Zygote intrafallopian transfer as a successful treatment for unexplained infertility*

FERTILITY AND STERILITY

Vol. 52, No.2, August 1989 Printed in U. S .A.

Copyright " 1989 The American Fertil ity Society

Zygote intrafallopian transfer as a successful treatment for unexplained infertility* Paul Devroey, M.D.t Catherine Staessen, M.Sc. Michel Camus, M.D.

Esther De Grauwe, M.D. Arjoko Wisanto, M .D . Andre C. Van Steirteghem, M.D., Ph.D.

Center for Reproductive Medicine, Vrije Universiteit Brussel, Brussels, Belgium

This study describes the zygote intrafallopian transfer treatment in patients with unexplained infertility. After retrieval, the oocytes were inseminated with 80,000 progressive motile sperm cells per milliliter. If fertilization occurred, a maximum of three zygotes were replaced by laparoscopy in the fimbria! end of one healthy fallopian tube. A pregnancy rate of 48.1 % per zygote intrafallopian transfer replacement was obtained. Seventeen pregnancies are actually ongoing, two patients delivered, and seven patients miscarried. Even after replacing ~ maximum of three zygotes, there were 6 twin and 2 triplet pregnancies. Fertil Steril 52:246, 1989

In 1986, we reported the first pregnancy after replacing three zygotes in one healthy fallopian tube in a female patient with sperm antibodies.1 This approach involves the video laparoscopic transfer of fertilized oocytes in an attempt to mimic the early embryonic development in the fallopian tube. Subsequently, more pregnancies have been re ported.2-4 The results of 61 zygote intrafallopian transfer treatment cycles in 54 patients with longstanding unexplained infertility are reported. Furthermore, the fate of the supernumerary cryopreserved zygotes and embryos are analyzed.

MATERIALS AND METHODS

From June 1987 until June 1988, 54 patients with longstanding unexplained infertility were treated in our center in 61 cycles by zygote intrafallopian transfer.

Received October 17, 1988; revised and accepted April 21, 1989. * Supported by gra n t 3.0036.85 from t he Belgian Fund for Medica l Scien tific R esearch. t Reprint requests: Paul Devroey, M .D., Center for R eproductive M edicine, Academic H ospital Vrije U niversiteit Brussel, Laarbeeklaan 101, B -1090 Brussels, Belgium.

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Patient Description

The mean.age of the women was 31.8 ± 4.5 years (range, 24 to 42 years), and the mean age of the male partner was 34.1 ± 6.2 years (range, 23 to 52 years). Forty-four patients had primary infertility since an average of 6.5 ± 3.6 years (range, 1 to 13 years) and 10 patients had secondary infertility since 4.5 ± 3.1 years (range, 2 to 9 years). All preliminary examinations in these couples were normal: repeated semen analyses, hysterosalpingogram, laparoscopy, postcoital test, and a full endocrine evaluation. Ovarian stimulation was done by the combination of a gonadotropin-releasing hormone analog (Buserelin, Suprefact, Hoechst Frankfurt, West Germany) and human menopausal gonadotropin (hMG) (Humegon, Organon Oss, Netherlands; Pergonal, Serono, line Zyma, Belgium) in 55 cycles or by the association of clomiphene citrate (Pergotime, Serono, line Zyma, Belgium) and hMG in 6 cycles. Both types of stimulation regimens have been extensively described. 5_8 Oocyte Retrieval

Thirty-six hours after the inject ion of 10,000 IU human chorionic gonadotropin (Pregnyl, Organon Oss, or Profasi, Serono) a transvaginal ultrasono-

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graphically guided oocyte retrieval was performed. 9 •10

Table 1

Pregnancy Rate and Number of Zygotes Replaced Pregnancies

No. of zygotes

No. of trials

1 2 3

3 9 42

0 5 21"

54

26

FW.

