Are implantation and pregnancy outcome impaired in diethylstilbestrol-exposed women after in vitro fertilization and embryo transfer~*

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1 FERTILITY AND STERILITY Copyright The American Fertility Society Printed on acid-free paper in U.S.A. Are implantation and pregnancy outcome impaired in diethylstilbestrol-exposed women after in vitro fertilization and embryo transfer~* Vishvanath C. Karande, M.D. t+ Richard G. Lester, M.D. Suheil J. Muasher, M.D. t Debra L. Jonest Anibal A. Acosta, M.D.t Howard W. Jones, Jr., M.D.t The Howard and Georgeanna Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Forty-six infertile women with a history of diethylstilbestrol (DES) exposure in utero underwent 149 stimulation attempts for in vitro fertilization (IVF). The mean (±SE) number of preovulatory oocytes harvested at retrieval and transferred was 3.9 ± 3.3 (536/ 138) and 2.6 ± 1.4 (328/124). When compared with patients with tubal factor infertility and ~4 pre-embryos transferred, the clinical pregnancy rate (15.3% versus 22%) was not statistically different. However, the term/ongoing pregnancy rate was significantly lower in the study group (8% versus 16%). Comparison of the IVF outcome with different uterine anomalies as detected by hysterosalpingogram (n = 29) showed a trend for a worse prognosis in women with constrictions and a combination of T -shape and constrictions. It is concluded that implantation and pregnancy outcome are impaired in DES-exposed women after IVF. Fertil SteriI54:287, 1990 Diethylstilbestrol (DES) exposure in utero is associated with an adverse reproductive outcome, including spontaneous abortions, tubal pregnancies, and premature labor. 1 The data regarding the fertility rates in DES-exposed women, however, are still conflicting. Whereas Herbst et al. 2 found an increased incidence of infertility, Cousins et al.a and Barnes et al. 4 did not. Kaufman et al. 5 reported specific changes in the contour of the uterine cavity Received January 12, 1990; revised and accepted April 19, * Presented in part at the 38th Annual Meeting of The American College of Obstetricians and Gynecologists, San Francisco, California, May 7 to 10, t The Howard and Georgeanna Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia. :j: Reprint requests: Vishvanath C. Karande, M.D., The Howard and Georgeanna Jones Institute for Reproductive Medicine, 825 Fairfax Avenue, 6th Floor, Norfolk, Virginia Department of Radiology, Eastern Virginia Medical School. in these women. In a subsequent study6 they compared the hysterosalpingogram (HSG) appearance of the uterine cavity with the incidence of infertility; the presence of an abnormal HSG was not a factor in infertility. However, when specific abnormalities were evaluated, an increased difficulty with conceiving was noted in women with constriction of the upper uterine cavity and a combination of a T -shaped uterus with constriction of the upper uterine cavity. The study lacked details of the infertility evaluation and the subsequent outcome of the pregnancies; therefore the impact of the uterine anomalies on implantation and the ongoing pregnancy rate is still unclear. We have reported our in vitro fertilization (IVF) results in 17 DES-exposed infertile women. 7 The preliminary data (pregnancy rate of 23.5% per patient or 19% per embryo transfer) were encouraging. In this study, the experience with a larger group of patients is reported. To analyze the functional efficiency ofthe uteri, we further divided the Karande et al. IVF outcome in DES-exposed women 287

2 patients into groups based on their HSG appearance, and the IVF outcomes for the different groups were compared. MATERIALS AND METHODS All patients with a history of DES exposure seen in the Norfolk program from January 1981 to July 1989 were included in the study group. All had ovulatory menstrual cycles, as judged by basal body temperature charts, timed endometrial biopsies, and/or midluteal phase serum progesterone determinations. The history of DES exposure in utero was either volunteered by the patient and/or confirmed by the patient's mother or her physician's records after the patient's HSG or pelvic examination revealed abnormalities associated with DES exposure in utero. Twenty-nine available HSGs were assessed independently by a radiologist (R.G.L.), and the IVF outcomes for the groups were compared. The HSGs were classified as T -shaped, hypoplastic, wide lower segment, constrictions, irregular margins, and combination of T -shaped with constrictions, as described by Kaufman et a1. 6 (Fig. 1). All patients underwent pure gonadotropin stimulation and monitoring by established protocols.8 Oocyte retrieval was performed laparoscopically9 or transvaginally by published techniques. 