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Immunosuppression supports implantation of zona pellucida dissected human embryos
Vol. 53, No.4, April 1990
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright" 1990 The American Fertility Society
Immunosuppression supports implantation of zona pellucida dissected human embryos
Jacques Cohen, Ph.D.*t:l: Henry Malter, B.Sc.t Carlene Elsner, M.D.*
Hilton Kort, M.D.* Joe Massey, M.D.* Mary Pat Mayer, RN.*
Reproductive Biology Associates, and Gamete and Embryo Research Laboratory, Emory University School of Medicine, Atlanta, Georgia
The effect of low dose immunosuppression with methylprednisolone during the first 4 days after oocyte retrieval on potential immune cell invasion of partially zona dissected embryos in utero was investigated in alternate in vitro fertilization patients (n = 32). The incidence of pregnancy was significantly higher in patients receiving methylprednisolone (7 of 18,39%) than in control patients (1 of 14,7%). Twenty-eight percent (11 of 39) of the embryos replaced in the corticosteroid treated patients implanted, whereas only 7% (2 of 31) of embryos in control patients had a fetal heart beat. There were no side effects reported in any ofthe patients receiving corticosteroids. It can be concluded that methylprednisolone supports implantation of embryos with small holes in their zonae. However, the actual mechanisms of corticosteroid support on the interaction between immune cells and micromanipulated embryos are not well understood. Fertil Steril53:662, 1990
The ZOna pellucida provides several protective functions for the cleaving embryo. In mammals, this outer glycoprotein layer prevents dispersal of blastomeres and reduces direct contact between the embryo and foreign cells such as the epithelial lining ofthe reproductive tract, immune cells, spermatozoa, or the cells of other embryos.1-3 In addition, the ZOna facilitates passage of the embryo through the fallopian tube. Its presence is not essential for continued development Once compaction has occurred. 4 However, in precompacted embryos the zona must be present and intact. Although 15% of all oocytes obtained for in vitro fertilization (IVF) are not fertilized because of a Received August 15, 1989; revised and accepted December 5, 1989. * Reproductive Biology Associates. t Department of Gynecology and Obstetrics, Gamete and Embryo Research Laboratory, Emory University School of Medicine. " Reprint requests and present address: Jacques Cohen, Ph.D., Center for Reproductive Medicine and Infertility, Cornell University Medical College, Helmsley Medical Tower, Room 306, 505 East 70 Street, New York, New York 10021.
*
662
Cohen et al.
zOna pellucida dysfunction, there is little known about the nature of abnormalities in human zonae pellucidae. 5 In addition, ZOna thinning during the first cleavage divisions appears to be an important factor correlated with hatching and implantation. Embryos with uniformly thick ZOnae rarely implant. 6 Successful application of assisted fertilization, embryo splitting, and biopsy procedures necessitates opening of the zona pellucida. Application of these methods in precompacted embryos of rabbit, bovine, and sheep may cause cell death as a result of immune cell penetration through the artificial gap.7 An elegant way of avoiding contact with immune cells in the female reproductive tract was introduced by Willadsen,S who applied an insoluble agar layer around the micromanipulated embryo. The agar embedding sealed any holes in the ZOna pellucida, thus allowing for normal development in the reproductive tract. Hence, invasive procedures involving dissection of ZOnae in embryos of larger domestic species are either applied after compaction or after agar embedding. Several problems
Immunosuppression and partial zona dissection
Fertility and Sterility
need to be addressed before the application of such methods in micro manipulated human embryos. The use of a temporary recipient or host uterus is impractical for many reasons. The ingredients of the agar or coating may need to be adjusted to ensure timely disintegration of the embedding material before blastocyst expansion. Precompacted human embryos with small incisions in their zonae are currently replaced without physical protection. 9 ,10 The possibility of immune invasion in such embryos can not be excluded even w hen the incisions are smaller than 5 J,tm, after partial zona dissection.lO This method is aimed at mechanical opening of the zona pellucida allowing spermatozoa from infertile men to traverse into the perivitelline space. However, uterine polymorphonuclear leucocytes or segmented neutrophils may change in size and shape, possibly penetrating the precompacted embryo through the narrow incision. l l The effect of immunosuppression with methylprednisolone on possible immune cell invasion was therefore investigated in alternate IVF patients who had zona dissected embryos replaced. MATERIALS AND METHODS
