- Email: [email protected]
Successful pregnancies from the transfer of pronucleate embryos in an outpatient in vitro fertilization program
Vol. 44, No.2, August 1985 Printed in U.8A.
FERTILITY AND STERILITY Copyright ~ 1985 The American Fertility Society
Successful pregnancies from the transfer of pronucleate embryos in an outpatient in vitro fertilization program
Kamal K. Ahuja, Ph.D. *t:I: William Smith, D.M.U.t Michael Tucker, Ph.D. t Ian Craft, F.R.C.S.t In Vitro Fertilization Laboratory, Cromwell Hospital, London, and Medical Research Council, Carshalton, Surrey, United Kingdom
Twenty-nine infertile women were given clomiphene citrate (100 mg/day, days 3 to 7), human menopausal gonadotropin (150 1U/day, days 7 to 12 or 13), and human chorionic gonadotropin (2000 1U) for the induction of ovulation. Eggs were collected by laparoscopy and preincubated for 6 hours before being cultured with spermatozoa for fertilization. Approximately 16 to 18 hours after insemination, the eggs that showed two pronuclei were transferred to the patient's uterus. As confirmed by the ultrasonic appearance of a gestational sac, six women who received pronucleate embryos became pregnant, and three pregnancies resulted in normal full-term deliveries. These results confirm that, unlike requirements for most laboratory and farm animals, the requirements of synchrony between the preimplantation human uterus and developing embryos are not very stringent. In vitro fertilization treatment procedures can thus be made simpler by the transfer of embryos at the pronucleate stage. Fertil Steril44:181, 1985
In vitro fertilization (IVF) and embryo transfer is now an accepted form of treatment for some types of male and female infertility. In most clinics the treatment requires the preovulatory monitoring of the plasma and urinary hormones to measure ovarian follicular growth, the fertilization of laparoscopically collected oocytes by husband's or donor spermatozoa, the culture of resulting embryos to the 4- to 8-cell stage, and finally the placement of these embryos in the patient's uterus. In many centers women are still Received January 21, 1985; revised and accepted April 12, 1985. *Reprint requests: Dr. K. K. Ahuja, IVF Laboratory, Humana Hospital, Wellington Place, London NW8 9LE, United Kingdom. tIVF Laboratory, Cromwell Hospital. *Medical Research Council. Vol. 44, No.2, August 1985
required to be hospitalized for 6 to 9 days for the facilitation of serial blood and urine collection for hormone assays and for awaiting fertilization of the eggs and subsequent embryo transfer. This is an expensive and stressful part of the treatment, especially because success rates are quite unpredictable and significantly lower than the natural pregnancy, even with the transfer of more than one embryo. Our understanding of early human development needs to be more complete before this method of treatment becomes simpler and more widely available to all infertile couples. It has been our policy to monitor ovarian follicular development by ultrasound scanning on a purely outpatient basis without the use of conventional hormone assays. Patients attending the clinic require an average of only three ultrasound scans during the preovulatory phase in order for us to time egg collection. More recently we have Abujaet al. Transfer of pro nucleate embryos
181
• i
a
ill; •
Table 1. Comparison Between Fertilization and Implantation Rates in Two Different Culture Media Medium
B3 + human serum albumin Earle's + cord serum
No. of patients
Total no. of eggs collected (eggs/patient)
% Fertilization
% Implantation"
(proportion)
18
81 (4.5)
66.7
22.3 (4/18)
11
26 (2.4)
80.7
18.9 (2/11)
aConfirmed by ultrasound.
shown that in the follicular phase, the endometrium undergoes distinct qualitative and quantitative changes that can be monitored ultrasonically and, in conjunction with the follicular measurements only, can further optimize the timing of human chorionic gonadotropin (hCG).l In this communication, preliminary data are presented that suggest that the outpatient procedure can be simplified further if the replacement of embryos is carried out before they have undergone syngamy.
after insemination, the loosely attached cumulus cells were removed from the oocytes by gentle pipetting and checked for the presence of pronuclei. The oocytes containing two pronuclei were transferred directly to the patient's uterus in 8 to 10 fll of fresh culture medium. No blood or urine sampling was performed, and no hormone support was given during the luteal phase of the cycle. All patients were treated on an outpatient/daystay basis. All resulting pregnancies were confirmed by the ultrasonic appearance of a gestational sac.
