484
stimulation. Basal follicle stimulating hormone (FSH) levels may be used to predict those patients who cannot be effectively stimulated (12). Patients with a basal F S H greater than 18 IU/L have been excluded in the current program, being categorized as having occult ovarian failure. In order to compensate for the reduced implantation rate in women over 40 years, the number of eggs or embryos may be increased. This strategy is limited by the reduced number of eggs available. If pregnancy rates are increased by giving higher doses of progesterone in donor egg patients over 40 years (10), then the same therapy may be effective in women using their own eggs or embryos. Donor egg IVF or GIFT is the most successful therapy for women wishing to conceive over 40 years. The success rate per treatment cycle has been similar to that in younger women, 30% (13). The low natural fertility and markedly reduced success of artificial reproductive technology in women over 40 years suggests that conception is an urgent problem, particularly as fertility declines rapidly between 40 and 50 years of age. Full investigation of fertility status can be offered immediately so that any defects can be treated as soon as possible. If the patient fails to conceive, GIFT or IVF procedures may be offered without delay. The reasons for the difficulty in conceiving should be explained to the patient and the alternatives such as the use of donor eggs considered by the patient (14). Most couples take some time to understand, consider, and proceed to the use of donor eggs so it is preferable that this procedure be offered early in the management of the infertile patient over 40 years wishing to conceive. It is incorrect to define an arbitrary period of time when an older patient is infertile, as any woman over 40 years is likely to be subfertile, and fewer than 25% can be expected to conceive in 1 year. REFERENCES 1. Gray RH: Social influences in fertility at later ages of reproduction. J Biosoc Sci Suppl 1979;6:97-115 2. MacLeod J, Gold RZ: The male factor in fertility and sterility. VI. Semen quality and certain other factors in relations to ease of conception. Fertil Steril 1953;4:10-33 3. Vessey M, Doll R, Peto R, Johnson B, Wiggins P: A longterm follow up study of women using different methods of contraception--an interim report. J Biosoc Sci t976;8:373427 4. Adams CE: In Reproductive Senescence in Reproduction in Mammals, CR Austin, RV Short (eds). London, Cambridge University Press, 1984, p 212
SHORT COMMUNICATIONS
5. Doring GK: The incidence of anovutar cycles in women. J Reprod Fertil 1969;Suppl 6:77-81. 6. Adams CE: In Reproductive Senescence in Reproduction in Mammals, CR Austin, RV Short (eds). London, Cambridge University Press, 1984, p 218 7. Piette C, de Mougon J, Bachelot A, Spira A: IVF influence of women's age on pregnancy rates. Hum Reprod 1990;5(1): 56-59 8. Wood C, Riley R: IVF. Melbourne, Hill of Content, 1992, pp 53-56 9. Berget PA, Williams MA, Grunfeld F, Fox J, Hofmann GE, Navot D: Age related decrease in fertility potential is attributable to occyte quality rather than implantation failure. American Fertility Society 1991, abstr 0.062, $27 10. Meldrum R: Younger eggs for older women. Presentation at Serono Symposium on Oocyte Donation, Monash University, Melbourne, 1991 11. Baker TG: Population of germ ceils in the human ovary. Am J Obstet Gynaecol 1900;100:746-761 12. Cameron IT, O'Shea FC, Rolland JM, Hughes EG, De Kretser DM, Healy DL: Occult ovarian failure: A syndrome of infertility, regular menses and elevated FSH concentrations. J Clin Endocrinol Metab 1987;67(6): 1190-1194 13. Meldrum DR, Marr B, Stubbs C, Wisot A, Hamilton M, Heit R: Oocyte donation--A new approach for the poor prognosis patient. 1990 Abstracts of 46th American Fertility Society Meeting, Oct 1990, 0-024 14. Osborn J: Life table analysis of GII~q?results---effects of age: Information sheet for patients entering Monash programme. Melbourne, 1991
Carl Wood 1 n a n Calderon Amanda Crombie Infertility Medical Centre 62 Erin Street Richmond, Victoria 3121, Australia 1 To whom correspondence should be addressed at Department of Obstetrics and Gynaecology, Monash University, 246 Clayton Road, Clayton, Victoria 3168, Australia.
DETROIT, MICHIGAN Tetraploidy Associated with Human Pronuclear Embryo Cryopreservation: A Case Report
Submitted: June 5, 1992 Accepted: July 20, 1992
INTRODUCTION Advances in the fields of reproduction and cryobiology now allow routine freezing of human embryos Journal o f Assisted Reproduction and Genetics, VoL 9, No. 5, 1992
SHORT COMMUNICATIONS
that result from in vitro fertilization. Many programs cryopreserve embryos at the pronuclear stage, immediately following oocyte fertilization with formation of the second polar body and both pronuclei, but before completion of the first cleavage division. Worldwide experience has shown such cryopreservation and transfer to be a safe and relatively efficient process in terms of both viable embryo recovery and per transfer pregnancy rates (1,2). However, an increased risk of pregnancy wastage or congenital abnormality resulting from such embryo manipulation has not been completely excluded, since at this stage the embryo may be susceptible to damage resulting from handling, dehydration and cryopreservation. This case documents an association between pronuclear stage cryopreservation and tetraploidy, with subsequent spontaneous abortion.