Thirty-seven male factor couples, each with a long duration of infertility, requested partial zona dissection between May 1988 and June 1989. Forty-four cycles for oocyte collection were performed (7 couples were treated twice). Previous semen analyses were consistently abnormal; 8 men were oligozoospermic «10 X 106 /mL), 13 had asthenozoospermia «20% motile and/or abnormal forward progression of all spermatozoa), and the remaining 16 had combinations of both, usually with high numbers of abnormally shaped spermatozoa (>70%). Several men had retrograde ejaculation, several had previous attempts at varicocele repair, 1 had testicular teratoma, and several were unable to conceive due to failed reversed vasectomies. The majority of patients had idiopathic male infertility. The methods for follicular stimulation and transvaginal ultrasound oocyte collection have been described elsewhere. 12 One of the following three antibiotics was used in each patient immediately after egg collection: (1) 1 g of cephalosporin (Ancef; Smith, Kline and French, Philadelphia, PA), (2) 100 mg doxycycline-hyclate (Vibramycin; Pfizer, New York, NY), and (3) 1 g of cephamycin (Mefoxin; Merck, Sharpe and Dohme, West Point, PA). These antibiotics are given routinely to IVF patients in our program as prophylaxis against inVol. 53, No.4, April 1990
fection after the retrieval procedure. The half time of these broad spectrum antibiotics vary from <2 hours to 1 day. In addition, all patients routinely received 250 mg tetracycline (Barr Labs, Pomona, NY) four times daily for 2 days starting on the evening of the egg retrieval. Methods for sperm preparation, the PZD-protocol, tool making, and micromanipulation have been described previously. 10 Methylprednisolone (Medrol; The Upjohn Co., Kalamazoo, MI) and tetracycline (Barr Labs) were administered to alternate patients who had monospermic fertilization after zona dissection. Methylprednisolone (16 mg/d) treatment was commenced on the evening after oocyte collection and ceased 4 days later. The same patients received 2 more days of tetracycline (250 mg four times daily) in addition to the dosage mentioned above. The other patients (control) were not aware of this medication trial and did not receive methylprednisolone and the additional 2 days of tetracycline. Four patients had zona dissected embryos replaced and were not allocated to receive medication. They did not become pregnant and returned for a second attempt of partial zona dissection. These patients received medication during this second cycle. The treatment group (n = 18) was therefore larger than the control group (n = 14). RESULTS
Thirty-two patients had between one and three zona dissected embryos replaced. Eight clinical pregnancies (defined by fetal heart activity) were obtained in the 24 patients who had more than one embryo for replacement. Twin pregnancies occurred only after replacement of three embryos. Five of the eight clinical pregnancies had ultrasound evidence of twins with cardiac activity in both gestational sacs. In a patient with Cushing's syndrome, both sacs were reabsorbed in the first trimester after cardiac activity had been documented. One patient had evidence of the vanishing twin syndrome, but subsequently delivered a healthy girl. Another patient miscarried a single fetus. Two pregnancies are still progressing normally and 4 patients delivered six babies. Only one twin pregnancy was obtained in the control group (Table 1). Two zona dissected and one zona-intact embryo were replaced in this patient. She delivered nonidentical twins. The incidence of embryonic implantation of zona dissected embryos in patients receiving medication was 28%, possibly indicating that the hatching process is fa-
Cohen et al.
Immunosuppression and partial zona dissection
663
r
Table 1
Methylprednisolone and Tetracycline Administration to Patients Receiving PZD-Embryos and Effects on Implantation
Number of embryos replaced 1 2 3 Total
Incidence of implantation in patients
Incidence of implantation per embryo
M/T" treatment
M/T" treatment
Control
0/5 2/5 5/8
0/3 0/5 1/6
7/18 (39)b
1/14 (7)b
0/5 2/10 9/24 11/39 (28)C
Control 0/3 0/10 2/18 2/31 (7)c
"M, methylprednisolone; T, tetracycline. b Fisher's exact test, P < 0.05; value in parentheses expressed as percent.