MATERIALS AND METHODS
RESULTS
The study involved 29 consecutive couples participating in our IVF program. Fifteen patients had irreparable tubal damage, 11 patients had idiopathic infertility of many years' duration, and 3 patients were treated because of severe oligospermia. The treatment was carried out on the basis of one attempt per patient. Multiple ovulation was induced by 100 mg clomiphene citrate (Clomid, Merrell Pharmaceuticals, Slough, UK) given daily from days 3 to 7 of the cycle and 150 IU of human menopausal gonadotropin (Pergonal, Serono Laboratories, Ltd., Welwyn Garden City, UK) given daily from day 7; the follicular growth was monitored ultrasonically with a Diasonics mechanical sector scanner (model DRF 1, Diasonics, Inc., Sunnyvale, CA) as described previously. When the leading follicle reached 18 mm in diameter, 2000 IU ofhCG (Profasi, Serono Laboratories, Inc.) was given intramuscularly; 00cytes were aspirated from follicles laparoscopically 36 hours later.2 The techniques used for IVF and culture of embryos and the details of replacing embryos in the uterus have been described previously.2 Briefly, the oocytes were preincubated in 1 ml of Earle's medium that contained 10% cord serum or Menezo's B3 medium that contained 20% (wt/vol) human serum albumin (Sigma Chemicals, London, UK) at 37°C in 5% CO 2 in air for 6 hours before insemination with the husband's sperm in specially designed glass dishes. About 16 to 18 hours
Of 107 preovulatory oocytes collected from 29 patients, 75 (70%) had two pronuclei when examined 16 to 18 hours after insemination. All pronucleate embryos were transferred, and 6 of 29 patients (20.6%) became pregnant. Fertilization rates were slightly higher in the Earle's medium, compared with the B3 medium (Table 1), although the possibility that this difference may be the result of fewer eggs collected per patient in the Earle's + cord serum group cannot be ruled out at the present time. The pregnancy rates did not exhibit any statistically significant differences between the two groups. All pregnancies were established in patients between 22 and 34 years of age; five of the six pregnancies were obtained when four or more embryos were transferred (Table 2), further strengthening our long-held belief" that until suitable techniques are available to determine the viability of preimplantation human embryos,
182
Ahuja et al.
Transfer of pronucleate embryos
Table 2. Relation Between Pregnancy Rate and Number of Pronucleate Embryos Replaced No. of embryos
1 2 3 4 5 6
No. of patients
Clinical pregnancies confirmed by ultrasound
9
o
7
1
6
o
3 2 2
2
1 2
Fertility and Sterility
fil
the number of embryos to be transferred should not be restricted to two or three. This pregnancy rate compares quite favorably with the 15% to 20% success rate obtained in previous studies when 2- to 8-cell embryos were transferred. 1, 4, 5 Pregnancies were established in all three treatment groups, two pregnancies in patients suffering from tubal damage, three pregnancies in patients treated for idiopathic infertility, and one pregnancy in the patients with severe oligospermia. Pregnancies resulting from the transfer of two and six embryos were lost between 6 and 8 weeks after transfer. A detailed cytologic and hormonal analysis of the luteal phase of these pregnancies was not carried out. The remaining three singleton pregnancies have resulted in normal full-term deliveries. DISCUSSION
In the majority of IVF clinics, freshly fertilized ova are cultured for 24 to 48 hours before replacement in the uterus. The rationale behind this practice is twofold. Firstly, it allows the achievement of a closer synchrony between the preimplantation uterus and developing embryos. The entry of the fertilized ovum into the uterus is controlled in a species-specific manner. In most species it normally occurs at the morula-to-blastocyst stages when, under the influence of circulating hormones, the uterus is not lethal or hostile to the embryo.6 In natural cycles, the human embryo arrives in the uterine cavity 3 to 4 days after ovulation. 7 In the present study, successful intrauterine pregnancies resulting from the replacement of embryos approximately 18 to 20 hours after laparoscopy (i.e., when the endometrium is 60 to 70 hours behind) suggest there is a rather weak or uncontrolled synchrony between the preimplantation uterus and developing embryos in a stimulated cycle. Even though the uterus is not fully receptive, it is capable of supporting the growth of early embryos until the initiation of implantation. Whether this explanation is applicable to the unstimulated endometrium is a matter for future research, although based on histologic data some conflicting reports have already appeared in the literature. s,9 The lack of uterine hostility toward pronucleate embryos may also indicate that, at least during the luteal phase, there are similarities between the oviductal and uterine environments. This is further supported by the similar protein profiles in Vol. 44, No.2, August 1985
Table 3. Analysis of 100 Ultrasonically Confirmed Pregnancies Resulting from the IVF Program at Cromwell Hospital Stages of embryonic developmenta
No. of pregnancies
1 cell 2 cell 3 cell 4 cell Mixed 2-8 cell
2 8 1
41
48
aForty to 44 hours after insemination.