CASE PRESENTATION A 30-year-old nulligravid Caucasian woman began controlled ovarian hyperstimulation for an in vitro fertilization (IVF) procedure as treatment for unexplained primary infertility. Urofollitropin (Metrodin, Serono Laboratories, Inc., Norwall, MA), 150 IU, and human menopausal gonadotropin (Pergonal, Serono Laboratories, Inc., Norwall, MA), 150 IU, were administered daily in two divided intramuscular (im) doses beginning on cycle day (CD) 3, at which time the serum estradiol level was 46 pg/ml, follicle stimulating hormone (FSH) was 8.3 mIU/ml, and luteinizing hormone (LH) was 11.3 mIU/ml. By CD 6 the estradiol level had risen to 179 pg/ml, and daily estradiol and ultrasound monitoring was begun 2 days later. By CD 11 a total of 12 follicles greater than 14 mm was noted on both ovaries, with two follicles measuring 18 mm in average dimension. Estradiol level at that time was 1820 pg/ml. Human chorionic gonadotropin (Profasi, Serono Laboratories, Inc., Norwall, MA), 10,000 IU, was given im at 9:30 i'M. Transvaginal oocyte retrieval was begun 34.5 hr later under intravenous sedation supplemented with 1% lidocaine infiltration of the vagina for local anesthesia. Eleven preovulatory oocytes were recovered. The sperm sample obtained that day had a volume of 2.0 ml with 110 x 106 spermatozoa/ml, 75% motility, and 55% normal morphology. A two-step wash and centrifugation, followed by a 90-min swim-up, yielded a sample with 85% motility and 35 x 10 6 Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
485
spermatozoa/ml. Each oocyte was inseminated with 150,000 motile spermatozoa per our usual protocol. When first examined 18 to 20 hr after insemination, all oocytes were noted to have two pronuclei (PN), and 10 of the 11 additionally had two polar bodies (PB) visible. The couple had previously given written consent for cryopreservation of "exc e s s " embryos remaining after fresh transfer. Seven pronuclear embryos (each with two PN and two PB) were cryopreserved in three plastic straws beginning 26 hr after retrieval. Embryos were first washed in human tubal fluid supplemented with 15% patient serum, followed by stepwise dehydration to 1.5 M propanediol containing 2.0 M sucrose. Initial cooling in a Kryo 10 Programmable Freezer (T.S. Scientific, Perkasie, PA) from room temperature to -7°C occurred at a rate of 2°/min, after which embryos were seeded with a precooled forceps. Further cooling to -32°C was at a rate of 0.3°/rain. After holding for 1 hr at -32°C, embryos were plunged into liquid nitrogen for storage. Twenty-four hours later, i.e., on the day of embryo transfer, the embryo with two PN but no second PB was clearly atretic and discarded. Cleavage was noted in the three remaining embryos. Fifty hours after retrieval a two-cell embryo with unequal blastomeres, a four-cell embryo with minor fragmentation, and a five-cell embryo with major fragmentation were transferred to the uterus transcervically without complication. Daily intramuscular progesterone supplementation was continued until 14 days after embryo transfer, at which time the patient's human chorionic gonadotropin (hCG) level was below 5 mIU/ml serum. Progesterone was discontinued and a menstrual period followed. The couple declined endometrial assessment using late luteal-phase biopsy prior to replacement of cryopreserved embryos. During the first cycle after retrieval a spontaneous LH surge was detected by urinary sampling every 4 hr. Five embryos were thawed 27 days after cryopreservation by equilibration in a 30°C water bath. Propanediol and sucrose were sequentially removed as the embryos were rehydrated. Two of the embryos were atretic after recovery, while the remaining three appeared normal. Cleavage was documented prior to transfer, and 23 hr after thawing was initiated a two-cell, a three- to four-cell, and a six-cell embryo were transferred transcervically to the uterine cavity. Twelve days after transfer the hCG level was 45 mIU/ml, and pregnancy was confirmed two days later with an hCG level that had risen to 110 mIU/ml. Daily
486
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intramuscular progesterone supplementation was continued. Serial hCG assay initially suggested a normal pregnancy, and two concordant gestational sacs were identified sonographically 25 days after transfer. However, over the subsequent 24 days no fetal pole was demonstrated and the gestational sacs stopped growing. A diagnosis of blighted twin gestation was made, but the patient declined dilatation and curettage, instead agreeing to observation with the expectation of spontaneous complete abortion. hCG levels declined over the next 30 days and the onset of bleeding and cramping was quickly followed by passage of tissue. Bleeding and cramping then stopped and the cervix and uterus involuted. hCG levels eventually declined to and remained below the assay detection limit (5 mlU/ml). Pathologic examination of the products of conception showed no embryonic derivatives. Portions of each gestational sac were sent for chromosome analysis. Cells
1
6
13
2
failed to grow from one sample, while the karyotype on the other was 92,XXXX (Fig. 1).
