Journal o f in Vitro Fertilization and Embryo Transfer, Vol. 8, No. 5, 1991
LOS ANGELES, CALIFORNIA
rate mouse experiments was performed, along with internal controls for each bioassay. Our purpose was to document the safety of this product as a lighting adjunct for laboratory and clinical use in in vitro fertilization (IVF) programs.
The Effect of Chemiluminescent Light Exposure on the in Vitro Development of Mouse Embryos
MATERIALS AND METHODS
Embryo Collection The collection of one-cell embryos was performed as previously described (4). Briefly, 6-weekold B6CBAF l female mice were superovulated with l0 IU pregnant mare's serum gonadotropin (PMSG; Sigma Chemical Company, St. Louis, MO) and l0 IU human chorionic gonadotropin (hCG; Serono Corporation, Randolf, MA) intraperitoneally injected 48 hr apart. Each female was next mated with one CD-1 male mouse at the time of hCG injection. Twenty hours after hCG, the female mice were sacrificed by cervical dislocation, and one-cell embryos were collected. Following dispersion of the cumulus with hyaluronidase 0.1%, 10 to 20 embryos were transferred to each study culture dish. Embryos that failed to develop to the two-cell stage after 24 hr in culture were excluded from the study. Undeveloped and degenerating zygotes were removed from the culture dishes.
Submitted: February 15, 1991 Accepted: June 4, 1991
INTRODUCTION Chemiluminescent lighting has recently been introduced for use in gynecologic procedures. A chemical lightstick (Cyanamid, American Cyanamid Company, Wayne, N J) containing an oxalate ester may be activated and attached onto a plastic adaptor inside a speculum blade (Speculite, Trylon Ltd., Harbor City, CA) to provide diffuse, shadowless illumination of the cervix and vagina without generating heat. The enhanced view facilitates the performance of procedures involving the uterine cervix. This property makes the lighting device especially attractive for use in the performance of embryo transfer, where excellent visualization of the ectocervix is essential. Visible fluorescent lighting has been implicated as a possible inhibitor of embryogenesis (1-3). For this reason, human embryos grown in culture following in vitro fertilization are often shielded from exposure to direct light. Similarly, direct lighting is usually kept to a minimum during the transfer of the embryos from the incubator to the patient. To test for possible detrimental effects of chemiluminescent light exposure upon cleavage and blastulation of mouse embryos in vitro, we cultured one-cell embryos in vitro after prolonged exposure to a chemiluminescent light source. One-cell embryos were assayed in the presence of either a white or a gold chemiluminescent light. A series of sepa0740-7769/91/1000-0290506.50/09 1991 Plenum Publishing Corporation
Medium Preparation Ham's F-10 solution (HF-10, GIBCO, Grand Island, NY) identical to that used in our IVF program was prepared as previously described (5) and supplemented with bovine serum albumin (BSA; fraction V, Sigma), 5 mg/ml. The solution was then aliquoted to separate beakers to be tested with either a gold light, a white light, or no light (control group).
Experimental Design After hyaluronidase treatment, cumulus-free, one-cell embryos were washed three times in con290
trol media and then transferred into culture dishes. The dishes exposed to the lights were kept shielded within the incubator so as not to expose the control embryos. Lightsticks were placed adjacent to each individual dish, approximately 1 min after light activation in order to avoid any burst effect. Lights were allowed to sit unperturbed in this position to permit a prolonged exposure to luminescence. The white light illuminates for approximately 30 to 45 min, and the gold light 10 to 15 min before complete extinction. Control and illuminated embryos were shielded from one another and placed in the same incubators. Two separate experiments were performed in order to study the effect of both white and gold chemiluminescent lights upon mouse blastulation. All runs were performed in quadruplicate and included controls. Assays were performed approximately 4 weeks apart. The number of two-cell embryos developing to blastocysts after 96 hr in culture was recorded. The cumulative percentage of blastocysts developing from two-cell embryos was calculated for each experiment and compared to controls not exposed to light but otherwise identically cultured. Data from control and exposed embryos were pairwise compared with the nonparametric Mann-Whitney test. Each experimental replicate was analyzed independently. RESULTS Of 450 one-cell embryos collected, 96 embryos (21.3%) did not develop to the two-cell stage and were excluded from the study. This is consistent with our previous experience using this assay (4). There were no significant differences (P > 0.05) in the percentage of one-cell embryos developing to the two-cell stage between the control groups and those groups exposed to light (Table I). The 354 two-cell embryos were used in the analysis. No significant differences were noted in the percentage of two-cell embryos developing to blastocysts at 96 hr for embryos exposed to either the white or the gold chemiluminescent light source as compared to controis in any of the assays performed. DISCUSSION Controversy has arisen regarding the possible toxic effect of radiant light upon early embryos.
