Human Reproduction vol.6 no.3 pp.446-449, 1991
Analysis of ectopic pregnancies resulting from in-vitro fertilization and embryo transfer*
V.C.Karande, J.T.Flood, N.Heard, L.Veeck and S.J.Muasher1 The Howard and Georeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 825 Fairfax Avenue, 6th Floor, Norfolk, VA, 23507, USA. 'To whom correspondence should be addressed
The purpose of this study was to analyse the risk factors, stimulation characteristics, site and outcome of pregnancy and future fecundity of patients who develop ectopic pregnancies after in-vitro fertilization (TVF). Of 3145 transfer cycles between January 1981 and July 1989, 27 (3.3%) of the resulting 825 pregnancies were ectopic. There was a significantly greater incidence of a prior ectopic pregnancy in the study group compared to the controls. Compared to matched controls with intrauterine pregnancies, the study group had significantly higher peak oestradiol levels. Twentyone ectopic pregnancies were ampullary, two were interstitial, one-was abdominal, one was cervical and two were heterotopic. Sixteen of the patients subsequently underwent 40IVF attempts with a pregnancy rate of 28% per transfer. We conclude that patients with a prior ectopic pregnancy are at risk for an FVF ectopic pregnancy. The subsequent FVF outcome of those who develop ectopic pregnancies after IVF is encouraging. Key words: ectopic/IVF/tubal pregnancy/risk factors/fecundity
Introduction The first pregnancy reported with in-vitro fertilization (TVF) was an ectopic (Steptoe and Edwards, 1976). Since then, there have been several reports of ectopic pregnancies occurring after IVF (Seppala, 1985; Cohen et al., 1986, 1988; Martinez and Trounson, 1986; Correy etal., 1988). Most of these reports are based on a small number of patients, or on collaborative results between centres, with inherent variables in personnel, stimulation protocols and transfer techniques which could influence the results and conclusions. Data on the risk factors for developing an IVF ectopic are conflicting. Martinez and Trounson (1986) could not identify any risk factors, while Cohen et al. (1988) identified the number of patent tubes at the time of transfer as a risk factor. This study analyses the risk factors, stimulation •Presented in pan at the 45th Annual Meeting of the American Fertility Society. San Francisco, California, November 11-16. 1989
446
characteristics, site and outcome of pregnancy and future fecundity of IVF patients who develop ectopic pregnancies in a large and well-established FVF programme. Materials and methods In a retrospective analysis of patients having transfer cycles after IVF between January 1981 and July 1989 (Norfolk series 1 to 35), patients with an ectopic pregnancy were selected for review. The following data were recorded: patient's age, aetiology of infertility, history of a prior ectopic pregnancy, patency of the Fallopian tubes from a recent hysterosalpingogram (HSG), stimulation protocol used, peak oestradiol (E2) levels and method of oocyte retrieval. The details of the transfer were also recorded, including the number of pre-embryos transferred, the degree of difficulty of the transfer and the volume of culture medium used. The site of the ectopic pregnancy, the condition of the tube and the surgical treatment were noted. The outcome of pregnancy in study patients who returned for subsequent IVF attempts was also analysed. Serial quantitation of human chorionic gonadotrophin (HCG) in study group patients with single tubal pregnancies (n = 22) was compared with one control group consisting of a similar number of patients with normal pregnancies (n = 25), and another group consisting of first trimester spontaneous abortions (n = 25) occurring at the same time as one of the study group patients. All patients undergoing stimulation between June 1988 and July 1989 were selected as a control group to compare patient characteristics and HSG findings. For comparison of the stimulation characteristics, a case-control group was necessary. For each patient in the study group, we selected a patient from the same series with a normal intrauterine pregnancy as a matched control. The control had an identical stimulation protocol, was of similar age and had a similar aetiology of infertility. AH patients had undergone stimulation with pure gonadotrophin and monitoring by established protocols (Rosenwaks and Muasher, 1986). Oocyte retrieval was performed laparoscopicaUy (Jones et al., 1982) or transvaginally using published techniques (Flood et al., 1989). Preincubation of oocytes, sperm preparation, embryology laboratory procedures and embryo transfer apparatus and technique were employed as previously described (Veeck etal., 1983; Veeck, 1986; Garcia, 1986). The transfers were classified according to the degree of difficulty, i.e. as smooth or difficult. E2 levels were determined by a commercially available radioimmunoassay (RIA) (Pantex; Santa Monica, CA) and total variation within the assay was < 7.9%. Human chorionic gonadotrophin levels were also determined by a commercially © Oxford University Press
Ectopic pregnancy and IVF/ET
available R1A (Dade; Cambridge, MA) and were standardized according to the First International Reference Preparation. The total variation within the assay was 9, 6.3 and 6.8% at 20, 61 and 270 mlU/ml, respectively. Statistical comparison of the study group and the control groups was done by x2 analysis and unpaired f-tests where appropriate. Comparison between the stimulation characteristics of the study group and the matched controls was done by stepwise regression analysis. A value of P < 0.05 was considered to be significant. The ability of predictor variables to correctly assign outcomes was assessed by discriminant analysis. Results Between January 1981 and July 1989 (Norfolk series 1-35), 1798 patients underwent 3145 transfer cycles. Of the 825 resultant pregnancies, 27 (3.3%) were ectopic and one patient had two ectopic pregnancies after IVF (see details below), accounting for 27 cycles in 26 patients. The average age