DISCUSSION Tetraploidy has been reported in 4-6% of spontaneous abortuses in which karyotypic abnormalities are identified (3). Although rare tetraploid mosaic infants have been described (4), the vast majority of tetraploid pregnancies apparently end in early spontaneous abortion. In vitro, tetraploidy often results in cleavage arrest early in embryonic development, approximately at the eight-cell stage (5). The incidence of polyploidy in conception resulting from assisted reproduction appears to be low (6,7). Plachot (6) identified a single tetraploid in 21 abnormal karyotypes found in 34 spontaneous abortions conceived by IVF without embryo cryopreservation. In addition, a single tetraploid spon-
4
3
7
|
14
15
9
5
I0
II
16
17
12
18
19 20 21 22 X Y Fig. 1. Photomicrograph of metaphase karyotype obtained from the products of conception of one gestational sac, stained with trypsin-Giemsa. Analysis of polymorphic markers, acrocentric satellites, and centromeric regions suggests duplication of both the maternal and the paternal haploid complements. Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
SHORT COMMUNICATIONS
taneous abortion has been reported among the 2811 clinical pregnancies conceived through in vitro fertilization in 1989 (1). In that same report, 234 pregnancies were conceived from 2124 frozen embryo transfer cycles, resulting in 172 (8%) live births, and a 92,XXYY chromosomal abnormality resulting in spontaneous abortion was noted. To our knowledge, the case reported herein therefore represents only the second in the literature documenting tetraploidy in association with human embryo cryopreservation and the first for which detailed information is available. The mechanisms by which polyploidy may occur are listed in Table I. Of these possibilities, several features suggest mitotic dysfunction as the etiology of tetraploidy in this case. We observed two polar bodies during the development of each of the seven oocytes which were cryopreserved, suggesting that the tetraploidy was not the result of digyny (incorporation of polar bodies) or suppressed maternal meiosis. The presence of only two pronuclei argues against polyspermic fertilization. Parthenogenic activation of a diploid oocyte can be excluded by careful analysis of the karyotype, which shows sufficient differences in the chromosomes to exclude a common ancestor. Most importantly, analysis of polymorphic markers on chromosomes 3, 9, and 16, the acrocentric satellites, and centromeric regions suggests duplication of each haploid maternal and paternal chromosome complement. This implies normal duplication but failure of anaphase and cytokinesis at the first mitotic division. In the handling of these embryos, development appeared normal up to the point when they were cryopreserved at the pronuclear stage° After chromosome duplication, we postulate that cryopreservation and/or thawing may have damaged the spindle apparatus of the zygote as it prepared for the first mitotic division. At this critical time disruption of the spindle could inappropriately signal completion of mitosis. Such an effect has been demon-
Table I. Potential Cytopathologic Mechanisms of Tetraploidy 1. Meiotic errors in germ-cell precursors leading to diploid gametes 2. Incorporation or suppression of the formatJion of one of the polar bodies (digyny) 3. Suppression of the 1st or 2nd maternal meiotic division 4. Fertilization of the ovum by two or more spermatozoa (polyspermy) 5. Initial mitotic dysfunction (nondisjunction) resulting in chromosome duplication but cleavage failure
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
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strated in animal embryos studied in vitro, where 15 to 60 min of cold treatment has been shown to disrupt the spindle apparatus by first depolymerizing continuous microtubules, followed subsequently by kinetochore fibers (8). In this situation, upon thawing and with the spindle disrupted, the zygote would conclude the first mitotic division without completing anaphase or cytokinesis, thereby yielding a tetraploid single cell embryo. Subsequent mitosis would then duplicate this 92,XXXX karyotype and establish a tetraploid pregnancy. It is of interest in this case that both gestational sacs resulted clinically and pathologically in a blighted ovum. Embryoblastic failure with development only of the extraembryonic compartment has been described (9) in other cases of paternal component polyploidy, which is known to favor trophoblastic development. In contrast, maternal component polyploidy favors embryoblast development, where an embryonic pole is present but the extraembryonic compartment is underdeveloped. Development exclusively of the trophoblast is consistent with the clinical course (blighted ovum) of this pregnancy, wherein a fetus was not identified using ultrasound, nor were embryonic derivatives found at the time of histologic examination. Finally, given the clinical and pathologic similarity of the two sacs, it is conceivable that the other sac from which cells failed to grow had an identical karyotype. This raises the possibility that the early tetraploid embryo may have completely divided into identical twins. We conclude that cryopreservation at the pronuclear stage may have damaged the delicate spindle apparatus of the early zygote, resulting in failed anaphase and cytokinesis. Since chromosome duplication had already taken place, this mitotic error resulted in tetraploidy. Cryopreservation of embryos which have developed beyond the pronuclear stage might prevent such cold-induced damage to these sensitive microtubular structures. Alternatively, meiosis I oocytes might be less susceptible to cryodamage and therefore preferable for storage of "excess" gametes derived from assisted reproductive technologies, although current technology precludes this possibility at the present time. Finally, if further investigation establishes that the risk of karyotypic abnormality associated with cryopreservation is sufficiently high, prenatal genetic diagnosis might be indicated to investigate pregnancies resulting from such embryo manipulation.