the human of tubal and uterine secretions,lO as well as of the ectopic implantation, a pathophysiologic event that occurs exclusively in the human. Secondly, the practice of culturing embryos for 24 to 48 hours before replacement ensures that the embryos that undergo retarded growth or cleavage arrest are detected and eliminated. The cleavage rates of human embryos that result'from IVF vary considerably, and rapidly dividing embryos are regarded as having a better potential to result in successful pregnancies. 11 From a retrospective analysis of the first 100 pregnancies that resulted in our own program (Table 3), we have also found that more than 80% of the embryos completed the first two or three cleavage divisions within 40 to 44 hours after insemination. However, some of the remaining embryos that suffered apparent cleavage arrest or retarded growth resulted in pregnancy. Clearly the cleaving ability of embryos fertilized in vitro is not necessarily a true indication of their ability to implant in utero. It is possible that some of the normally dividing embryos did not produce a pregnancy because they lost their potential to implant in utero because of prolonged culture in vitro. Some animal studies have shown that embryos fertilized in vitro do not grow as efficiently as in vivo embryos and that their potential to implant in hCG-synchronized recipients is impaired,12, 13 although one recent study in the mouse failed to find significant differences between the two groups.14 Culture conditions have also been shown to exert deleterious effects on hamster embryos,15, 16 which further supports the view that a variety of culture media used for human IVF may not reflect the true fallopian tube environment and may exert some adverse effects during the prolonged culture of human embryos. If the results obtained from this pilot study can be reproduced in larger randomized trials, it could be argued that a maximal pregnancy rate will result after the replacement of pronucleate embryos. In Ahuja et al.
Transfer of pronucleate embryos
183
141
-,
• ___ &2&
terms of simplifying the IVF technology to suit the outpatient environment, we would also regard the present method as superior to the previously reported method of replacing sperm/egg complexes in the uterus within 7 to 8 hours after laparoscopy,17, 18 because this obviates fertilization failure due to immunologic or idiopathic factors as being responsible for a failed treatment cycle. Acknowledgments. We thank David Whittingham, Ph.D., for a critical reading of the manuscript and Miss Henrietta Lee for typing it. REFERENCES 1. Smith W, Porter R, Ahuja K, Craft I: Ultrasonic assessment of endometrial changes in stimulated cycles in an in vitro fertilization and embryo transfer programme. J In Vitro Fertil Embryo Trans 1:233, 1984 2. Craft I: In vitro fertilization-clinical methodology. Br J Hosp Med 31:90, 1984 3. Craft I, Porter R, Green S, Tucker M, Smith B, Twigg H, Ahuja K, Whittingham DG: Success of fertility, embryo number and in-vitro fertilization. Lancet 1:732, 1984 4. Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM, Steptoe PC, Webster JM: Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fertil Embryo Trans 1:23, 1984 5. Speirs A, Trounson A, Warner GM, Yovich JL, Saunders DM, Chen C: Summary of results. In Clinical In Vitro Fertilization, Edited by C Wood, A Trounson. Berlin, Springer-Verlag, 1984, p 157 6. Webb PD, Glasser SR: Implantation. In Human In Vitro Fertilization and Embryo Transfer, Edited by DP Wolf, MM Quigley. New York, Plenum Press, 1984, p 341 7. Croxatto HB, Ortiz ME, Diaz S, Hess R, Balmaceda J, Croxatto HD: Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following LH peak. Am J Obstet Gynecol 132:629, 1978
184
Ahuja et aI.