of patients was 33 ± 0.6 y (mean ± standard error). Eleven patients (42%) had primary infertility; of the remaining 15,10 had previous ectopic pregnancies, three had had previous term deliveries and six had had previous miscarriages. Twenty-three patients had a tubal factor, two patients had endometriosis, and one had diethylstilboestrol (DES) exposure as the primary aetiology of infertility. Secondary diagnosis were endometriosis in six patients, tubal disease in two, DES exposure in one and male factor in two. Twenty-five patients had a history of tubal surgery; proximal patency was found on HSG bilaterally in 16 patients and unilaterally in 10; one patient had bilateral proximal tubal obstruction. Only three (11 %) of the ectopic pregnancies occurred in a Fallopian tube which had not undergone tuba! surgery. For comparison of patient characteristics and HSG findings, 344 women who underwent 741 stimulation cycles between June 1988 and July 1989 served as controls. The primary diagnosis was tubal factor in 207 patients, male factor in 83, endometriosis in 30, anovulation in nine, DES exposure in two and idiopathic in 13. Of these, 175 (50.8%) had primary infertility. Of the remaining 169, 67 had a history of ectopic pregnancy, 38 had had term pregnancies and 40 had had spontaneous abortions. The characteristics of the control group were similar to the overall patient population in our programme (data not shown). There was a significantly higher history of ectopic pregnancy in the study group than in the control groups (10/26 versus 67/344; P < 0.05). Also, the incidence of tubal infertility was significantly higher in the study group than in controls (23/26 versus 207/344; P < 0.01). Of the 344 controls, 144 (42%) achieved a pregnancy. Comparison of the HSGs of the study group and these 344 patients revealed no significant differences in the proportions of patients with one, two or no patent tubes. The stimulation protocol of patients with IVF ectopic pregnancies consisted of a combination of two ampoules of follicle stimulating hormone (FSH) (Metrodin, Serono Laboratories, Randolph, MA) and two ampoules of human menopausal gonadotrophin (HMG) (Pergonal, Serono) in 16 patients. Eight patients received pure FSH stimulation; one received two ampoules of HMG. Two patients developed ectopic pregnancies after transfer of cryopreserved pre-embryos during a subsequent
natural cycle. All patients were normal (n = 13) or high (n = 12) responders with a peak Ej of 1113 ± 9 5 pg/ml. Fifteen patients had a laparoscopic and 10 patients an ultrasounddirected trans vaginal oocyte retrieval. The stimulation characteristics of the study group and the matched controls respectively were the following: peak E2; 1113 ± 105 versus 696 ± 76 pg/ml (P < 0.002); number of ampoules used, 20 ± 3 versus 18 ± 3 (NS); number of mature oocytes retrieved, 4.5 ± 0.8 versus 4.0 ± 0.7 (NS); number of pre-embryos transferred, 3.8 ± 0.3 versus 3.1 ± 0.3 (NS). Stepwise regression indicated that peak Ej but not stimulation protocol, number of pre-embryos transferred or history of tubal surgery significantly predicted ectopic pregnancy (F = 10.14; P = 0.0025; R2 = 15.7%). Using these same variables, discriminant analysis correctly classified outcome for 64% of ectopics and 76% of intrauterine pregnancies (P = 0.014). All transfers were without difficulty and were classified as 7.0001 6.000-
1 5.000 •J 4.000m 3.000 CO.
^ 2.000 1.000 0
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 Day
Fig. 1. /3-HCG levels until day 22 in patients with single tubal pregnancies (n = 22). • • , mean; A A , mean + standard deviation (SD); and • T , mean - SD, compared with those of normal singleton pregnancies (shaded area) (n = 25). Day 0 is day of retrieval. 2.500-, 2,000 1.500 1.000
o
2 500
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Fig. 2. 0-HCG levels until day 22 in patients with single tubal pregnancies (n = 22). • • , mean; A A , mean + standard deviation (SD); and • • , mean - SD, compared with those of spontaneous first trimester abortions (shaded area) (n = 25). Day 0 is day of retrieval. 447
V.C.Karande et al.
'smooth'. The volume of culture fluid transferred was 70 /xl in 22 patients, 40 jA in three and 90 ^1 in two. Twenty-one pregnancies were in the ampullary region of the tube (one patient had bilateral ampullary pregnancies), two in the interstitial region, one abdominal and one cervical. There were two heterotopic pregnancies (ampullary tubal + intrauterine), of which one delivered a normal term baby and the other is ongoing. Of the 25 tubal pregnancies, 13 had ruptured at diagnosis, eight were unruptured, and the status of four was unknown. Of the two interstitial pregnancies, one occurred in a patient with a previous bilateral salpingectomy and the other in a patient who had had a salpingectomy on the side of the ectopic. The surgical treatment was a salpingectomy in most cases with ampullary ectopic pregnancies (n = 24). The interstitial pregnancies were treated with wedge resection, the abdominal pregnancy necessitated removal of the pregnancy from the abdominal cavity and the patient with a cervical pregnancy had an emergency hysterectomy due to uncontrollable haemorrhage. Figure 1 quantitates HCG values of the study group (n = 22; only single tubal pregnancies were included) compared with 25 normal singleton pregnancies and Figure 2 compares HCG levels with those in 25 singleton pregnancies that miscarried. There were no significant differences in the HCG values up to day 18 after transfer in the study group compared with the normal singleton pregnancies and those that miscarried. Subsequent HCG values were consistently higher in the normal pregnancies. Sixteen patients subsequently underwent 40 IVF attempts in 36 transfer cycles. Ten pregnancies resulted (27.8% per transfer), of which four delivered at term, three miscarried and two are ongoing. One patient with a previous IVF ectopic pregnancy which was treated with salpingostomy had a heterotopic pregnancy, with the tubal pregnancy occurring on the same side.