488
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REFERENCES 1. Medical Research International, Society for Assisted Reproductive Technology, American Fertility Society: In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1989 results from the IVF-ET Registry. Fertil Steril 1991;55(1):14-22. 2. Brinsden P, Macnamee MC, Wick K: Pregnancy rates following frozen thawed embryo replacement are as good as those following fresh embryo replacement. Proceedings 1991 AFS meeting. Fertil Steril 1991;abstr 0-038, S17 3. Sheppard DM, Fisher RA, Lawler SD, Poley S: Tetraploid conceptus with three paternal contributions. Hum Genet 1982;62:317-374 4. Pitt D, Leversha M, Sinfield C, Campbell P, Anderson R, Bryan D, Rogers J: Tetraploidy in a liveborn infant with spina bifida and other anomalies. J Med Gen 1981;18:309311 5. Plachot M, de Grouchy J, Junca AM, Mandelbaum J, SalatBaroux J, Cohen J: Chromosome analysis of human oocytes and embryos: Does delayed fertilization increase chromosome imbalance? Hum Reprod 1988;3:125-127 6. Plachot M: Chromosome analysis of spontaneous abortions after IVF. A European study. Hum Reprod 1989;4(4):425429 7. Le Porrier N, Thepot F, Camier B, Herlicoviez M, Herlicoviez D, Obry E, Sauvalle A, Alliet J: Etude cytogenetique des produits de fausses couches prococes apres fecondation in vitro. Contra Fertil Sex 1988;16:652-653 8. Magistrini M, Szollosi D: Effects of cold and of isopropylN-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980;22(2):699-707 9. Surti U, Szulman AE, Wagner K, Leppert M, O'Brien JJ: Tetraploid partial hydatiform moles: Two cases with a triploid paternal contribution and a 92,XXXY karyotype. Hum Genet 1986;72:15-21
Kenneth A. Ginsburg1 Anthony G. Sacco Merrilie F. Rousseau Charla M. Blacker Kamran S. Moghissi Division of Reproductive Endocrinology and Infertility Department of Obstetrics and Gynecology Wayne State University School of Medicine and Hutzel Hospital Detroit, Michigan Mark P. Johnson Division of Reproductive Genetics Department of Obstetrics and Gynecology Wayne State University School of Medicine and Hutzel Hospital Detroit, Michigan 1 To whom correspondence should be addressed at Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Hutzel Hospital, 4707 St. Antoine, Detroit, Michigan 48201.
JEDDAH,
SAUDI ARABIA
A n E v a l u a t i o n o f t h e E f f e c t o f the Anesthetic Agent P r o f o f o l ( D i p r i v a n ) on the O u t c o m e o f H u m a n in Vitro Fertilization
Submitted: July 25, 1992 Accepted: August 3, 1992
MATERIALS AND METHODS Twenty patients who were scheduled for ultrasound-guided follicular aspiration were studied. They were informed of the purpose of the study and they all gave their informed consent. All 20 women were undergoing in vitro fertilization (IVF) for either tubal disease (15 women) or unexplained infertility (5 women). The women were between 23 and 36 years of age. Ovarian stimulation was carried out with intramuscular human menopausal gonadotropin (hMG) in doses of 150 to 300 U daily from the second day of the menstrual cycle and continued until the morning of human chorionic gonadotropin (hCG) administration. Follicular growth was monitored by daily ultrasound scans from day 9 of the menstrual cycle. Daily serial determination of serum luteinizing hormone (LH) and estradiol (E2) were commenced on day 9 of the menstrual cycle until the day of hCG administration. When the mean follicular diameter of at least two follicles was 19 mm, close monitoring of serum L H every 6 hr was commenced prior to administration of 5000 U of hCG 34 to 36 hr to the scheduled follicular aspiration procedure. All patients were admitted and seen at least 3 hr prior to the operation for assessment of cardiopulmonary fitness for anesthetic. History of allergy was inquired and no patients were included if they had any known history of allergic reaction to medication. No preanesthetic medication was required in any of these patients. All patients had fasted for at least 8 hr prior to admission. A dose of 1 to 1.5 mg/kg body weight of Fentanyl for pain relief was given intravenously. This was followed by an initial intravenous bolus of Diprivan emulsion administered in a dose of 2.5 mg/kg body Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