Transfer of pronucleate embryos
8. Garcia JE, Acosta AA, Hsiu J-G, Jones HW Jr: Advanced endometrial maturation after ovulation induction with human menopausal gonadotropinihuman chorionic gonadotropin for in vitro fertilization. Fertil Steril 41:31, 1984 9. Abate V: The endometrium at the time of transfer. Presented at the Third World Congress ofln Vitro Fertilization and Embryo Transfer, Helsinki, May 14 to 17, 1984. Abstract, p 221 10. Beier HM, Beier-Hellwig K, Delbos R: Hormones and proteins involved in uterine preparation for implantation. In Fertilization of the Human Egg In Vitro, Edited by HM Beier, HR Lindner. Berlin, Springer-Verlag, 1983, p 307 11. Mohr L, Trounson A: In vitro fertilization and embryonic growth. In Clinical In Vitro Fertilization, Edited by C Wood, A Trounson. Berlin, Springer-Verlag, 1984, Chap 8, p 99 12. -Leroy F, Massip A, Puissant F, Camus M, Vandurzwalmen P: Effect of in vitro culture, freezing and transfer on mouse embryonic capacity for implantation. Arch Androl 11:184, 1983 13. Shalgi R: Developmental capacity of rat embryos produced by in vivo or in vitro fertilization. Gamete Res 10:77,1984 14. Glenister P, Wood M, Whittingham D: Unpublished results, personal communication 15. Whittingham DG, Bavister BD: Development of hamster eggs fertilized in vitro or in vivo. J Reprod Fertil 38:489, 1974 16. Farrel PS, Bavister BD: Short-term exposure of two-cell hamster embryos to culture media is detrimental to viability. Bioi Reprod 31:109, 1984 17. Craft I, McLeod F, Green S, Djahanbakhch 0, Bernard A, Twigg H: Human pregnancy following oocyte and sperm transfer to the uterus. Lancet 1:1031, 1982 18. Asch RH, Ellsworth LR, Balmaceda JP, Wong DC: Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet 2:1034, 1984
Fertility and Sterility
FERTILITY AND STERILITY Copyright ~ 1985 The American Fertility Society
Successful pregnancies from the transfer of pronucleate embryos in an outpatient in vitro fertilization program
Kamal K. Ahuja, Ph.D. *t:I: William Smith, D.M.U.t Michael Tucker, Ph.D. t Ian Craft, F.R.C.S.t In Vitro Fertilization Laboratory, Cromwell Hospital, London, and Medical Research Council, Carshalton, Surrey, United Kingdom
Twenty-nine infertile women were given clomiphene citrate (100 mg/day, days 3 to 7), human menopausal gonadotropin (150 1U/day, days 7 to 12 or 13), and human chorionic gonadotropin (2000 1U) for the induction of ovulation. Eggs were collected by laparoscopy and preincubated for 6 hours before being cultured with spermatozoa for fertilization. Approximately 16 to 18 hours after insemination, the eggs that showed two pronuclei were transferred to the patient's uterus. As confirmed by the ultrasonic appearance of a gestational sac, six women who received pronucleate embryos became pregnant, and three pregnancies resulted in normal full-term deliveries. These results confirm that, unlike requirements for most laboratory and farm animals, the requirements of synchrony between the preimplantation human uterus and developing embryos are not very stringent. In vitro fertilization treatment procedures can thus be made simpler by the transfer of embryos at the pronucleate stage. Fertil Steril44:181, 1985
In vitro fertilization (IVF) and embryo transfer is now an accepted form of treatment for some types of male and female infertility. In most clinics the treatment requires the preovulatory monitoring of the plasma and urinary hormones to measure ovarian follicular growth, the fertilization of laparoscopically collected oocytes by husband's or donor spermatozoa, the culture of resulting embryos to the 4- to 8-cell stage, and finally the placement of these embryos in the patient's uterus. In many centers women are still Received January 21, 1985; revised and accepted April 12, 1985. *Reprint requests: Dr. K. K. Ahuja, IVF Laboratory, Humana Hospital, Wellington Place, London NW8 9LE, United Kingdom. tIVF Laboratory, Cromwell Hospital. *Medical Research Council. Vol. 44, No.2, August 1985