Discussion Although the incidence of ectopic pregnancies in this report (3.3%) is comparable with that of other reports (Seppala, 1985; Cohen etal., 1986, 1988; Martinez and Trounson, 1986; Correy et al., 1988), it is somewhat high compared with the general population (Mishell, 1987). The study patients were in the 'good' prognosis category for achieving an IVF pregnancy. They were young, all normal or high responders to gonadotrophin, and only two had a male factor as a secondary cause of their infertility. This suggests that there are common factors predisposing to both tubal and uterine pregnancy. A previous report emphasizing the importance of a woman's reproductive history suggested that the pregnancy rate per cycle of IVF treatment was higher in women who had a previous conception (Peek et al., 1985). This study, however, identifies a history of ectopic pregnancy as a risk factor for developing an IVF ectopic. The pre-embryo has a tendency to enter the tube more often than previously suspected (Correy et al., 1988). We can only speculate that perhaps in these patients the pre-embryo has a propensity to implant in the tube during natural conception as well as embryo transfer. The increased incidence of tubal factor infertility in the study group is probably related to the prior ectopic pregnancy. 448
A recent study (Cohen et al., 1986) indicated that the number of 'permeable' tubes identified on HSG is a risk factor for ectopic pregnancy. In this study, the incidence of ectopic pregnancy increased from 3.9 or 5.0% (with no or two permeable tubes respectively) to 12.3% (with one permeable tube). We found no such correlation. Two different control groups were necessary. In order to compare patient characteristics and HSG findings, for logistical reasons, we included all patients stimulated during a period of one year. This was to determine whether there were any special characteristics in patients who subsequently developed IVF ectopics. Any selection bias introduced by this method should be offset by the large number of patients and the fact that their characteristics were representative of our overall patient population in terms of diagnosis and other IVF characteristics. The control group thus forms a representative sample of our overall population. The second control group consisted of matched patients with intrauterine pregnancies for comparison of stimulation characteristics. Since this group of patients was controlled for age and aetiology, it could not be used to compare patient characteristics. The only significant difference was in the peak E2 level. Steroid hormones have been reported to alter tubal function with in vitro preparations, and E2 was shown to elicit a high contractile response in tubal musculature Lindblom et al., 1980). This is believed to be via the potentiation of the activation of adrenoceptors (Hershlag et al., 1989). Although the study group showed a significantly higher peak E2 level, this is not a prerequisite for developing an ectopic as we have observed two ectopics after the transfer of cryopreserved pre-embryos during a natural cycle when E2 levels were relatively low. Standard protocols were used for controlled ovarian hyperstimulation, oocyte retrieval and embryo transfer. The volume of culture medium used at transfer (70 /xl in most cases) was routine. All transfers were classified as 'smooth'. Thus, none of these parameters was useful in predicting the risk of ectopic pregnancy. Follow-up care of the patients was usually provided by their referring physicians. It is of some interest that the tube ruptured in 13 patients despite follow-up with serial quantitation of HCG values. Often this was due to reluctance to intervene with a 'precious' pregnancy until the diagnosis was 'confirmed'. In some cases (especially the heterotopic pregnancies), rupture was due to failure of diagnosis because an intrauterine sac had been visualized and normally rising titres of HCG were observed. Confino et al. (1986) reported that a single HCG value (50 mlU/ml) on day 9 after embryo transfer differentiates a normal from an abnormal pregnancy. We could not confirm this finding. It is our experience that normal pregnancies sometimes have an HCG value < 50 mlU/ml on day 9 after embryo transfer. Previous reports have analysed the predictive value of serial HCG titres in diagnosing IVF ectopics (Confino et al., 1986; Okamoto et al., 1987). Although titres below the 25th percentile are fairly accurate, they are often misleading in cases of heterotopic or twin tubal pregnancies (Dor et al., 1984; Sondheimer et al., 1985). This finding emphasizes the importance of a high clinical suspicion in diagnosing these cases. Progesterone (P) levels have been used to differentiate normal