C Fisher's exact test, P < 0.05; value in parentheses expressed as percent.
cilitated in these patients. The incidence of implantation of embryos from nonmale factor IVF patients in our program during the same period was 12%. There were no side effects reported in any of the patients receiving corticosteroids.
diminishes the presence of uterine lymphocytes thereby allowing the embryo to develop normally. The incisions after partial zona dissection are narrow and it is unlikely that monocytes or macrophages will invade through these gaps as is the case in microsurgically treated precompacted rabbit and sheep embryos in which the blastomere number was reduced or altered. 7 This technique requires the presence of very large gaps in the zona pellucida, often exceeding 30 p.m. Direct invasion of immune cells through the incisions in the zona pellucida may possibly occur in embryos in which the gap increased after replacement. This could be caused by contractions of the fallopian tube or excessive swelling of the blastomeres. 9 Blastomeres of zona dissected embryos cultured on bovine endometrial fibroblasts may swell considerably causing the incision to open. 15 This same phenomenon may occur in the fallopian tube when noncocultured zona dissected embryos are replaced. Immunosuppression probably has an advantage as it decreases peripheral immune cells, especially segmented neutrophils. Several other mechanisms may explain the current findings. Segmented neutrophils are able to change shape markedly, possibly invading the perivitelline space by ameboid-type movements through the incision in the zona pellucida. Despite their diameter of 13 p.m they are designed to insinuate and push their way through narrow gaps between endothelial lining cells of capillaries. They are attracted to their target by chemical signals. Embryo replacement might be causing minor trauma to the endometrium resulting in an increase in the number of segmented neutrophils. Moreover, it is possible that microbial organisms or foreign cells are incorporated with the embryos attracting even larger numbers of immune cells. Immune cells may also affect the embryos by excreting digestive enzymes in the vicinity of the gap
DISCUSSION
Methylprednisolone administered to IVF patients for 4 days after oocyte retrieval supports implantation when embryos have small holes in their zonae pellucidae. Implantation of zona dissected embryos in immunosuppressed patients was four times higher than in those patients who did not receive exogenous corticosteroids. Broad spectrum antibiotics were administered to both immunosuppressed and control patients prophylactically, but immunosuppressed patients received 2 additional days of tetracycline. It is unlikely that this explains the differences in implantation rates between the two groups, as others have found that zona-drilled embryos rarely implanted in patients who received antibiotics only.13 However, micromanipulation may compromise some protective properties of the zona pellucida. Administration of antibiotics to the recipients of micro manipulated embryos is recommended, considering that vaginal microbial organisms may occasionally be carried into the uterine cavity by means of the replacement catheter. Further insight into the actual mechanisms of immunosuppression in IVF patients is needed to understand the interaction between immune cells and the micromanipulated embryo. In general, administration of exogenous corticosteroids for long periods will initially induce a paradoxical rise in blood lymphocytes lasting several weeks. 14 This phenomenon represents a compartmental shift from til'lsues to blood that subsides as the body pool of lymphocytes diminishes after prolonged treatment. It can be postulated that the current therapy 664
Cohen et al.
Immunosuppression and partial zona dissection
Fertility and Sterility
in the zona pellucida. Immunosuppression may also support implantation of IVF embryos with intact zonae, by reducing the occurrence of an inflammatory response after insertion of the replacement catheter. However, the incidence of implantation of zona intact embryos replaced in immunosuppressed patients using the protocol described here did not increase significantly in a recent study performed in this program. 16 Thirtyseven patients received corticosteroids and the zonae oftheir oocytes and embryos were not micromanipulated. Clinical pregnancy could be confirmed in only 24% of these patients and 12% ofthe embryos implanted.
5.
6.
7. 8.
9.
10.
Acknowledgments. We are grateful to Graham Wright, B.Sc., Ms. Sharon Wiker, Leigh Inge, B.Sc., William Graves, Ph.D., Dorothy Mitchell, M.D., Beth Talansky, Ph.D., Mina Alikani, M.Sc., Ed Fugger, Ph.D., and Andy Toledo, M.D. for their support of this study.
REFERENCES
12.
13. 14.