stimulation. Basal follicle stimulating hormone (FSH) levels may be used to predict those patients who cannot be effectively stimulated (12). Patients with a basal F S H greater than 18 IU/L have been excluded in the current program, being categorized as having occult ovarian failure. In order to compensate for the reduced implantation rate in women over 40 years, the number of eggs or embryos may be increased. This strategy is limited by the reduced number of eggs available. If pregnancy rates are increased by giving higher doses of progesterone in donor egg patients over 40 years (10), then the same therapy may be effective in women using their own eggs or embryos. Donor egg IVF or GIFT is the most successful therapy for women wishing to conceive over 40 years. The success rate per treatment cycle has been similar to that in younger women, 30% (13). The low natural fertility and markedly reduced success of artificial reproductive technology in women over 40 years suggests that conception is an urgent problem, particularly as fertility declines rapidly between 40 and 50 years of age. Full investigation of fertility status can be offered immediately so that any defects can be treated as soon as possible. If the patient fails to conceive, GIFT or IVF procedures may be offered without delay. The reasons for the difficulty in conceiving should be explained to the patient and the alternatives such as the use of donor eggs considered by the patient (14). Most couples take some time to understand, consider, and proceed to the use of donor eggs so it is preferable that this procedure be offered early in the management of the infertile patient over 40 years wishing to conceive. It is incorrect to define an arbitrary period of time when an older patient is infertile, as any woman over 40 years is likely to be subfertile, and fewer than 25% can be expected to conceive in 1 year. REFERENCES 1. Gray RH: Social influences in fertility at later ages of reproduction. J Biosoc Sci Suppl 1979;6:97-115 2. MacLeod J, Gold RZ: The male factor in fertility and sterility. VI. Semen quality and certain other factors in relations to ease of conception. Fertil Steril 1953;4:10-33 3. Vessey M, Doll R, Peto R, Johnson B, Wiggins P: A longterm follow up study of women using different methods of contraception--an interim report. J Biosoc Sci t976;8:373427 4. Adams CE: In Reproductive Senescence in Reproduction in Mammals, CR Austin, RV Short (eds). London, Cambridge University Press, 1984, p 212
SHORT COMMUNICATIONS
5. Doring GK: The incidence of anovutar cycles in women. J Reprod Fertil 1969;Suppl 6:77-81. 6. Adams CE: In Reproductive Senescence in Reproduction in Mammals, CR Austin, RV Short (eds). London, Cambridge University Press, 1984, p 218 7. Piette C, de Mougon J, Bachelot A, Spira A: IVF influence of women's age on pregnancy rates. Hum Reprod 1990;5(1): 56-59 8. Wood C, Riley R: IVF. Melbourne, Hill of Content, 1992, pp 53-56 9. Berget PA, Williams MA, Grunfeld F, Fox J, Hofmann GE, Navot D: Age related decrease in fertility potential is attributable to occyte quality rather than implantation failure. American Fertility Society 1991, abstr 0.062, $27 10. Meldrum R: Younger eggs for older women. Presentation at Serono Symposium on Oocyte Donation, Monash University, Melbourne, 1991 11. Baker TG: Population of germ ceils in the human ovary. Am J Obstet Gynaecol 1900;100:746-761 12. Cameron IT, O'Shea FC, Rolland JM, Hughes EG, De Kretser DM, Healy DL: Occult ovarian failure: A syndrome of infertility, regular menses and elevated FSH concentrations. J Clin Endocrinol Metab 1987;67(6): 1190-1194 13. Meldrum DR, Marr B, Stubbs C, Wisot A, Hamilton M, Heit R: Oocyte donation--A new approach for the poor prognosis patient. 1990 Abstracts of 46th American Fertility Society Meeting, Oct 1990, 0-024 14. Osborn J: Life table analysis of GII~q?results---effects of age: Information sheet for patients entering Monash programme. Melbourne, 1991
Carl Wood 1 n a n Calderon Amanda Crombie Infertility Medical Centre 62 Erin Street Richmond, Victoria 3121, Australia 1 To whom correspondence should be addressed at Department of Obstetrics and Gynaecology, Monash University, 246 Clayton Road, Clayton, Victoria 3168, Australia.
DETROIT, MICHIGAN Tetraploidy Associated with Human Pronuclear Embryo Cryopreservation: A Case Report
Submitted: June 5, 1992 Accepted: July 20, 1992
INTRODUCTION Advances in the fields of reproduction and cryobiology now allow routine freezing of human embryos Journal o f Assisted Reproduction and Genetics, VoL 9, No. 5, 1992
SHORT COMMUNICATIONS
that result from in vitro fertilization. Many programs cryopreserve embryos at the pronuclear stage, immediately following oocyte fertilization with formation of the second polar body and both pronuclei, but before completion of the first cleavage division. Worldwide experience has shown such cryopreservation and transfer to be a safe and relatively efficient process in terms of both viable embryo recovery and per transfer pregnancy rates (1,2). However, an increased risk of pregnancy wastage or congenital abnormality resulting from such embryo manipulation has not been completely excluded, since at this stage the embryo may be susceptible to damage resulting from handling, dehydration and cryopreservation. This case documents an association between pronuclear stage cryopreservation and tetraploidy, with subsequent spontaneous abortion.