Ectopic pregnancy and IVF/ET from abnormal pregnancies (Confino et al., 1986). Since we routinely supplement our patients with P post-transfer, we could not analyse the predictive value of P. The data from patients who returned for subsequent IVF attempts are encouraging and suggest that they have a good prognosis for subsequent pregnancy. Several attempts have been made to prevent IVF ectopic pregnancies. Steptoe et al. (1976) suggested bilateral salpingectomy; however, in this study as well as in others (Pavi et al., 1986), interstitial pregnancies occurred in patients without tubes. Yovich et al. (1985) used uterine length measured by ultrasound to guide placement of the pre-embryos and a transferred volume of < 50 id. Neither of these measures was effective. We conclude that IVF patients with a history of ectopic pregnancy are at risk for another ectopic pregnancy. Their stimulation characteristics are similar to those of patients developing intrauterine pregnancies except for higher peak E2 levels. Patients who develop ectopic pregnancies during IVF, however, have a good chance of achieving a normal pregnancy with subsequent IVF attempts and should be counselled accordingly. References Cohen.J., Mayaux,M.J., Guihard-Moscato,M.L. and Schwartz,D. (1986) In-vitro fertilization and embryo transfer: a collaborative study of 1163 pregnancies on the incidence and risk factors of ectopic pregnancies. Hum. Reprod., 1, 255-258. CohenJ., Mayaux,M.J. and Guihard-Moscato,M.L. (1988) Pregnancy outcomes after in vitro fertilization: a collaborative study on 2342 pregnancies. Ann. N.Y. Acad. Sci., 541, 1—6. Confino,E., Demir,R.H., Friberg,J. and Gleicher,N. (1986) The predictive value of hCG-|3 subunit levels in pregnancies achieved by in vitro fertilization and embryo transfer: an international collaborative study. Fertil. SteriL, 45, 526-531. Correy,J.F., Watkins.R.A., Bradfield.G.F., Gamer.S., Watson,S. and Gray,G. (1988) Spontaneous pregnancies and pregnancies as a result of treatment on an in vitro fertilization program terminating in ectopic pregnancies or spontaneous abortions. Fertil. SteriL, 50, 85-88. Dor.J., Rudak,E., Mashiach,S., Goldman.B. and Nebel.L. (1984) Unilateral tubal twin pregnancy following in vitro fertilization and embryo transfer. Fertil. SteriL, 42, 297-299. FloodJ.T., Muasher.S.J., Simmonetti.S., Kriener.D., Acosta.A.A. and Rosenwaks,Z. (1989) Comparison between laparoscopically and ultrasonographically guided transvaginal follicular aspiration methods in an in vitro fertilization program in the same patients using the same stimulation protocol. J. In Vitro FertiL Embryo Transfer, 6, 180—185. Garcia.J.E. (1986) Conceptus transfer. In Jones.H.W. Jr, Jones.G.S., Hodgen.G.D. and Rosenwaks.Z. (eds), In Vitro Fertilization-Norfolk. Williams and Wilkins, Baltimore, MD, p.215. Hershlag.A., Diamond.M.P. and DeChemey.A.H. (1989) Tubal physiology: an appraisal. J. Gynecol. Surg., 5, 3 - 2 5 . Jones,H.W.,Jr, Acosta.A.A. and Garcia.J.E. (1982) A technique for the aspiration of oocytes from human ovarian follicles. Fertil. SteriL, 37, 26-29. Lindblom.B., Hamburger,L. and Ljung,B. (1980) Contractile patterns of isolated oviductal smoodi muscles under different hormonal conditions. Fertil. SteriL, 33, 283-288. Martinez,F. and Trounson.A. (1986) An analysis of factors associated with ectopic pregnancy in a human in vitro fertilization program. Fertil. SteriL, 45, 79-87. Mishell,D.R.,Jr (1987) Ectopic pregnancy. In Droegemueller.W., Herbst.A.L., Mishell,D.R.,Jr. and Stenchever.M.A. (eds),
Comprehensive Gynecology. C.V. Mosby Company, St Louis, MO., p.406. Okamoto.S.H., Healy.D.L., Morrow,L.M., Rogers.P.A., Trounson.A.O. and Wood.E.C. (1987) Predictive value of plasma human chorionic gonadotrophin /3 subunit in diagnosing ectopic pregnancy after in vitro fertilization and embryo transfer. Br. Med. J., 294, 667-670. Pavi,C.N., Neuenschwander.E. and Gschwind.C. (1986) Interstitial pregnancy following bilateral salpingectomy and in vitro fertilizationembryo transfer. FertiL SteriL, 46, 701-702. Peek.J.C, Clark.A. and Graham.F.M. (1985) Conception rates after IVF in women with previous ectopic pregnancy. Getter) Lancet, U, 275. Rosenwaks.Z. and Muasher,S.J. (1986) Recruitment of fertilizable eggs. In Jones.H.W. Jr, Jones,G.S., Hodgen.G.D. and Rosenwaks.Z. (eds), In Vitro Fertilization-Norfolk. Williams and Wilkins, Baltimore, MD, p.30. Seppala.M. (1985) The world collaborative report on in vitro fertilization and embryo transfer: current state of the art in January, 1984. Ann. N.Y. Acad. Sci., 442, 558-566. Sondheimer.S.J., Tureck,R.W., Blasco.L., StraussJ.in, Arger.P. and. Mennuti.M. (1985) Simultaneous ectopic pregnancy with intrauterine twin gestations after in vitro fertilization and embryo transfer. Fertil. SteriL, 43, 313-316. Steptoe,P.C. and Edwards,R.G. (1976) Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet, i, 880-882. Veeck.L.L., Worthan,J.W.E.,Jr, Witmyer.J., Sandow,B.A., Acosta.A.A., Garcia.J.E., Jones.G.S. and Jones.H.W.,Jr (1983) Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil. SteriL, 39, 594-602. Veeck.L.L. (1986) Atlas of the Human Oocyte and Early Conceptus. Williams and Wilkins, Baltimore, MD. Yovich.J.L., Turner.S.R. and Murphy.A.J. (1985) Embryo transfer technique as a cause of ectopic pregnancies in in vitro fertilization. Fertil. SteriL, 44, 318-321. Received on June 8, 1990; accepted on November 15, 1990 •
449
Analysis of ectopic pregnancies resulting from in-vitro fertilization and embryo transfer*
V.C.Karande, J.T.Flood, N.Heard, L.Veeck and S.J.Muasher1 The Howard and Georeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 825 Fairfax Avenue, 6th Floor, Norfolk, VA, 23507, USA. 'To whom correspondence should be addressed
The purpose of this study was to analyse the risk factors, stimulation characteristics, site and outcome of pregnancy and future fecundity of patients who develop ectopic pregnancies after in-vitro fertilization (TVF). Of 3145 transfer cycles between January 1981 and July 1989, 27 (3.3%) of the resulting 825 pregnancies were ectopic. There was a significantly greater incidence of a prior ectopic pregnancy in the study group compared to the controls. Compared to matched controls with intrauterine pregnancies, the study group had significantly higher peak oestradiol levels. Twentyone ectopic pregnancies were ampullary, two were interstitial, one-was abdominal, one was cervical and two were heterotopic. Sixteen of the patients subsequently underwent 40IVF attempts with a pregnancy rate of 28% per transfer. We conclude that patients with a prior ectopic pregnancy are at risk for an FVF ectopic pregnancy. The subsequent FVF outcome of those who develop ectopic pregnancies after IVF is encouraging. Key words: ectopic/IVF/tubal pregnancy/risk factors/fecundity