1. Bronson RA, McLaren A: Transfer to mouse oviduct of eggs with and without the zona pellucida. J Reprod Fertil 22: 129,1970 2. Modlinski J A: The role of the zona pellucida in the development of mouse eggs in vivo. J Embryol Exp Morphol 23: 539, 1970 3. Moore NW, Adams CE, Rowson LEA: Developmental potential of single blastomeres of the rabbit egg. J Reprod Fertil 17:527, 1968 4. Trounson AO, Moore NW: The survival and development
Vol. 53, No.4, April 1990
11.
15.
16.
of sheep eggs following complete or partial removal of the zona pellucida. J Reprod FertiI41:97, 1974 Malter HE, Talansky BE, Gordon J, Cohen J: Monospermy and polyspermy after partial zona dissection of reinseminated human oocytes. Gamete Res 23:377, 1989 Cohen J, Inge KL, Suzman M, Wiker S, Wright G: Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril51:820, 1989 Willadsen SM: Micromanipulation of embryos of the large domestic species. Thesis. Copenhagen, 1982 Willadsen SM: A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature (Lond) 277:298, 1979 Ng SC, Bongso TA, Ratnam SS, Sathananthan AH, Chan CLK, Wong PC, Hagglund L, Anandakumar C, Wong YC, Goh VHH: Pregnancy after transfer of multiple sperm under the zona. Lancet 2:790, 1988 Malter HE, Cohen J: Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 51:139, 1989 Oliver JM, Berlin RD: Surface cytoskeletal events regulating membrane topography. Semin Hematol 20:282, 1983 Cohen J, DeVane GW, Elsner CW, Kort HI, Massey JB, Norbury S: Cryopreserved zygotes and embryos and endocrinologic factors in the replacement cycle. Fertil Steril 50: 61,1988 Fugger E, Warner C: Personal communications Jandl JH: Chronic lymphatic leukemia. In Blood, Edited by JH Jandl. Boston, Little Brown Co, 1987, p 769 Wiemer KE, Cohen J, Wiker SR, Malter HE, Wright G, Godke RA: Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation. Fertil Steril 52:503, 1989 Cohen J, Elsner C, Kort H, Massey J, Mayer MP, Wiemer K: Impairment of the hatching process following in vitro fertilization in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod. In press
Cohen et al.
Immunosuppression and partial zona dissection
665
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright" 1990 The American Fertility Society
Immunosuppression supports implantation of zona pellucida dissected human embryos
Jacques Cohen, Ph.D.*t:l: Henry Malter, B.Sc.t Carlene Elsner, M.D.*
Hilton Kort, M.D.* Joe Massey, M.D.* Mary Pat Mayer, RN.*
Reproductive Biology Associates, and Gamete and Embryo Research Laboratory, Emory University School of Medicine, Atlanta, Georgia
The effect of low dose immunosuppression with methylprednisolone during the first 4 days after oocyte retrieval on potential immune cell invasion of partially zona dissected embryos in utero was investigated in alternate in vitro fertilization patients (n = 32). The incidence of pregnancy was significantly higher in patients receiving methylprednisolone (7 of 18,39%) than in control patients (1 of 14,7%). Twenty-eight percent (11 of 39) of the embryos replaced in the corticosteroid treated patients implanted, whereas only 7% (2 of 31) of embryos in control patients had a fetal heart beat. There were no side effects reported in any ofthe patients receiving corticosteroids. It can be concluded that methylprednisolone supports implantation of embryos with small holes in their zonae. However, the actual mechanisms of corticosteroid support on the interaction between immune cells and micromanipulated embryos are not well understood. Fertil Steril53:662, 1990
The ZOna pellucida provides several protective functions for the cleaving embryo. In mammals, this outer glycoprotein layer prevents dispersal of blastomeres and reduces direct contact between the embryo and foreign cells such as the epithelial lining ofthe reproductive tract, immune cells, spermatozoa, or the cells of other embryos.1-3 In addition, the ZOna facilitates passage of the embryo through the fallopian tube. Its presence is not essential for continued development Once compaction has occurred. 4 However, in precompacted embryos the zona must be present and intact. Although 15% of all oocytes obtained for in vitro fertilization (IVF) are not fertilized because of a Received August 15, 1989; revised and accepted December 5, 1989. * Reproductive Biology Associates. t Department of Gynecology and Obstetrics, Gamete and Embryo Research Laboratory, Emory University School of Medicine. " Reprint requests and present address: Jacques Cohen, Ph.D., Center for Reproductive Medicine and Infertility, Cornell University Medical College, Helmsley Medical Tower, Room 306, 505 East 70 Street, New York, New York 10021.