Culture Conditions

Oocytes were collected and cultured in Earle's medium supplemented with 8% of heat-inactivated patient's serum. Each oocyte was incubated in a 25JLI drop of medium under paraffin oil (37°C, 5% CO 2 , 5% O2 ,90% N 2 ). Fresh semen samples were collected at the time of oocyte retrieval. After washing, sperm cells were selected by a conventional swim-up procedure. After 2 to 6 hours of incubation, 2,000 spermatozoa (80,000/ml) were added. Fertilization was assessed 18 hours after insemination. The surrounding cumulus cells were gently removed mechanically and the oocytes were evaluated under an inverted microscope (200X) for the presence of two pronuclei. If at least one oocyte with regular smooth cytoplasm and two distinct pronuclei containing nucleoli was observed, the decision to perform a zygote intrafallopian transfer procedure was taken. Zygote Intrafallopian Transfer

A maximum of three zygotes were loaded in a translucent Teflon catheter, 30 cm long, 0.75 mm diameter, with end hole, fitted with a Luer lock plastic hub and with a 1 ml glass Hamilton syringe at one end (K-KEI-2011, William A. Cook, Melbourne, Australia Pty Ltd., Melbourne, Australia). The catheter was rinsed several times in Earle's culture medium supplemented with 75% inactivated patient's serum. The sequence of loading was as follows: transfer medium, 5 JLI air, a maximum of three zygotes in 15 JLI medium, 5 JLI air, and 5 JLI medium. Video translaparoscopic intrafallopian transfer was performed under general anesthesia. The loaded transfer catheter was inserted through a 10cm long trocar (1.3 mm diameter, Labotect, GmbH, Labor-Technik, Gottingen, West Germany) and placed 3 cm in the right or left fallopian tube. A maximum of three zygotes than were gently injected in only one healthy fallopian tube and the catheter checked to ensure that it was completely emptied. Cryopreservation of Supernumerary Zygotes

n -

The supernumerary zygotes were cryopreserved at the pronucleate stage with 1,2 propanediol (PROH). In some cases, the zygotes were kept in

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(%)

(0%) (55%) (50%) (48.1%)

" Including six twins and two triplets.

culture until the 4- to 8-cell stage and subsequently frozen with dimethyl sulphoxide (DMSO).11-14 Statistical Analysis

All relevant data were entered in a spread sheet on an Apple Macintosh PC (Apple Computer, Cupertino, CAl and a chi-square test was applied at the 5% level of significance. RESULTS

In 61 treatment cycles scheduled for zygote intrafallopian transfer, 664 oocytes were retrieved (mean, 10.9; median, 8.75; range, 1 to 38); 598 (90%) of the retrieved oocytes were mature. After insemination, 467 oocytes (65.8%) were fertilized, including 27 polyspermic zygotes. The zygote intrafallopian transfer procedure was done in 54 cycles. In 7 cycles, zygote intrafallopian transfer could not be done because of fertilization failure in 4, the impossibility to perform the intratubal transfer in 1, and a severe risk of hyperstimulation in 2. Per ZIFT one to three zygotes (mean, 2.7) were replaced, and 26 pregnancies were established, per transfer. The pregnancy rate per number of replaced zygotes is summarized in Table 1. After replacing 2 to 3 zygotes, half of the patients conceived. Seventeen pregnancies are actually ongoing, including 6 sets of twins and 2 triplets' so far two patients delivered two healthy childre~; seve~ had early pregnancy wastage: three were preclinical, and in four patients, who aborted, the pregnancy had been confirmed by ultrasound. No ectopic pregnancies were observed. Thirty-six out of 147 replaced zygotes implanted (24%). The ongoing pregnancy rate per retrieval was 31.1 % (19/61), and per replacement 35.9% (19/54). To limit the risk of multiple pregnancies, no more than 3 zygotes were replaced and 263 supernumerary zygotes were further cultured in vitro. Sixty-five were cryopreserved at the pronucleate Unexplained infertility and zygote intrafallopian transfer

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Table 2 Pregnancy and Implantation Rate After IVF, GIFT, and Zygote Intrafallopian Transfer in Patients with Unexplained Infertility Zygote intrafallopian transfer

No. of

no.

Transfers Pregnancies Replaced Zygotes Embryos Oocytes Implanted embryos a

54 a 26 a 147

no.