Introduction The first pregnancy reported with in-vitro fertilization (TVF) was an ectopic (Steptoe and Edwards, 1976). Since then, there have been several reports of ectopic pregnancies occurring after IVF (Seppala, 1985; Cohen et al., 1986, 1988; Martinez and Trounson, 1986; Correy etal., 1988). Most of these reports are based on a small number of patients, or on collaborative results between centres, with inherent variables in personnel, stimulation protocols and transfer techniques which could influence the results and conclusions. Data on the risk factors for developing an IVF ectopic are conflicting. Martinez and Trounson (1986) could not identify any risk factors, while Cohen et al. (1988) identified the number of patent tubes at the time of transfer as a risk factor. This study analyses the risk factors, stimulation •Presented in pan at the 45th Annual Meeting of the American Fertility Society. San Francisco, California, November 11-16. 1989
446
characteristics, site and outcome of pregnancy and future fecundity of IVF patients who develop ectopic pregnancies in a large and well-established FVF programme. Materials and methods In a retrospective analysis of patients having transfer cycles after IVF between January 1981 and July 1989 (Norfolk series 1 to 35), patients with an ectopic pregnancy were selected for review. The following data were recorded: patient's age, aetiology of infertility, history of a prior ectopic pregnancy, patency of the Fallopian tubes from a recent hysterosalpingogram (HSG), stimulation protocol used, peak oestradiol (E2) levels and method of oocyte retrieval. The details of the transfer were also recorded, including the number of pre-embryos transferred, the degree of difficulty of the transfer and the volume of culture medium used. The site of the ectopic pregnancy, the condition of the tube and the surgical treatment were noted. The outcome of pregnancy in study patients who returned for subsequent IVF attempts was also analysed. Serial quantitation of human chorionic gonadotrophin (HCG) in study group patients with single tubal pregnancies (n = 22) was compared with one control group consisting of a similar number of patients with normal pregnancies (n = 25), and another group consisting of first trimester spontaneous abortions (n = 25) occurring at the same time as one of the study group patients. All patients undergoing stimulation between June 1988 and July 1989 were selected as a control group to compare patient characteristics and HSG findings. For comparison of the stimulation characteristics, a case-control group was necessary. For each patient in the study group, we selected a patient from the same series with a normal intrauterine pregnancy as a matched control. The control had an identical stimulation protocol, was of similar age and had a similar aetiology of infertility. AH patients had undergone stimulation with pure gonadotrophin and monitoring by established protocols (Rosenwaks and Muasher, 1986). Oocyte retrieval was performed laparoscopicaUy (Jones et al., 1982) or transvaginally using published techniques (Flood et al., 1989). Preincubation of oocytes, sperm preparation, embryology laboratory procedures and embryo transfer apparatus and technique were employed as previously described (Veeck etal., 1983; Veeck, 1986; Garcia, 1986). The transfers were classified according to the degree of difficulty, i.e. as smooth or difficult. E2 levels were determined by a commercially available radioimmunoassay (RIA) (Pantex; Santa Monica, CA) and total variation within the assay was < 7.9%. Human chorionic gonadotrophin levels were also determined by a commercially © Oxford University Press
Ectopic pregnancy and IVF/ET
available R1A (Dade; Cambridge, MA) and were standardized according to the First International Reference Preparation. The total variation within the assay was 9, 6.3 and 6.8% at 20, 61 and 270 mlU/ml, respectively. Statistical comparison of the study group and the control groups was done by x2 analysis and unpaired f-tests where appropriate. Comparison between the stimulation characteristics of the study group and the matched controls was done by stepwise regression analysis. A value of P < 0.05 was considered to be significant. The ability of predictor variables to correctly assign outcomes was assessed by discriminant analysis. Results Between January 1981 and July 1989 (Norfolk series 1-35), 1798 patients underwent 3145 transfer cycles. Of the 825 resultant pregnancies, 27 (3.3%) were ectopic and one patient had two ectopic pregnancies after IVF (see details below), accounting for 27 cycles in 26 patients. The average age of patients was 33 ± 0.6 y (mean ± standard error). Eleven patients (42%) had primary infertility; of the remaining 15,10 had previous ectopic pregnancies, three had had previous term deliveries and six had had previous miscarriages. Twenty-three patients had a tubal factor, two patients had endometriosis, and one had diethylstilboestrol (DES) exposure as the primary aetiology of infertility. Secondary diagnosis were endometriosis in six patients, tubal disease in two, DES exposure in one and male factor