succeed by avoiding passage of sperm through the female genital tract and by concentrating isolated fractions of viable sperm at the fertilization site. Based on the present results, GIFT appears to be an attractive alternative toivf in cases in which the female partner of the infertile couple has at least one intact fallopian tube. Additionally, it should be emphasized that GIFT and IVF are not mutually exclusive and may be complementary. Unexpected surgical findings and/or the recovery of immature oocytes may necessitate performance of IVF rather than GIFT in some cases. Moreover, we are. currently initiating a program in which we will attempt GIFT and use any extra oocytes to perform IVF. If embryos are formed, they will be cryopreserved22 for future use in nonstimulated cycles. An international, multicenter study of GIFT is currently in progress and should provide information regarding the incidence of multiple gestations and ectopic pregnancies, enabling improvement of our current protocol. We are maintaining a register of the karyotypes of the first 0 newborns resulting from GIFT. Because of the lack of contact of the unfertilized oocyte and sperm in vitro and the absence of any possibility of manipulation of a human embryo, the GIFT technique may well be more acceptable than other techniques to some cultural and religious groups in society. REFERENCES 1. Steptoe PC, Edwards RG: Birth after the reimplantation of a human embryo. Lancet 2:33, Sher G, Knutzen VK, Stratton CJ, Montakhab MM, AIlenson SG: In vitro sperm capacitation and transcervical intrauterine insemination for the treatment of refractory infertility: Phase 1. Fertil Steril 1:20, Buster JE, Bustillo M, Thorneycroft IH, Simon JA, Boyers SP, Marshall JR, Louw JA, Seed RW, Seed RG: Nonsurgical transfer of in vivo fertilised donated ova to five infertile women: report of two pregnancies. Lancet 1:81, Craft I, McLeod F, Green S, Djahanbakhch 0, Bernard A, Twigg H, SmithW, Lindsay K, Edmonds K: Human pregnancy following oocyte and sperm transfer to the uterus. Lancet 1:31, Asch RH, Ellsworth LR, Balmaceda JP, Wong PC: Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet 2:3, 198. Asch RH, Balmaceda JP, Ellsworth LR, Wong PC: Gamete intrafallopian transfer (GIFT): a new treatment for infertility. Int J Fertil 30:1, Moghissi KS, Wallach EE: Unexplained infertility. Fertil Steril 39:5, Asch RH, Rojas FJ: The effects of RU8 on the luteal phase of the rhesus monkey. J Steroid Biochem 22:227, Ericsson RJ: Isolation and storage of progressively motile human sperm. Andrologia 9:111, Wong PC, Balmaceda JP, Blanco JD, Gibbs RS, Asch RH: Sperm washing and swim-up technique using antibiotics removes microbes from human semen. Fertil Steril5:97, Blasco L: Clinical tests of sperm fertilizing ability. Fertil Steril1:177, Asch RH: Laparoscopic recovery of sperm from peritoneal fluid in patients with negative or poor Sims-Huhner test. Fertil Steril 27:1111, Trounson AO, Leeton JF, Wood C, Webb J, Kovacs G: The investigation of idiopathic infertility by in vitro fertilization. Fertil Steril 3:31, Wong PC, Heitman T:Balmaceda JP, Ellsworth LH, Asch RH: Pregnancies following gamete intra-fallopian transfer (GIFT) in the Macaca TfUl,latta (Abstract P-11l). Pre, sented at the Forty-First Annual Meeting of The American Fertility Society, Chicago, IL, September 28 to October 2, Published by The American Fertility Society, Birmingham, AL, 1985, p Stone SC: Peritoneal recovery of sperm in patients with infertility associated with inadequate cervical mucus. Fertil Steril 0:802, Rogers SF, Jacobs WM: Infertility and endometriosis: conservative surgical approach. Fertil Steril19:529, Croxatto HB, Ortiz ME, Diaz S, Hess R, Balmaceda J, Croxatto H-D: Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following luteinizing hormone peak. Am J Obstet Gynecol 132:29, Drake TS, O'Brien WF, Ramwell PW, Metz SA: Peritoneal fluid thromboxane B2 and -keto-prostaglandin Flo< in endometriosis. Am J Obstet Gynecol :01, Werlin LB, dizerega GS, Hodgen GD: Endometriosis: effect on ovulation, ovum pickup, and transport in monkeys: an interim report (Abstr). Fertil Steril 35:23S, Asch et ai. Preliminary experiences with GIFT Fertility and Bterility

6 20. Dhont M, Serreyn R, Duvivier P, Vanluchene E, De Boever J, Vandekerckhove D: Ovulation stigma and concentration of progesterone and estradiol in peritoneal fluid: relation with fertility and endometriosis. Fertil Steril 1:872, Stanger JD, Y ovich JL: Failure of human oocyte release at ovulation. Fertil Steril 1:827, Trounson A, Mohr L: Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707, 1983 Vol. 5, No.3, March 198 Asch et ai. Preliminary experiences with GIFT 371