CASE PRESENTATION A 30-year-old nulligravid Caucasian woman began controlled ovarian hyperstimulation for an in vitro fertilization (IVF) procedure as treatment for unexplained primary infertility. Urofollitropin (Metrodin, Serono Laboratories, Inc., Norwall, MA), 150 IU, and human menopausal gonadotropin (Pergonal, Serono Laboratories, Inc., Norwall, MA), 150 IU, were administered daily in two divided intramuscular (im) doses beginning on cycle day (CD) 3, at which time the serum estradiol level was 46 pg/ml, follicle stimulating hormone (FSH) was 8.3 mIU/ml, and luteinizing hormone (LH) was 11.3 mIU/ml. By CD 6 the estradiol level had risen to 179 pg/ml, and daily estradiol and ultrasound monitoring was begun 2 days later. By CD 11 a total of 12 follicles greater than 14 mm was noted on both ovaries, with two follicles measuring 18 mm in average dimension. Estradiol level at that time was 1820 pg/ml. Human chorionic gonadotropin (Profasi, Serono Laboratories, Inc., Norwall, MA), 10,000 IU, was given im at 9:30 i'M. Transvaginal oocyte retrieval was begun 34.5 hr later under intravenous sedation supplemented with 1% lidocaine infiltration of the vagina for local anesthesia. Eleven preovulatory oocytes were recovered. The sperm sample obtained that day had a volume of 2.0 ml with 110 x 106 spermatozoa/ml, 75% motility, and 55% normal morphology. A two-step wash and centrifugation, followed by a 90-min swim-up, yielded a sample with 85% motility and 35 x 10 6 Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
485
spermatozoa/ml. Each oocyte was inseminated with 150,000 motile spermatozoa per our usual protocol. When first examined 18 to 20 hr after insemination, all oocytes were noted to have two pronuclei (PN), and 10 of the 11 additionally had two polar bodies (PB) visible. The couple had previously given written consent for cryopreservation of "exc e s s " embryos remaining after fresh transfer. Seven pronuclear embryos (each with two PN and two PB) were cryopreserved in three plastic straws beginning 26 hr after retrieval. Embryos were first washed in human tubal fluid supplemented with 15% patient serum, followed by stepwise dehydration to 1.5 M propanediol containing 2.0 M sucrose. Initial cooling in a Kryo 10 Programmable Freezer (T.S. Scientific, Perkasie, PA) from room temperature to -7°C occurred at a rate of 2°/min, after which embryos were seeded with a precooled forceps. Further cooling to -32°C was at a rate of 0.3°/rain. After holding for 1 hr at -32°C, embryos were plunged into liquid nitrogen for storage. Twenty-four hours later, i.e., on the day of embryo transfer, the embryo with two PN but no second PB was clearly atretic and discarded. Cleavage was noted in the three remaining embryos. Fifty hours after retrieval a two-cell embryo with unequal blastomeres, a four-cell embryo with minor fragmentation, and a five-cell embryo with major fragmentation were transferred to the uterus transcervically without complication. Daily intramuscular progesterone supplementation was continued until 14 days after embryo transfer, at which time the patient's human chorionic gonadotropin (hCG) level was below 5 mIU/ml serum. Progesterone was discontinued and a menstrual period followed. The couple declined endometrial assessment using late luteal-phase biopsy prior to replacement of cryopreserved embryos. During the first cycle after retrieval a spontaneous LH surge was detected by urinary sampling every 4 hr. Five embryos were thawed 27 days after cryopreservation by equilibration in a 30°C water bath. Propanediol and sucrose were sequentially removed as the embryos were rehydrated. Two of the embryos were atretic after recovery, while the remaining three appeared normal. Cleavage was documented prior to transfer, and 23 hr after thawing was initiated a two-cell, a three- to four-cell, and a six-cell embryo were transferred transcervically to the uterine cavity. Twelve days after transfer the hCG level was 45 mIU/ml, and pregnancy was confirmed two days later with an hCG level that had risen to 110 mIU/ml. Daily
486
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intramuscular progesterone supplementation was continued. Serial hCG assay initially suggested a normal pregnancy, and two concordant gestational sacs were identified sonographically 25 days after transfer. However, over the subsequent 24 days no fetal pole was demonstrated and the gestational sacs stopped growing. A diagnosis of blighted twin gestation was made, but the patient declined dilatation and curettage, instead agreeing to observation with the expectation of spontaneous complete abortion. hCG levels declined over the next 30 days and the onset of bleeding and cramping was quickly followed by passage of tissue. Bleeding and cramping then stopped and the cervix and uterus involuted. hCG levels eventually declined to and remained below the assay detection limit (5 mlU/ml). Pathologic examination of the products of conception showed no embryonic derivatives. Portions of each gestational sac were sent for chromosome analysis. Cells
1
6
13
2
failed to grow from one sample, while the karyotype on the other was 92,XXXX (Fig. 1).