in two. Twenty-five patients had a history of tubal surgery; proximal patency was found on HSG bilaterally in 16 patients and unilaterally in 10; one patient had bilateral proximal tubal obstruction. Only three (11 %) of the ectopic pregnancies occurred in a Fallopian tube which had not undergone tuba! surgery. For comparison of patient characteristics and HSG findings, 344 women who underwent 741 stimulation cycles between June 1988 and July 1989 served as controls. The primary diagnosis was tubal factor in 207 patients, male factor in 83, endometriosis in 30, anovulation in nine, DES exposure in two and idiopathic in 13. Of these, 175 (50.8%) had primary infertility. Of the remaining 169, 67 had a history of ectopic pregnancy, 38 had had term pregnancies and 40 had had spontaneous abortions. The characteristics of the control group were similar to the overall patient population in our programme (data not shown). There was a significantly higher history of ectopic pregnancy in the study group than in the control groups (10/26 versus 67/344; P < 0.05). Also, the incidence of tubal infertility was significantly higher in the study group than in controls (23/26 versus 207/344; P < 0.01). Of the 344 controls, 144 (42%) achieved a pregnancy. Comparison of the HSGs of the study group and these 344 patients revealed no significant differences in the proportions of patients with one, two or no patent tubes. The stimulation protocol of patients with IVF ectopic pregnancies consisted of a combination of two ampoules of follicle stimulating hormone (FSH) (Metrodin, Serono Laboratories, Randolph, MA) and two ampoules of human menopausal gonadotrophin (HMG) (Pergonal, Serono) in 16 patients. Eight patients received pure FSH stimulation; one received two ampoules of HMG. Two patients developed ectopic pregnancies after transfer of cryopreserved pre-embryos during a subsequent
natural cycle. All patients were normal (n = 13) or high (n = 12) responders with a peak Ej of 1113 ± 9 5 pg/ml. Fifteen patients had a laparoscopic and 10 patients an ultrasounddirected trans vaginal oocyte retrieval. The stimulation characteristics of the study group and the matched controls respectively were the following: peak E2; 1113 ± 105 versus 696 ± 76 pg/ml (P < 0.002); number of ampoules used, 20 ± 3 versus 18 ± 3 (NS); number of mature oocytes retrieved, 4.5 ± 0.8 versus 4.0 ± 0.7 (NS); number of pre-embryos transferred, 3.8 ± 0.3 versus 3.1 ± 0.3 (NS). Stepwise regression indicated that peak Ej but not stimulation protocol, number of pre-embryos transferred or history of tubal surgery significantly predicted ectopic pregnancy (F = 10.14; P = 0.0025; R2 = 15.7%). Using these same variables, discriminant analysis correctly classified outcome for 64% of ectopics and 76% of intrauterine pregnancies (P = 0.014). All transfers were without difficulty and were classified as 7.0001 6.000-
1 5.000 •J 4.000m 3.000 CO.
^ 2.000 1.000 0
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 Day
Fig. 1. /3-HCG levels until day 22 in patients with single tubal pregnancies (n = 22). • • , mean; A A , mean + standard deviation (SD); and • T , mean - SD, compared with those of normal singleton pregnancies (shaded area) (n = 25). Day 0 is day of retrieval. 2.500-, 2,000 1.500 1.000
o
2 500
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Fig. 2. 0-HCG levels until day 22 in patients with single tubal pregnancies (n = 22). • • , mean; A A , mean + standard deviation (SD); and • • , mean - SD, compared with those of spontaneous first trimester abortions (shaded area) (n = 25). Day 0 is day of retrieval. 447
V.C.Karande et al.
'smooth'. The volume of culture fluid transferred was 70 /xl in 22 patients, 40 jA in three and 90 ^1 in two. Twenty-one pregnancies were in the ampullary region of the tube (one patient had bilateral ampullary pregnancies), two in the interstitial region, one abdominal and one cervical. There were two heterotopic pregnancies (ampullary tubal + intrauterine), of which one delivered a normal term baby and the other is ongoing. Of the 25 tubal pregnancies, 13 had ruptured at diagnosis, eight were unruptured, and the status of four was unknown. Of the two interstitial pregnancies, one occurred in a patient with a previous bilateral salpingectomy and the other in a patient who had had a salpingectomy on the side of the ectopic. The surgical treatment was a salpingectomy in most cases with ampullary ectopic pregnancies (n = 24). The interstitial pregnancies were treated with wedge resection, the abdominal pregnancy necessitated removal of the pregnancy from the abdominal cavity and the patient with a cervical pregnancy had an emergency hysterectomy due to uncontrollable haemorrhage. Figure 1 quantitates HCG values of the study group (n = 22; only single tubal pregnancies were included) compared with 25 normal singleton pregnancies and Figure 2 compares HCG levels with those in 25 singleton pregnancies that miscarried. There were no significant differences in the HCG values up to day 18 after transfer in the study group compared with the normal singleton pregnancies and those that miscarried. Subsequent HCG values were consistently higher in the normal pregnancies. Sixteen patients subsequently underwent 40 IVF attempts in 36 transfer cycles. Ten pregnancies resulted (27.8% per transfer), of which four delivered at term, three miscarried and two are ongoing. One patient with a previous IVF ectopic pregnancy which was treated with salpingostomy had a heterotopic pregnancy, with the tubal pregnancy occurring on the same side.