*
662
Cohen et al.
zOna pellucida dysfunction, there is little known about the nature of abnormalities in human zonae pellucidae. 5 In addition, ZOna thinning during the first cleavage divisions appears to be an important factor correlated with hatching and implantation. Embryos with uniformly thick ZOnae rarely implant. 6 Successful application of assisted fertilization, embryo splitting, and biopsy procedures necessitates opening of the zona pellucida. Application of these methods in precompacted embryos of rabbit, bovine, and sheep may cause cell death as a result of immune cell penetration through the artificial gap.7 An elegant way of avoiding contact with immune cells in the female reproductive tract was introduced by Willadsen,S who applied an insoluble agar layer around the micromanipulated embryo. The agar embedding sealed any holes in the ZOna pellucida, thus allowing for normal development in the reproductive tract. Hence, invasive procedures involving dissection of ZOnae in embryos of larger domestic species are either applied after compaction or after agar embedding. Several problems
Immunosuppression and partial zona dissection
Fertility and Sterility
need to be addressed before the application of such methods in micro manipulated human embryos. The use of a temporary recipient or host uterus is impractical for many reasons. The ingredients of the agar or coating may need to be adjusted to ensure timely disintegration of the embedding material before blastocyst expansion. Precompacted human embryos with small incisions in their zonae are currently replaced without physical protection. 9 ,10 The possibility of immune invasion in such embryos can not be excluded even w hen the incisions are smaller than 5 J,tm, after partial zona dissection.lO This method is aimed at mechanical opening of the zona pellucida allowing spermatozoa from infertile men to traverse into the perivitelline space. However, uterine polymorphonuclear leucocytes or segmented neutrophils may change in size and shape, possibly penetrating the precompacted embryo through the narrow incision. l l The effect of immunosuppression with methylprednisolone on possible immune cell invasion was therefore investigated in alternate IVF patients who had zona dissected embryos replaced. MATERIALS AND METHODS
Thirty-seven male factor couples, each with a long duration of infertility, requested partial zona dissection between May 1988 and June 1989. Forty-four cycles for oocyte collection were performed (7 couples were treated twice). Previous semen analyses were consistently abnormal; 8 men were oligozoospermic «10 X 106 /mL), 13 had asthenozoospermia «20% motile and/or abnormal forward progression of all spermatozoa), and the remaining 16 had combinations of both, usually with high numbers of abnormally shaped spermatozoa (>70%). Several men had retrograde ejaculation, several had previous attempts at varicocele repair, 1 had testicular teratoma, and several were unable to conceive due to failed reversed vasectomies. The majority of patients had idiopathic male infertility. The methods for follicular stimulation and transvaginal ultrasound oocyte collection have been described elsewhere. 12 One of the following three antibiotics was used in each patient immediately after egg collection: (1) 1 g of cephalosporin (Ancef; Smith, Kline and French, Philadelphia, PA), (2) 100 mg doxycycline-hyclate (Vibramycin; Pfizer, New York, NY), and (3) 1 g of cephamycin (Mefoxin; Merck, Sharpe and Dohme, West Point, PA). These antibiotics are given routinely to IVF patients in our program as prophylaxis against inVol. 53, No.4, April 1990
fection after the retrieval procedure. The half time of these broad spectrum antibiotics vary from <2 hours to 1 day. In addition, all patients routinely received 250 mg tetracycline (Barr Labs, Pomona, NY) four times daily for 2 days starting on the evening of the egg retrieval. Methods for sperm preparation, the PZD-protocol, tool making, and micromanipulation have been described previously. 10 Methylprednisolone (Medrol; The Upjohn Co., Kalamazoo, MI) and tetracycline (Barr Labs) were administered to alternate patients who had monospermic fertilization after zona dissection. Methylprednisolone (16 mg/d) treatment was commenced on the evening after oocyte collection and ceased 4 days later. The same patients received 2 more days of tetracycline (250 mg four times daily) in addition to the dosage mentioned above. The other patients (control) were not aware of this medication trial and did not receive methylprednisolone and the additional 2 days of tetracycline. Four patients had zona dissected embryos replaced and were not allocated to receive medication. They did not become pregnant and returned for a second attempt of partial zona dissection. These patients received medication during this second cycle. The treatment group (n = 18) was therefore larger than the control group (n = 14). RESULTS