required to be hospitalized for 6 to 9 days for the facilitation of serial blood and urine collection for hormone assays and for awaiting fertilization of the eggs and subsequent embryo transfer. This is an expensive and stressful part of the treatment, especially because success rates are quite unpredictable and significantly lower than the natural pregnancy, even with the transfer of more than one embryo. Our understanding of early human development needs to be more complete before this method of treatment becomes simpler and more widely available to all infertile couples. It has been our policy to monitor ovarian follicular development by ultrasound scanning on a purely outpatient basis without the use of conventional hormone assays. Patients attending the clinic require an average of only three ultrasound scans during the preovulatory phase in order for us to time egg collection. More recently we have Abujaet al. Transfer of pro nucleate embryos
181
• i
a
ill; •
Table 1. Comparison Between Fertilization and Implantation Rates in Two Different Culture Media Medium
B3 + human serum albumin Earle's + cord serum
No. of patients
Total no. of eggs collected (eggs/patient)
% Fertilization
% Implantation"
(proportion)
18
81 (4.5)
66.7
22.3 (4/18)
11
26 (2.4)
80.7
18.9 (2/11)
aConfirmed by ultrasound.
shown that in the follicular phase, the endometrium undergoes distinct qualitative and quantitative changes that can be monitored ultrasonically and, in conjunction with the follicular measurements only, can further optimize the timing of human chorionic gonadotropin (hCG).l In this communication, preliminary data are presented that suggest that the outpatient procedure can be simplified further if the replacement of embryos is carried out before they have undergone syngamy.
after insemination, the loosely attached cumulus cells were removed from the oocytes by gentle pipetting and checked for the presence of pronuclei. The oocytes containing two pronuclei were transferred directly to the patient's uterus in 8 to 10 fll of fresh culture medium. No blood or urine sampling was performed, and no hormone support was given during the luteal phase of the cycle. All patients were treated on an outpatient/daystay basis. All resulting pregnancies were confirmed by the ultrasonic appearance of a gestational sac.
MATERIALS AND METHODS
RESULTS
The study involved 29 consecutive couples participating in our IVF program. Fifteen patients had irreparable tubal damage, 11 patients had idiopathic infertility of many years' duration, and 3 patients were treated because of severe oligospermia. The treatment was carried out on the basis of one attempt per patient. Multiple ovulation was induced by 100 mg clomiphene citrate (Clomid, Merrell Pharmaceuticals, Slough, UK) given daily from days 3 to 7 of the cycle and 150 IU of human menopausal gonadotropin (Pergonal, Serono Laboratories, Ltd., Welwyn Garden City, UK) given daily from day 7; the follicular growth was monitored ultrasonically with a Diasonics mechanical sector scanner (model DRF 1, Diasonics, Inc., Sunnyvale, CA) as described previously. When the leading follicle reached 18 mm in diameter, 2000 IU ofhCG (Profasi, Serono Laboratories, Inc.) was given intramuscularly; 00cytes were aspirated from follicles laparoscopically 36 hours later.2 The techniques used for IVF and culture of embryos and the details of replacing embryos in the uterus have been described previously.2 Briefly, the oocytes were preincubated in 1 ml of Earle's medium that contained 10% cord serum or Menezo's B3 medium that contained 20% (wt/vol) human serum albumin (Sigma Chemicals, London, UK) at 37°C in 5% CO 2 in air for 6 hours before insemination with the husband's sperm in specially designed glass dishes. About 16 to 18 hours
Of 107 preovulatory oocytes collected from 29 patients, 75 (70%) had two pronuclei when examined 16 to 18 hours after insemination. All pronucleate embryos were transferred, and 6 of 29 patients (20.6%) became pregnant. Fertilization rates were slightly higher in the Earle's medium, compared with the B3 medium (Table 1), although the possibility that this difference may be the result of fewer eggs collected per patient in the Earle's + cord serum group cannot be ruled out at the present time. The pregnancy rates did not exhibit any statistically significant differences between the two groups. All pregnancies were established in patients between 22 and 34 years of age; five of the six pregnancies were obtained when four or more embryos were transferred (Table 2), further strengthening our long-held belief" that until suitable techniques are available to determine the viability of preimplantation human embryos,
182
Ahuja et al.