Table I. The Effect of Chemiluminescent Light Exposure Upon Blastulation of Mouse Embryos Grown in Vitro
Hirao and Yanagimachi (1) first reported on detrimental effects to unfertilized hamster eggs induced by short-wavelength (<480-nm) light exposure in vitro. Later, Fischer et al. (2) revealed stagedependent susceptibility of rabbit embryos exposed to direct visible light. Exposure of day 1 rabbit embryos to as little as 1 hr of illumination at wavelengths of 320 to 740 nm resulted in diminished thymidine incorporation and ultrastructural evidence of cell damage as viewed by electron microscopy (3). Detrimental effects include delayed cleavage, diminished cell proliferation, and cellular degeneration. Such damage may be further compounded by exposure of embryos to other biophysical stresses such as temperature, pH or osmolarity changes. Based upon these results and those of the earlier investigators (1), it appears prudent to minimize light exposure to early developing embryos. Although not well characterized, it is believed that light of less than 500-nm wavelength is most harmful. The one-ceU mouse embryo bioassay has been extensively used as a standard model for toxicity in the evaluation of media used for human IVF (4). The development of embryos in the one-cell mouse embryo culture system has been demonstrated to be highly sensitive to changes in the culture milieu. Furthermore, we have shown that the one-ceU stage is the period of development when the early conceptus is most sensitive to toxic effects. The assay evaluates the developmental capacity of preimplantation embryos, with damage expressed as a disturbance in cleavage and growth capacity. No impairment of cell proliferation or retarded development
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 8, No. 5, 1991
was present in embryos exposed to either of the chemiluminescent light sources. Given the short duration of light exposure and the characteristics of the chemiluminescent light source (spectral output curve peaks >600 nm), it is unlikely that a significant risk of toxicity exists. The chemiluminescent light source may be utilized in human in vitro fertilization and embryo transfer procedures. Applications for the device include provision of light for embryo handling and embryo transfer. Also, a chemiluminescent light provides practical illumination of the cervix during the performance of intrauterine inseminations, postcoital testing, and vaginal preps prior to and following transvaginal aspirations. Such lighting eliminates intravaginal shadowing and facilitates the placement of catheters. Based upon these toxicity studies there appears to be no reason to preclude its use as a lighting adjunct for procedures involving embryos.
REFERENCES 1. Hirao Y, Yanagimachi R: Detrimental effect of visible light on meiosis of mammalian eggs in vitro. J Exp Zool 1978; 206:365-370 2. Fischer B, Schumacher A, Hegele-Hartung C, Beier HM: Potential risk of light and room temperature exposure on preimplantation embryos. Fertil Steril 1988;50:938-944 3. Schumacher A, Fischer B: Influence of visible light and room temperature on cell proliferation in preimplantation rabbit embryos. J Reprod Fertil 1988;84:197-204 4. Davidson A, Vermesh M, Lobo R, Paulson R: Mouse embryo culture as quality control for human in vitro fertilization: The one cell versus the two cell model. Fertil Steril 1988;49:516-521 5. Sauer M, Paulson R, Macaso T, Francis M, Lobo R: Establishment of a nonanonymous donor oocyte program: Preliminary experience at the University of Southern California. Fertil Steril 1989;52:433-436
Mark V. Sauer 1 Mary Francis Thelma Macaso Richard J. Paulson Department of Obstetrics & Gynecology Division of Reproductive Endocrinology University of Southern California Los Angeles, California 90024 1To whom correspondence should be addressed at Women's Hospital, Room L946, 1240 North Mission Road, Los Angeles, California 90033.
Bilateral T u b a l E c t o p i c P r e g n a n c y A f t e r in Vitro Fertilization and E m b r y o Transfer
Submitted: February 26, 1991 Accepted: June 4, 1991
INTRODUCTION The first successful pregnancy and uneventually ectopic pregnancy after in vitro fertilization (IVF) of a preovulatory human oocyte and subsequent embryo transfer (ET) in 1976 was reported by Steptoe and Edwards (1). Since then, the techniques of IVF and ET have been widely and successfully used to treat infertile women, particularly those with tubal diseases. There have been a few other reports of ectopic pregnancy following IVF-ET (2,4), ectopic gestation was reported at an incidence of 10% of the established pregnancies in one center (3). In recent years, the therapeutic use of clomiphene citrate or menotropins for ovulation induction has greatly increased the incidence of multiple ovulation and thus may increase the risk of ectopic pregnancy (5). We report a bilateral tubal ectopic pregnancy following IVF-ET.
CASE REPORT The patient is a 31-year-old married woman with 10 years of primary infertility due to pelvic inflammatory disease. Following an initial diagnosis of bilateral hydrosalpinges with severe pelvic adhesions, a previous reconstructive tubal operation was performed by Dr. Sun, which included salpingolysis and salpingostomy during the operation. However, pregnancy failed to occur. In June 1990, the patient was referred from Lii Ching Sheau Hospital to our IVF-ET program for further management; and on
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 8, No. 5, 1991