Discussion Although the incidence of ectopic pregnancies in this report (3.3%) is comparable with that of other reports (Seppala, 1985; Cohen etal., 1986, 1988; Martinez and Trounson, 1986; Correy et al., 1988), it is somewhat high compared with the general population (Mishell, 1987). The study patients were in the 'good' prognosis category for achieving an IVF pregnancy. They were young, all normal or high responders to gonadotrophin, and only two had a male factor as a secondary cause of their infertility. This suggests that there are common factors predisposing to both tubal and uterine pregnancy. A previous report emphasizing the importance of a woman's reproductive history suggested that the pregnancy rate per cycle of IVF treatment was higher in women who had a previous conception (Peek et al., 1985). This study, however, identifies a history of ectopic pregnancy as a risk factor for developing an IVF ectopic. The pre-embryo has a tendency to enter the tube more often than previously suspected (Correy et al., 1988). We can only speculate that perhaps in these patients the pre-embryo has a propensity to implant in the tube during natural conception as well as embryo transfer. The increased incidence of tubal factor infertility in the study group is probably related to the prior ectopic pregnancy. 448
A recent study (Cohen et al., 1986) indicated that the number of 'permeable' tubes identified on HSG is a risk factor for ectopic pregnancy. In this study, the incidence of ectopic pregnancy increased from 3.9 or 5.0% (with no or two permeable tubes respectively) to 12.3% (with one permeable tube). We found no such correlation. Two different control groups were necessary. In order to compare patient characteristics and HSG findings, for logistical reasons, we included all patients stimulated during a period of one year. This was to determine whether there were any special characteristics in patients who subsequently developed IVF ectopics. Any selection bias introduced by this method should be offset by the large number of patients and the fact that their characteristics were representative of our overall patient population in terms of diagnosis and other IVF characteristics. The control group thus forms a representative sample of our overall population. The second control group consisted of matched patients with intrauterine pregnancies for comparison of stimulation characteristics. Since this group of patients was controlled for age and aetiology, it could not be used to compare patient characteristics. The only significant difference was in the peak E2 level. Steroid hormones have been reported to alter tubal function with in vitro preparations, and E2 was shown to elicit a high contractile response in tubal musculature Lindblom et al., 1980). This is believed to be via the potentiation of the activation of adrenoceptors (Hershlag et al., 1989). Although the study group showed a significantly higher peak E2 level, this is not a prerequisite for developing an ectopic as we have observed two ectopics after the transfer of cryopreserved pre-embryos during a natural cycle when E2 levels were relatively low. Standard protocols were used for controlled ovarian hyperstimulation, oocyte retrieval and embryo transfer. The volume of culture medium used at transfer (70 /xl in most cases) was routine. All transfers were classified as 'smooth'. Thus, none of these parameters was useful in predicting the risk of ectopic pregnancy. Follow-up care of the patients was usually provided by their referring physicians. It is of some interest that the tube ruptured in 13 patients despite follow-up with serial quantitation of HCG values. Often this was due to reluctance to intervene with a 'precious' pregnancy until the diagnosis was 'confirmed'. In some cases (especially the heterotopic pregnancies), rupture was due to failure of diagnosis because an intrauterine sac had been visualized and normally rising titres of HCG were observed. Confino et al. (1986) reported that a single HCG value (50 mlU/ml) on day 9 after embryo transfer differentiates a normal from an abnormal pregnancy. We could not confirm this finding. It is our experience that normal pregnancies sometimes have an HCG value < 50 mlU/ml on day 9 after embryo transfer. Previous reports have analysed the predictive value of serial HCG titres in diagnosing IVF ectopics (Confino et al., 1986; Okamoto et al., 1987). Although titres below the 25th percentile are fairly accurate, they are often misleading in cases of heterotopic or twin tubal pregnancies (Dor et al., 1984; Sondheimer et al., 1985). This finding emphasizes the importance of a high clinical suspicion in diagnosing these cases. Progesterone (P) levels have been used to differentiate normal