Transfer of pronucleate embryos
Table 2. Relation Between Pregnancy Rate and Number of Pronucleate Embryos Replaced No. of embryos
1 2 3 4 5 6
No. of patients
Clinical pregnancies confirmed by ultrasound
9
o
7
1
6
o
3 2 2
2
1 2
Fertility and Sterility
fil
the number of embryos to be transferred should not be restricted to two or three. This pregnancy rate compares quite favorably with the 15% to 20% success rate obtained in previous studies when 2- to 8-cell embryos were transferred. 1, 4, 5 Pregnancies were established in all three treatment groups, two pregnancies in patients suffering from tubal damage, three pregnancies in patients treated for idiopathic infertility, and one pregnancy in the patients with severe oligospermia. Pregnancies resulting from the transfer of two and six embryos were lost between 6 and 8 weeks after transfer. A detailed cytologic and hormonal analysis of the luteal phase of these pregnancies was not carried out. The remaining three singleton pregnancies have resulted in normal full-term deliveries. DISCUSSION
In the majority of IVF clinics, freshly fertilized ova are cultured for 24 to 48 hours before replacement in the uterus. The rationale behind this practice is twofold. Firstly, it allows the achievement of a closer synchrony between the preimplantation uterus and developing embryos. The entry of the fertilized ovum into the uterus is controlled in a species-specific manner. In most species it normally occurs at the morula-to-blastocyst stages when, under the influence of circulating hormones, the uterus is not lethal or hostile to the embryo.6 In natural cycles, the human embryo arrives in the uterine cavity 3 to 4 days after ovulation. 7 In the present study, successful intrauterine pregnancies resulting from the replacement of embryos approximately 18 to 20 hours after laparoscopy (i.e., when the endometrium is 60 to 70 hours behind) suggest there is a rather weak or uncontrolled synchrony between the preimplantation uterus and developing embryos in a stimulated cycle. Even though the uterus is not fully receptive, it is capable of supporting the growth of early embryos until the initiation of implantation. Whether this explanation is applicable to the unstimulated endometrium is a matter for future research, although based on histologic data some conflicting reports have already appeared in the literature. s,9 The lack of uterine hostility toward pronucleate embryos may also indicate that, at least during the luteal phase, there are similarities between the oviductal and uterine environments. This is further supported by the similar protein profiles in Vol. 44, No.2, August 1985
Table 3. Analysis of 100 Ultrasonically Confirmed Pregnancies Resulting from the IVF Program at Cromwell Hospital Stages of embryonic developmenta
No. of pregnancies
1 cell 2 cell 3 cell 4 cell Mixed 2-8 cell
2 8 1
41
48
aForty to 44 hours after insemination.
the human of tubal and uterine secretions,lO as well as of the ectopic implantation, a pathophysiologic event that occurs exclusively in the human. Secondly, the practice of culturing embryos for 24 to 48 hours before replacement ensures that the embryos that undergo retarded growth or cleavage arrest are detected and eliminated. The cleavage rates of human embryos that result'from IVF vary considerably, and rapidly dividing embryos are regarded as having a better potential to result in successful pregnancies. 11 From a retrospective analysis of the first 100 pregnancies that resulted in our own program (Table 3), we have also found that more than 80% of the embryos completed the first two or three cleavage divisions within 40 to 44 hours after insemination. However, some of the remaining embryos that suffered apparent cleavage arrest or retarded growth resulted in pregnancy. Clearly the cleaving ability of embryos fertilized in vitro is not necessarily a true indication of their ability to implant in utero. It is possible that some of the normally dividing embryos did not produce a pregnancy because they lost their potential to implant in utero because of prolonged culture in vitro. Some animal studies have shown that embryos fertilized in vitro do not grow as efficiently as in vivo embryos and that their potential to implant in hCG-synchronized recipients is impaired,12, 13 although one recent study in the mouse failed to find significant differences between the two groups.14 Culture conditions have also been shown to exert deleterious effects on hamster embryos,15, 16 which further supports the view that a variety of culture media used for human IVF may not reflect the true fallopian tube environment and may exert some adverse effects during the prolonged culture of human embryos. If the results obtained from this pilot study can be reproduced in larger randomized trials, it could be argued that a maximal pregnancy rate will result after the replacement of pronucleate embryos. In Ahuja et al.