Thirty-two patients had between one and three zona dissected embryos replaced. Eight clinical pregnancies (defined by fetal heart activity) were obtained in the 24 patients who had more than one embryo for replacement. Twin pregnancies occurred only after replacement of three embryos. Five of the eight clinical pregnancies had ultrasound evidence of twins with cardiac activity in both gestational sacs. In a patient with Cushing's syndrome, both sacs were reabsorbed in the first trimester after cardiac activity had been documented. One patient had evidence of the vanishing twin syndrome, but subsequently delivered a healthy girl. Another patient miscarried a single fetus. Two pregnancies are still progressing normally and 4 patients delivered six babies. Only one twin pregnancy was obtained in the control group (Table 1). Two zona dissected and one zona-intact embryo were replaced in this patient. She delivered nonidentical twins. The incidence of embryonic implantation of zona dissected embryos in patients receiving medication was 28%, possibly indicating that the hatching process is fa-
Cohen et al.
Immunosuppression and partial zona dissection
663
r
Table 1
Methylprednisolone and Tetracycline Administration to Patients Receiving PZD-Embryos and Effects on Implantation
Number of embryos replaced 1 2 3 Total
Incidence of implantation in patients
Incidence of implantation per embryo
M/T" treatment
M/T" treatment
Control
0/5 2/5 5/8
0/3 0/5 1/6
7/18 (39)b
1/14 (7)b
0/5 2/10 9/24 11/39 (28)C
Control 0/3 0/10 2/18 2/31 (7)c
"M, methylprednisolone; T, tetracycline. b Fisher's exact test, P < 0.05; value in parentheses expressed as percent.
C Fisher's exact test, P < 0.05; value in parentheses expressed as percent.
cilitated in these patients. The incidence of implantation of embryos from nonmale factor IVF patients in our program during the same period was 12%. There were no side effects reported in any of the patients receiving corticosteroids.
diminishes the presence of uterine lymphocytes thereby allowing the embryo to develop normally. The incisions after partial zona dissection are narrow and it is unlikely that monocytes or macrophages will invade through these gaps as is the case in microsurgically treated precompacted rabbit and sheep embryos in which the blastomere number was reduced or altered. 7 This technique requires the presence of very large gaps in the zona pellucida, often exceeding 30 p.m. Direct invasion of immune cells through the incisions in the zona pellucida may possibly occur in embryos in which the gap increased after replacement. This could be caused by contractions of the fallopian tube or excessive swelling of the blastomeres. 9 Blastomeres of zona dissected embryos cultured on bovine endometrial fibroblasts may swell considerably causing the incision to open. 15 This same phenomenon may occur in the fallopian tube when noncocultured zona dissected embryos are replaced. Immunosuppression probably has an advantage as it decreases peripheral immune cells, especially segmented neutrophils. Several other mechanisms may explain the current findings. Segmented neutrophils are able to change shape markedly, possibly invading the perivitelline space by ameboid-type movements through the incision in the zona pellucida. Despite their diameter of 13 p.m they are designed to insinuate and push their way through narrow gaps between endothelial lining cells of capillaries. They are attracted to their target by chemical signals. Embryo replacement might be causing minor trauma to the endometrium resulting in an increase in the number of segmented neutrophils. Moreover, it is possible that microbial organisms or foreign cells are incorporated with the embryos attracting even larger numbers of immune cells. Immune cells may also affect the embryos by excreting digestive enzymes in the vicinity of the gap
DISCUSSION
Methylprednisolone administered to IVF patients for 4 days after oocyte retrieval supports implantation when embryos have small holes in their zonae pellucidae. Implantation of zona dissected embryos in immunosuppressed patients was four times higher than in those patients who did not receive exogenous corticosteroids. Broad spectrum antibiotics were administered to both immunosuppressed and control patients prophylactically, but immunosuppressed patients received 2 additional days of tetracycline. It is unlikely that this explains the differences in implantation rates between the two groups, as others have found that zona-drilled embryos rarely implanted in patients who received antibiotics only.13 However, micromanipulation may compromise some protective properties of the zona pellucida. Administration of antibiotics to the recipients of micro manipulated embryos is recommended, considering that vaginal microbial organisms may occasionally be carried into the uterine cavity by means of the replacement catheter. Further insight into the actual mechanisms of immunosuppression in IVF patients is needed to understand the interaction between immune cells and the micromanipulated embryo. In general, administration of exogenous corticosteroids for long periods will initially induce a paradoxical rise in blood lymphocytes lasting several weeks. 14 This phenomenon represents a compartmental shift from til'lsues to blood that subsides as the body pool of lymphocytes diminishes after prolonged treatment. It can be postulated that the current therapy 664
Cohen et al.