Ectopic pregnancy and IVF/ET from abnormal pregnancies (Confino et al., 1986). Since we routinely supplement our patients with P post-transfer, we could not analyse the predictive value of P. The data from patients who returned for subsequent IVF attempts are encouraging and suggest that they have a good prognosis for subsequent pregnancy. Several attempts have been made to prevent IVF ectopic pregnancies. Steptoe et al. (1976) suggested bilateral salpingectomy; however, in this study as well as in others (Pavi et al., 1986), interstitial pregnancies occurred in patients without tubes. Yovich et al. (1985) used uterine length measured by ultrasound to guide placement of the pre-embryos and a transferred volume of < 50 id. Neither of these measures was effective. We conclude that IVF patients with a history of ectopic pregnancy are at risk for another ectopic pregnancy. Their stimulation characteristics are similar to those of patients developing intrauterine pregnancies except for higher peak E2 levels. Patients who develop ectopic pregnancies during IVF, however, have a good chance of achieving a normal pregnancy with subsequent IVF attempts and should be counselled accordingly. References Cohen.J., Mayaux,M.J., Guihard-Moscato,M.L. and Schwartz,D. (1986) In-vitro fertilization and embryo transfer: a collaborative study of 1163 pregnancies on the incidence and risk factors of ectopic pregnancies. Hum. Reprod., 1, 255-258. CohenJ., Mayaux,M.J. and Guihard-Moscato,M.L. (1988) Pregnancy outcomes after in vitro fertilization: a collaborative study on 2342 pregnancies. Ann. N.Y. Acad. Sci., 541, 1—6. Confino,E., Demir,R.H., Friberg,J. and Gleicher,N. (1986) The predictive value of hCG-|3 subunit levels in pregnancies achieved by in vitro fertilization and embryo transfer: an international collaborative study. Fertil. SteriL, 45, 526-531. Correy,J.F., Watkins.R.A., Bradfield.G.F., Gamer.S., Watson,S. and Gray,G. (1988) Spontaneous pregnancies and pregnancies as a result of treatment on an in vitro fertilization program terminating in ectopic pregnancies or spontaneous abortions. Fertil. SteriL, 50, 85-88. Dor.J., Rudak,E., Mashiach,S., Goldman.B. and Nebel.L. (1984) Unilateral tubal twin pregnancy following in vitro fertilization and embryo transfer. Fertil. SteriL, 42, 297-299. FloodJ.T., Muasher.S.J., Simmonetti.S., Kriener.D., Acosta.A.A. and Rosenwaks,Z. (1989) Comparison between laparoscopically and ultrasonographically guided transvaginal follicular aspiration methods in an in vitro fertilization program in the same patients using the same stimulation protocol. J. In Vitro FertiL Embryo Transfer, 6, 180—185. Garcia.J.E. (1986) Conceptus transfer. In Jones.H.W. Jr, Jones.G.S., Hodgen.G.D. and Rosenwaks.Z. (eds), In Vitro Fertilization-Norfolk. Williams and Wilkins, Baltimore, MD, p.215. Hershlag.A., Diamond.M.P. and DeChemey.A.H. (1989) Tubal physiology: an appraisal. J. Gynecol. Surg., 5, 3 - 2 5 . Jones,H.W.,Jr, Acosta.A.A. and Garcia.J.E. (1982) A technique for the aspiration of oocytes from human ovarian follicles. Fertil. SteriL, 37, 26-29. Lindblom.B., Hamburger,L. and Ljung,B. (1980) Contractile patterns of isolated oviductal smoodi muscles under different hormonal conditions. Fertil. SteriL, 33, 283-288. Martinez,F. and Trounson.A. (1986) An analysis of factors associated with ectopic pregnancy in a human in vitro fertilization program. Fertil. SteriL, 45, 79-87. Mishell,D.R.,Jr (1987) Ectopic pregnancy. In Droegemueller.W., Herbst.A.L., Mishell,D.R.,Jr. and Stenchever.M.A. (eds),
Comprehensive Gynecology. C.V. Mosby Company, St Louis, MO., p.406. Okamoto.S.H., Healy.D.L., Morrow,L.M., Rogers.P.A., Trounson.A.O. and Wood.E.C. (1987) Predictive value of plasma human chorionic gonadotrophin /3 subunit in diagnosing ectopic pregnancy after in vitro fertilization and embryo transfer. Br. Med. J., 294, 667-670. Pavi,C.N., Neuenschwander.E. and Gschwind.C. (1986) Interstitial pregnancy following bilateral salpingectomy and in vitro fertilizationembryo transfer. FertiL SteriL, 46, 701-702. Peek.J.C, Clark.A. and Graham.F.M. (1985) Conception rates after IVF in women with previous ectopic pregnancy. Getter) Lancet, U, 275. Rosenwaks.Z. and Muasher,S.J. (1986) Recruitment of fertilizable eggs. In Jones.H.W. Jr, Jones,G.S., Hodgen.G.D. and Rosenwaks.Z. (eds), In Vitro Fertilization-Norfolk. Williams and Wilkins, Baltimore, MD, p.30. Seppala.M. (1985) The world collaborative report on in vitro fertilization and embryo transfer: current state of the art in January, 1984. Ann. N.Y. Acad. Sci., 442, 558-566. Sondheimer.S.J., Tureck,R.W., Blasco.L., StraussJ.in, Arger.P. and. Mennuti.M. (1985) Simultaneous ectopic pregnancy with intrauterine twin gestations after in vitro fertilization and embryo transfer. Fertil. SteriL, 43, 313-316. Steptoe,P.C. and Edwards,R.G. (1976) Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet, i, 880-882. Veeck.L.L., Worthan,J.W.E.,Jr, Witmyer.J., Sandow,B.A., Acosta.A.A., Garcia.J.E., Jones.G.S. and Jones.H.W.,Jr (1983) Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil. SteriL, 39, 594-602. Veeck.L.L. (1986) Atlas of the Human Oocyte and Early Conceptus. Williams and Wilkins, Baltimore, MD. Yovich.J.L., Turner.S.R. and Murphy.A.J. (1985) Embryo transfer technique as a cause of ectopic pregnancies in in vitro fertilization. Fertil. SteriL, 44, 318-321. Received on June 8, 1990; accepted on November 15, 1990 •
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