DISCUSSION Tetraploidy has been reported in 4-6% of spontaneous abortuses in which karyotypic abnormalities are identified (3). Although rare tetraploid mosaic infants have been described (4), the vast majority of tetraploid pregnancies apparently end in early spontaneous abortion. In vitro, tetraploidy often results in cleavage arrest early in embryonic development, approximately at the eight-cell stage (5). The incidence of polyploidy in conception resulting from assisted reproduction appears to be low (6,7). Plachot (6) identified a single tetraploid in 21 abnormal karyotypes found in 34 spontaneous abortions conceived by IVF without embryo cryopreservation. In addition, a single tetraploid spon-
4
3
7
|
14
15
9
5
I0
II
16
17
12
18
19 20 21 22 X Y Fig. 1. Photomicrograph of metaphase karyotype obtained from the products of conception of one gestational sac, stained with trypsin-Giemsa. Analysis of polymorphic markers, acrocentric satellites, and centromeric regions suggests duplication of both the maternal and the paternal haploid complements. Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
SHORT COMMUNICATIONS
taneous abortion has been reported among the 2811 clinical pregnancies conceived through in vitro fertilization in 1989 (1). In that same report, 234 pregnancies were conceived from 2124 frozen embryo transfer cycles, resulting in 172 (8%) live births, and a 92,XXYY chromosomal abnormality resulting in spontaneous abortion was noted. To our knowledge, the case reported herein therefore represents only the second in the literature documenting tetraploidy in association with human embryo cryopreservation and the first for which detailed information is available. The mechanisms by which polyploidy may occur are listed in Table I. Of these possibilities, several features suggest mitotic dysfunction as the etiology of tetraploidy in this case. We observed two polar bodies during the development of each of the seven oocytes which were cryopreserved, suggesting that the tetraploidy was not the result of digyny (incorporation of polar bodies) or suppressed maternal meiosis. The presence of only two pronuclei argues against polyspermic fertilization. Parthenogenic activation of a diploid oocyte can be excluded by careful analysis of the karyotype, which shows sufficient differences in the chromosomes to exclude a common ancestor. Most importantly, analysis of polymorphic markers on chromosomes 3, 9, and 16, the acrocentric satellites, and centromeric regions suggests duplication of each haploid maternal and paternal chromosome complement. This implies normal duplication but failure of anaphase and cytokinesis at the first mitotic division. In the handling of these embryos, development appeared normal up to the point when they were cryopreserved at the pronuclear stage° After chromosome duplication, we postulate that cryopreservation and/or thawing may have damaged the spindle apparatus of the zygote as it prepared for the first mitotic division. At this critical time disruption of the spindle could inappropriately signal completion of mitosis. Such an effect has been demon-
Table I. Potential Cytopathologic Mechanisms of Tetraploidy 1. Meiotic errors in germ-cell precursors leading to diploid gametes 2. Incorporation or suppression of the formatJion of one of the polar bodies (digyny) 3. Suppression of the 1st or 2nd maternal meiotic division 4. Fertilization of the ovum by two or more spermatozoa (polyspermy) 5. Initial mitotic dysfunction (nondisjunction) resulting in chromosome duplication but cleavage failure
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strated in animal embryos studied in vitro, where 15 to 60 min of cold treatment has been shown to disrupt the spindle apparatus by first depolymerizing continuous microtubules, followed subsequently by kinetochore fibers (8). In this situation, upon thawing and with the spindle disrupted, the zygote would conclude the first mitotic division without completing anaphase or cytokinesis, thereby yielding a tetraploid single cell embryo. Subsequent mitosis would then duplicate this 92,XXXX karyotype and establish a tetraploid pregnancy. It is of interest in this case that both gestational sacs resulted clinically and pathologically in a blighted ovum. Embryoblastic failure with development only of the extraembryonic compartment has been described (9) in other cases of paternal component polyploidy, which is known to favor trophoblastic development. In contrast, maternal component polyploidy favors embryoblast development, where an embryonic pole is present but the extraembryonic compartment is underdeveloped. Development exclusively of the trophoblast is consistent with the clinical course (blighted ovum) of this pregnancy, wherein a fetus was not identified using ultrasound, nor were embryonic derivatives found at the time of histologic examination. Finally, given the clinical and pathologic similarity of the two sacs, it is conceivable that the other sac from which cells failed to grow had an identical karyotype. This raises the possibility that the early tetraploid embryo may have completely divided into identical twins. We conclude that cryopreservation at the pronuclear stage may have damaged the delicate spindle apparatus of the early zygote, resulting in failed anaphase and cytokinesis. Since chromosome duplication had already taken place, this mitotic error resulted in tetraploidy. Cryopreservation of embryos which have developed beyond the pronuclear stage might prevent such cold-induced damage to these sensitive microtubular structures. Alternatively, meiosis I oocytes might be less susceptible to cryodamage and therefore preferable for storage of "excess" gametes derived from assisted reproductive technologies, although current technology precludes this possibility at the present time. Finally, if further investigation establishes that the risk of karyotypic abnormality associated with cryopreservation is sufficiently high, prenatal genetic diagnosis might be indicated to investigate pregnancies resulting from such embryo manipulation.