Immunosuppression and partial zona dissection
Fertility and Sterility
in the zona pellucida. Immunosuppression may also support implantation of IVF embryos with intact zonae, by reducing the occurrence of an inflammatory response after insertion of the replacement catheter. However, the incidence of implantation of zona intact embryos replaced in immunosuppressed patients using the protocol described here did not increase significantly in a recent study performed in this program. 16 Thirtyseven patients received corticosteroids and the zonae oftheir oocytes and embryos were not micromanipulated. Clinical pregnancy could be confirmed in only 24% of these patients and 12% ofthe embryos implanted.
5.
6.
7. 8.
9.
10.
Acknowledgments. We are grateful to Graham Wright, B.Sc., Ms. Sharon Wiker, Leigh Inge, B.Sc., William Graves, Ph.D., Dorothy Mitchell, M.D., Beth Talansky, Ph.D., Mina Alikani, M.Sc., Ed Fugger, Ph.D., and Andy Toledo, M.D. for their support of this study.
REFERENCES
12.
13. 14.
1. Bronson RA, McLaren A: Transfer to mouse oviduct of eggs with and without the zona pellucida. J Reprod Fertil 22: 129,1970 2. Modlinski J A: The role of the zona pellucida in the development of mouse eggs in vivo. J Embryol Exp Morphol 23: 539, 1970 3. Moore NW, Adams CE, Rowson LEA: Developmental potential of single blastomeres of the rabbit egg. J Reprod Fertil 17:527, 1968 4. Trounson AO, Moore NW: The survival and development
Vol. 53, No.4, April 1990
11.
15.
16.
of sheep eggs following complete or partial removal of the zona pellucida. J Reprod FertiI41:97, 1974 Malter HE, Talansky BE, Gordon J, Cohen J: Monospermy and polyspermy after partial zona dissection of reinseminated human oocytes. Gamete Res 23:377, 1989 Cohen J, Inge KL, Suzman M, Wiker S, Wright G: Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril51:820, 1989 Willadsen SM: Micromanipulation of embryos of the large domestic species. Thesis. Copenhagen, 1982 Willadsen SM: A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature (Lond) 277:298, 1979 Ng SC, Bongso TA, Ratnam SS, Sathananthan AH, Chan CLK, Wong PC, Hagglund L, Anandakumar C, Wong YC, Goh VHH: Pregnancy after transfer of multiple sperm under the zona. Lancet 2:790, 1988 Malter HE, Cohen J: Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 51:139, 1989 Oliver JM, Berlin RD: Surface cytoskeletal events regulating membrane topography. Semin Hematol 20:282, 1983 Cohen J, DeVane GW, Elsner CW, Kort HI, Massey JB, Norbury S: Cryopreserved zygotes and embryos and endocrinologic factors in the replacement cycle. Fertil Steril 50: 61,1988 Fugger E, Warner C: Personal communications Jandl JH: Chronic lymphatic leukemia. In Blood, Edited by JH Jandl. Boston, Little Brown Co, 1987, p 769 Wiemer KE, Cohen J, Wiker SR, Malter HE, Wright G, Godke RA: Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation. Fertil Steril 52:503, 1989 Cohen J, Elsner C, Kort H, Massey J, Mayer MP, Wiemer K: Impairment of the hatching process following in vitro fertilization in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod. In press
Cohen et al.
Immunosuppression and partial zona dissection
665