1o Preincubation of oocytes, sperm preparation, culturing conditions and techniques, embryology laboratory procedures, and embryo transfer (ET) apparatus and technique were employed as describedy-13. In selecting a control group, we needed patients similar to the study group except for the shape of the uterine cavity. Since most of the study patients had tubal factor infertility, all tubal factor patients during the study period who had a similar number of pre-embryos transferred (sa) served as controls. They were of the same age as the study group and underwent IVF at the same time. The technique of stimulation, retrieval, and transfer was also similar. The data of the pregnancy outcome in the study group and the controls were further analyzed, and only transfer cycles with :s;;4 pre-embryos developing from preovulatory oocytes were considered. The pregnancy rates for each group were compared with the number of pre-embryos transferred. The implantation rate was compared by analyzing the number of sacs visualized by ultrasound (US) (6 weeks after transfer) and the ongoing/term preg- 288 Karande et al. IVF outcome in DES-exposed women Figure 1 Hysterosalpingogram appearance of diethylstilbestrol-exposed women: (A) T-shaped; (B) Hypoplastic; (C) Wide lower segment; (D) Cornual constrictions; (E) Irregular margins; (F) Combination of constriction and T-shaped uterus. nancy rate in each group. Preclinical pregnancies were not included in any analyses. Statistical analysis was done using either a Student's t-test, a Fisher's exact test, or a x2 analysis, where appropriate. A P-value of <0.05 was considered significant. RESULTS From January 1981 to July 1989, 46 patients with a history of DES exposure in utero underwent 149 stimulation attempts for IVF (range 1 to 17 attempts). The mean (±SD) age of the patients was 32.7 ± 2.8 years (range 26 to 41 years). The mean Fertility and Sterility

3 duration of infertility was 4.6 ± 2.7 years (range 1 to 15 years). Nineteen patients had primary infertility. Of the 27 patients with secondary infertility, 24 had a history of ~ 1 ectopic pregnancy. Only 2 patients had a previous term pregnancy (1 had a baby with Potter's syndrome), and 1 had a premature delivery at 24 weeks. Eight patients had previous first trimester miscarriages. The etiology of infertility was pure tubal factor in 32 patients, endometriosis in 4, tubal plus endometriosis in 1, male factor in 1, hostile cervical mucus in 3, and idiopathic in 5. On examination, 31 patients had structural abnormalities of the upper vagina and cervix (shallow or absent fornices of the vagina, cervical hood, collar, etc.). The control group (n = 1,685) consisted of all patients with tubal factor infertility who underwent IVF during the study period and had ~4 pre-embryos transferred. Their mean age was 34.2 ± 3.6 years (not significantly different from the study group). There were no significant differences (P> 0.05) in the mean number of preovulatory oocytes retrieved (3.9 ± 3.3 [536/138] in the study group versus 3.5 ± 2.8 [5,945/1,685] in the controls) or transferred (2.6 ± 1.4 [328/124] in the study group versus 2.4 ± 1.1 [4,020/1,685] in the controls). There were 19 clinical pregnancies in the study group. Of these, 3 were ectopic (1 was tubal, 1 cervical [requiring a hysterectomy secondary to uncontrollable hemorrhage at 14 weeks], and 1 in a patient with an acute abdomen at 8 weeks; at laparotomy the only abnormal finding was a huge retroperitoneal hematoma with chorionic villi). Five pregnancies resulted in first trimester miscarriages, 8 were delivered at term, and 2 are ongoing. There was 1 preterm delivery at 23 weeks because of an incompetent cervix, and the fetus did not survive. There were no multifetal pregnancies. None of the patients had a cervical cerclage, though some were empirically administered 250 mg of 17-hydroxyprogesterone in oil intramuscularly per week until 30 weeks of gestation. The control group had 373 clinical pregnancies (235 normal term deliveries, 7 abnormal deliveries, 87 clinical miscarriages, 18 ectopics, and 26 ongoing pregnancies) and included 52 (13.9%) multifetal pregnancies. The clinical pregnancy rate of 15.3% (19/124) in the study group was not significantly different (P = 0.06) from the control group at 22% (373/1,685). The term/ongoing pregnancy rate per transfer in the study group of 8% (10/124) was significantly lower (P < 0.02) than the control group 16% (268/1,685). The data of the pregnancy outcome in the study group and the controls were further analyzed, and only transfer cycles with ~4 pre-embryos developing from preovulatory oocytes were considered (Table 1). Eleven cycles from the study group had ~5 pre-embryos transferred (57 pre-embryos transferred with 1 clinical pregnancy) and were excluded from this analysis. In a comparison of the study group with the controls, the clinical pregnancy rate per transfer (15.9% versus 22.1%, P> 0.05) and the clinical