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REFERENCES 1. Medical Research International, Society for Assisted Reproductive Technology, American Fertility Society: In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1989 results from the IVF-ET Registry. Fertil Steril 1991;55(1):14-22. 2. Brinsden P, Macnamee MC, Wick K: Pregnancy rates following frozen thawed embryo replacement are as good as those following fresh embryo replacement. Proceedings 1991 AFS meeting. Fertil Steril 1991;abstr 0-038, S17 3. Sheppard DM, Fisher RA, Lawler SD, Poley S: Tetraploid conceptus with three paternal contributions. Hum Genet 1982;62:317-374 4. Pitt D, Leversha M, Sinfield C, Campbell P, Anderson R, Bryan D, Rogers J: Tetraploidy in a liveborn infant with spina bifida and other anomalies. J Med Gen 1981;18:309311 5. Plachot M, de Grouchy J, Junca AM, Mandelbaum J, SalatBaroux J, Cohen J: Chromosome analysis of human oocytes and embryos: Does delayed fertilization increase chromosome imbalance? Hum Reprod 1988;3:125-127 6. Plachot M: Chromosome analysis of spontaneous abortions after IVF. A European study. Hum Reprod 1989;4(4):425429 7. Le Porrier N, Thepot F, Camier B, Herlicoviez M, Herlicoviez D, Obry E, Sauvalle A, Alliet J: Etude cytogenetique des produits de fausses couches prococes apres fecondation in vitro. Contra Fertil Sex 1988;16:652-653 8. Magistrini M, Szollosi D: Effects of cold and of isopropylN-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980;22(2):699-707 9. Surti U, Szulman AE, Wagner K, Leppert M, O'Brien JJ: Tetraploid partial hydatiform moles: Two cases with a triploid paternal contribution and a 92,XXXY karyotype. Hum Genet 1986;72:15-21
Kenneth A. Ginsburg1 Anthony G. Sacco Merrilie F. Rousseau Charla M. Blacker Kamran S. Moghissi Division of Reproductive Endocrinology and Infertility Department of Obstetrics and Gynecology Wayne State University School of Medicine and Hutzel Hospital Detroit, Michigan Mark P. Johnson Division of Reproductive Genetics Department of Obstetrics and Gynecology Wayne State University School of Medicine and Hutzel Hospital Detroit, Michigan 1 To whom correspondence should be addressed at Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Hutzel Hospital, 4707 St. Antoine, Detroit, Michigan 48201.
JEDDAH,
SAUDI ARABIA
A n E v a l u a t i o n o f t h e E f f e c t o f the Anesthetic Agent P r o f o f o l ( D i p r i v a n ) on the O u t c o m e o f H u m a n in Vitro Fertilization
Submitted: July 25, 1992 Accepted: August 3, 1992
MATERIALS AND METHODS Twenty patients who were scheduled for ultrasound-guided follicular aspiration were studied. They were informed of the purpose of the study and they all gave their informed consent. All 20 women were undergoing in vitro fertilization (IVF) for either tubal disease (15 women) or unexplained infertility (5 women). The women were between 23 and 36 years of age. Ovarian stimulation was carried out with intramuscular human menopausal gonadotropin (hMG) in doses of 150 to 300 U daily from the second day of the menstrual cycle and continued until the morning of human chorionic gonadotropin (hCG) administration. Follicular growth was monitored by daily ultrasound scans from day 9 of the menstrual cycle. Daily serial determination of serum luteinizing hormone (LH) and estradiol (E2) were commenced on day 9 of the menstrual cycle until the day of hCG administration. When the mean follicular diameter of at least two follicles was 19 mm, close monitoring of serum L H every 6 hr was commenced prior to administration of 5000 U of hCG 34 to 36 hr to the scheduled follicular aspiration procedure. All patients were admitted and seen at least 3 hr prior to the operation for assessment of cardiopulmonary fitness for anesthetic. History of allergy was inquired and no patients were included if they had any known history of allergic reaction to medication. No preanesthetic medication was required in any of these patients. All patients had fasted for at least 8 hr prior to admission. A dose of 1 to 1.5 mg/kg body weight of Fentanyl for pain relief was given intravenously. This was followed by an initial intravenous bolus of Diprivan emulsion administered in a dose of 2.5 mg/kg body Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