(%)

(48.1)

87 a 19 a

36 a ,b

(24.5)

24 a b

stage, the same day as the zygote intrafallopian transfer, using PROH as cryoprotectant; the remaining zygotes were further cultured for 24 hours, and 98 embryos were frozen at 4- or 8-cell stage with DMSO as cryoprotectant. In 16 of 26 pregnant patients, 110 supernumerary embryos are still cryopreserved for later use. In 16 of the nonpregnant patients, 53 embryos were frozen. So far, 19 multicellular cryopreserved embryos were thawed; 9 of them were found suitable for transcervical transfer, yielding two additional pregnancies of which one is ongoing and one ended in a miscarriage. Combining the zygote intrafallopian transfer and the transfer of frozen-thawed embryos, a pregnancy rate of 45.9% per oocyte retrieval and 51.8% per transfer was obtained so far. DISCUSSION

Our data showed that the pregnancy rate after the zygote intrafallopian transfer procedure was 42.7% per retrieval and 48.1% per replacement in patients with longstanding idiopathic infertility. The definitive pregnancy rate can only be evaluated after replacement of all cryopreserved embryos in subsequent natural cycles. Furthermore, 38% multiple pregnancies (six sets of twins and two sets of triplets) were observed in the group of patients in whom 3 zygotes were replaced (Table 1). It is remarkable that no multiple pregnancies were established when 2 zygotes were replaced. However, 5 out of 18 replaced zygotes implanted (28%) in 12 replacements (55%). These observations indicate that the pregnancy rate per transfer is similar after replacing 2 or 3 zygotes, respectively, 55% versus 50%. Furthermore, the implantation rate also was similar, replacing 2 or 3 zygotes, i.e., 28% versus 24%. These observations suggest that it could be advisable to further reduce Devroey et aI.

GIFT (%)

no.

(%)

(21.8)

50 15

(30)

(11.1)

149 18 b

217

P< 0.001.

248

IVF

(12.1)

P<0.05.

the number of replaced zygotes to 2 instead of 3 in order to diminish the risk of multiple pregnancies. In patients with unexplained infertility, the pregnancy and implantation rate after zygote intrafallopian transfer was significantly higher than after in vitro fertilization -embryo transfer (IVFET) (Table 2). Several arguments are available to understand the difference between the 11.1 % implantation rate after uterine transfer of a 4-cell embryo and the 24% implantation rate after tubal transfer of a zygote. The zygotes are probably not expelled from the fimbrial end of the fallopian tube, as it was reported for the 4-cell embryos after uterine replacement. 15 Furthermore, in zygote intrafallopian transfer, the early embryonic cleavages occur in the physiologic tubal milieu. This hypothesis also can be supported by the observation in the mouse that the I-cell stage is more sensitive than the 2-cell stage to environmental conditions such as pH, osmolality, and temperature. 16 Moreover, by replacing the zygotes into the fallopian tube, the cleaving embryos reach the uterine cavity at the appropriate time. However, to confirm the higher implantation rate after zygote intrafallopian transfer, controlled studies have to be performed such as the replacement of multicellular embryos in the fallopian tube, compared with uterine replacements of zygotes and multicellular embryos. Data confirming the superiority of tubal transfer in male infertility were published by Yovich et a1. 17 The pregnancy rate after zygote intrafallopian transfer was higher than after gamete intrafallopian transfer (GIFT), but the difference was not statistically different. In the zygote intrafallopian transfer procedure, the oocytes can be matured in vitro, abnormally fertilized oocytes are not replaced, and only fertilized oocytes are transferred. On the contrary, in the GIFT procedure, the 00-

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cytes cannot further complete maturation in vitro. IS Taking into account a 66% of fertilization rate of the oocytes, only two of three replaced 00cytes will be fertilized. The disadvantage of zygote intrafallopian transfer is the invasive two-step procedure: ultrasound-guided retrieval and laparoscopic replacement; however, when fertilization did not occur, the laparoscopy can be avoided. On the contrary, in the GIFT procedure, a laparoscopy has to be performed in all conditions. Seven of the 26 pregnancies (26%) ended in a clinical abortion. These results correlate with the findings of the abortion rate after GIFT and IVFET I9,20 Supernumerary zygotes require the availability of a cryopreservation program. So far, 2 additional pregnancies after replacing frozen and later thawed embryos were established. An additional number of pregnancies can be expected after the replacement of the frozen-thawed embryos. These favorable results of zygote intrafallopian transfer for the treatment of longstanding unexplained infertility will require definitive validation by a controlled study comparing zygote intrafallopian transfer and other methods of assisted procreation. Acknowledgments. We thank the nursing and technical staff for their skilful assistance, especially Ms. Pascale Henderix for collecting the data and Ms. Nadia Fenners for typing the manuscript.

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