miscarriage rate per pregnancy (44.4% versus 28.1 %, P> 0.05) were not statistically different. The ongoing pregnancy rate per transfer (8.8% versus 15.9%, P < 0.05) was significantly lower in the study group. In the study group, a total of 271 pre-embryos were transferred, and 20 sacs were detected in the 19 clinical pregnancies. One patient had a "disappearing twin" and delivered a singleton at term. In the control group, there were 4,020 pre-embryos transferred with 450 sacs detected on US. There were 64 sacs in the 64 patients with clinical pregnancies and 1 pre-embryo transferred, 108 sacs in the 91 pregnant patients with 2 pre-embryos transferred, 131 sacs in the 105 pregnant patients with 3 pre-embryos transferred, and 147 sacs in the 113 pregnant patients with 4 pre-embryos transferred. The implantation rate (number of sacs visualized on US per pre-embryo transferred) (7% versus 11.2%, P < 0.05) was significantly lower in the study group (Table 1). The implantation rate for the study group was even lower (6%) when the 11 cycles with ~5 pre-embryos transferred were included. No statistically significant differences could be noted between the two groups when the data for a similar number of pre-embryos transferred were compared (Table 1). Hysterosalpingograms were available in 29 patients. Twenty-two patients (81 %) had at-shaped' uterus, 4 (15%) had a hypoplastic, 5 (19%) had a wide lower segment, 15 (54%) had constriction (1 upper, 2 middle, 4 lower, and 8 cornual), and 6 patients (22%) had irregular margins (some patients had more than one abnormality). Two patients had a normal uterine cavity (they had lower genital tract abnormalities consistent with DES exposure). Four patients had intrauterine defects that were treated hysteroscopically before the IVF attempt. In comparison of the IVF outcome with HSG appearance of the uterus, there were no significant differences (P> 0.05) among the groups Karande et ai. IVF outcome in DES-exposed women 289

4 Table 1 Comparison of DES-Exposed Patients With s4 Pre-embryos Transferred and Controls No. of Clinical pregnancy Ongoing pregnancy Miscarriage rate Implantation rate pre-embryos No. of transfers per transfer per transfer per pregnancyb Sacs per embryo transferred 1 30 (455) 3.3 (14)C 2 30 (482) 23.3 (18.8)C 3 31 (391) 16.1 (27)C 4 22 (357) 22.7 (31.6) C Total 113 (1,685) 15.9 (22.1)C Eleven cycles with ;;0:5 pre-embryos transferred were excluded. b Includes ectopic pregnancies. C No significant difference when study group compared with controls; P > % % % % 0 (10.3)C 100 (26.6)C 3.3 (14)< 16.7 (13)C 28.6 (30.8)C 11.7 (11.2)C 9.7 (20.5)C 50 (23.8)C 5.3 (11.2)C 9.1 (21.8)C 40 (31)C 6.8 (10.3)C 8.8 (15.9)d 44.4 (28.1)C 7 (11.2)d d Statistically significant difference (P < 0.05) when study group compared with controls. (Table 2). However, there was a definite trend for patients with a hypoplastic uterus (none of which was T -shaped) to do better than the overall group (Table 2). On the other hand, patients with constrictions and a combination of at-shape with constriction did worse than others in spite of a similar number of preovulatory oocytes transferred (Table 2). The 2 patients with normal uteri underwent 3 IVF attempts, and no pregnancy resulted. One patient (tubal factor infertility) underwent 17 stimulations and 9 ETs. Of her 3 resultant pregnancies, 1 was ectopic, and 2 were first trimester miscarriages. She had a combination of a T -shaped uterus and a lower uterine constriction. DISCUSSION Between the late 1940s and 1971, an estimated 2 to 3 million women were prescribed DES during their pregnancies, thereby exposing to the drug in utero approximately 3 million progeny.1 Women from this population, now of reproductive age, often develop tubal factor infertility due to their propensity for tubal pregnancies. 1 Their performances during IVF are therefore a matter of interest and importance. Most of the women (81 %) with DES exposure in utero achieve at least one successful pregnancy.14 Our study group consisted of patients with primary infertility or a poor reproductive history (miscarriages, ectopic pregnancy, preterm delivery) and therefore represents only a subset of the overall population. The etiology of infertility in the study group was predominantly a tubal factor, and 24 of the 27 patients with secondary infertility had a history of at least one ectopic pregnancy. The mean number of preovulatory oocytes retrieved was similar to the control group, indicating that ovarian response to stimulation was not compromised in this group. The number of pre-embryos transferred also was similar. The clinical pregnancy rate per transfer (15.3% versus 22%) was almost statistically significant (P = 0.06), and the ongoing pregnancy rate per transfer (7.5% versus 16%) was significantly lower (P < 0.02) in the study group. This suggests that the abnormal shape negatively impacts on the functional efficiency of the uterus. To the best of our knowledge, Table 2 Correlation Between the Hysterosalpingogram Appearance of the Uterus and IVF Outcome (n = 27) Abnormality T-shaped Hypoplastic WideLSb Constrictions Irregular margins Combination C No. of patients 22 4 No. of attempts Pre-ovulatory oocytes per transfer Clinical pregnancies per transfer (%) Ongoing pregnancy rate (%) Seen singly or in combination with other abnormalities. b Lower uterine segment C Combination of T -shaped and constriction. 290 Karande et al. IVF outcome in DES-exposed women Fertility and Sterility