Transfer of pronucleate embryos
183
141
-,
• ___ &2&
terms of simplifying the IVF technology to suit the outpatient environment, we would also regard the present method as superior to the previously reported method of replacing sperm/egg complexes in the uterus within 7 to 8 hours after laparoscopy,17, 18 because this obviates fertilization failure due to immunologic or idiopathic factors as being responsible for a failed treatment cycle. Acknowledgments. We thank David Whittingham, Ph.D., for a critical reading of the manuscript and Miss Henrietta Lee for typing it. REFERENCES 1. Smith W, Porter R, Ahuja K, Craft I: Ultrasonic assessment of endometrial changes in stimulated cycles in an in vitro fertilization and embryo transfer programme. J In Vitro Fertil Embryo Trans 1:233, 1984 2. Craft I: In vitro fertilization-clinical methodology. Br J Hosp Med 31:90, 1984 3. Craft I, Porter R, Green S, Tucker M, Smith B, Twigg H, Ahuja K, Whittingham DG: Success of fertility, embryo number and in-vitro fertilization. Lancet 1:732, 1984 4. Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM, Steptoe PC, Webster JM: Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fertil Embryo Trans 1:23, 1984 5. Speirs A, Trounson A, Warner GM, Yovich JL, Saunders DM, Chen C: Summary of results. In Clinical In Vitro Fertilization, Edited by C Wood, A Trounson. Berlin, Springer-Verlag, 1984, p 157 6. Webb PD, Glasser SR: Implantation. In Human In Vitro Fertilization and Embryo Transfer, Edited by DP Wolf, MM Quigley. New York, Plenum Press, 1984, p 341 7. Croxatto HB, Ortiz ME, Diaz S, Hess R, Balmaceda J, Croxatto HD: Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following LH peak. Am J Obstet Gynecol 132:629, 1978
184
Ahuja et aI.
Transfer of pronucleate embryos
8. Garcia JE, Acosta AA, Hsiu J-G, Jones HW Jr: Advanced endometrial maturation after ovulation induction with human menopausal gonadotropinihuman chorionic gonadotropin for in vitro fertilization. Fertil Steril 41:31, 1984 9. Abate V: The endometrium at the time of transfer. Presented at the Third World Congress ofln Vitro Fertilization and Embryo Transfer, Helsinki, May 14 to 17, 1984. Abstract, p 221 10. Beier HM, Beier-Hellwig K, Delbos R: Hormones and proteins involved in uterine preparation for implantation. In Fertilization of the Human Egg In Vitro, Edited by HM Beier, HR Lindner. Berlin, Springer-Verlag, 1983, p 307 11. Mohr L, Trounson A: In vitro fertilization and embryonic growth. In Clinical In Vitro Fertilization, Edited by C Wood, A Trounson. Berlin, Springer-Verlag, 1984, Chap 8, p 99 12. -Leroy F, Massip A, Puissant F, Camus M, Vandurzwalmen P: Effect of in vitro culture, freezing and transfer on mouse embryonic capacity for implantation. Arch Androl 11:184, 1983 13. Shalgi R: Developmental capacity of rat embryos produced by in vivo or in vitro fertilization. Gamete Res 10:77,1984 14. Glenister P, Wood M, Whittingham D: Unpublished results, personal communication 15. Whittingham DG, Bavister BD: Development of hamster eggs fertilized in vitro or in vivo. J Reprod Fertil 38:489, 1974 16. Farrel PS, Bavister BD: Short-term exposure of two-cell hamster embryos to culture media is detrimental to viability. Bioi Reprod 31:109, 1984 17. Craft I, McLeod F, Green S, Djahanbakhch 0, Bernard A, Twigg H: Human pregnancy following oocyte and sperm transfer to the uterus. Lancet 1:1031, 1982 18. Asch RH, Ellsworth LR, Balmaceda JP, Wong DC: Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet 2:1034, 1984
Fertility and Sterility