5 this is the first report of a significantly reduced ongoing pregnancy rate with IVF in DES-exposed women and is different from our initial experience with this group.s Two patients underwent a total of three IVF attempts and did not achieve a pregnancy in spite of having normally shaped uterine cavities. It is difficult to attach any significance to this finding because of the small number involved. The HSGs were categorized into groups by an experienced radiologist to determine whether a particular abnormality had a worse prognosis. Although the differences in outcome among the groups did not achieve statistical significance when compared with the overall study group, some definite trends were noted. The hypoplastic uteri did better; the uteri with constrictions, especially when combined with a T -shape, did worse. To compare implantation rates and ongoing pregnancy rates, it was necessary to compare only those cycles with pre-embryos developing from preovulatory oocytes. Of the 124 such transfer cycles in the study group, 11 had >4 pre-embryos transferred (with 1 resultant pregnancy) and were not included. Although the clinical pregnancy rates were not different, the ongoing pregnancy rate per transfer was significantly lower (P < 0.05) in the study group. There was a high clinical miscarriage rate in the study group (44.4%). That the miscarriage rates were not statistically different is probably because of the small number of miscarriages in the study group. The implantation rate was significantly lower (P < 0.05) in the study group (7% versus 11 %) and even lower (6 %) if all cycles were included. When the data for the two groups were compared with a similar number of pre-embryos transferred, the study group consistently tended to do worse, although the differences were not statistically significant. We conclude that implantation and pregnancy outcome are impaired in DES-exposed women after IVF. Certain radiological abnormalities of the uterine cavity (constrictions, combination of T shape with constriction) tend to do worse, whereas others (hypoplastic) do better. Patients should be counseled accordingly. REFERENCES 1. Stillman RJ: In utero exposure to diethylstilbestrol: adverse effects on the reproductive tract and reproductive performance in male and female offspring. Am J Obstet GynecoI142:905, Herbst AL, Hubby MM, Blough RR, Azizi F: A comparison of pregnancy experience in DES-exposed and DES-unexposed daughters. J Reprod Med 24:62, Cousins L, Karp W, Lacey C, Lucas WE: Reproductive outcome of women exposed to diethylstilbestrol in utero. Obstet GynecoI56:70, Barnes AB, Colton T, Gunderson J, Noller KL, Tilley BC, Strama T, Townsend DE, Hatab P, O'Brien PC: Fertility and outcome of pregnancy on women exposed in utero to diethylstilbestrol. New Engl J Med 302:609, Kaufman RH, Binder GL, Gray PM, Jr, Adam E: Upper genital tract changes associated with exposure in utero to diethylstilbestrol. Am J Obstet GynecoI128:51, Kaufman RH, Adam E, Noller K, Irwin JF, Gray M: Upper genital tract changes and infertility in diethylstilbestrolexposed women. Am J Obstet GynecoI154:1312, Muasher SJ, Garcia JE, Jones HW, Jr: Experience with diethylstilbestrol-exposed infertile women in a program of in vitro fertilization. Fertil Steril 42:20, Rosenwaks Z, Muasher SJ: Recruitment offertilizable eggs. In In Vitro Fertilization-Norfolk, Edited by HW Jones, Jr, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p Jones HW, Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil SteriI37:26, FloodJT, Muasher SJ, Simonetti S, Kriener D, Acosta AA, Rosenwaks Z: 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 Fert Embryo Transfer 6:180, Veeck LL, Worthan JWE, Jr, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW, Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39:594, Veeck LL: Atlas of the Human Oocyte and Early Conceptus. Baltimore, Williams & Wilkins, Garcia JE: Conceptus transfer. In In Vitro Fertilization Norfolk, Edited by HW Jones, Jr, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p Herbst AL, Hubby MM, Azizi F, Makii MM: Reproductive and gynecologic surgical experience in diethylstilbestrolexposed daughters. Am J Obstet GynecoI141:1019, 1981 Karande et al. IVF outcome in DES-exposed women 291