Frozen-thawed embryo transfer is associated with a significantly reduced incidence of ectopic pregnancy

ORIGINAL ARTICLES: EARLY PREGNANCY Frozen-thawed embryo transfer is associated with a significantly reduced incidence of ectopic pregnancy Bruce S. Shapiro, M.D., Ph.D., a,b Said T. Daneshmand, M.D., a,b Laura De Leon, M.D., b Forest C. Garner, M.Sc., a,b Martha Aguirre, Ph.D., a and Cynthia Hudson, M.S. a a Fertility Center of Las Vegas and b Department of Obstetrics and Gynecology, University of Nevada School of Medicine, Las Vegas, Nevada Objective: To compare the incidence of ectopic pregnancy (EP) after fresh ET and thawed ET. Design: Retrospective cohort study. Setting: Private fertility center. Patient(s): This retrospective study included 2,150 blastocyst transfers, including all 1,460 fresh autologous blastocyst transfers and all 690 transfers of autologous blastocysts derived from post-thaw extended culture of thawed bipronuclear oocytes in the 8-year study period 2004 2011. Intervention(s): None. Main Outcome Measure(s): Visualized EP and treated persistent pregnancy of unknown location. Result(s): The rate of visualized EP was 1.5% in pregnancies in fresh autologous cycles, which was significantly more than the rate of 0 with autologous post-thaw extended culture. The rates of treated persistent pregnancy of unknown location were 2.5% and 0.3% in these two groups, respectively, a difference that was also statistically significant (relative risk 7.3, 95% confidence interval 1.7 31.0). Conclusion(s): Relative to fresh transfer, thawed ET was associated with significantly reduced incidence of EP. These findings are consistent with ovarian stimulation increasing the risk of EP. (Fertil Steril Ò 2012;98:1490 4. Ó2012 by American Society for Reproductive Medicine.) Key Words: Ectopic pregnancy, assisted reproduction, ovarian stimulation, embryo cryopreservation Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/shapirobs-embryo-cryopreservation-ectopic-pregnancy/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Ectopic pregnancy (EP) is approximately twice as frequent in pregnancies from assisted reproductive technologies (ART) as in spontaneous pregnancies (1). Reported rates of tubal EPs in ART range from 2% 5% (2, 3). Multiple hypotheses have been offered to explain the increased risk of EP in ART. These include specific attributes of ART patients, such as tubal Received June 19, 2012; revised July 17, 2012; accepted July 25, 2012; published online August 25, 2012. B.S.S. reports research grants from Merck Sharp & Dohme Inc., Ferring Pharmaceuticals Inc., Watson Pharmaceuticals Inc., and payment for lectures from Merck Sharp & Dohme Inc. (not related to this work). He also reports a consultancy with Cooper Surgical (not related to this work. S.T.D. reports research grants from Merck Sharp & Dohme Inc., Ferring Pharmaceuticals Inc., and Watson Pharmaceuticals Inc. (not related to this work). L.D.L. has nothing to disclose. F.C.G. reports research grants from Merck Sharp & Dohme Inc., Ferring Pharmaceuticals Inc., and Watson Pharmaceuticals Inc. (not related to this work). M.A. reports research grants from Merck Sharp & Dohme Inc., Ferring Pharmaceuticals Inc., and Watson Pharmaceuticals Inc. (not related to this work). C.H. reports research grants from Merck Sharp & Dohme Inc., Ferring Pharmaceuticals Inc., and Watson Pharmaceuticals Inc. (not related to this work). Reprint requests: Bruce S. Shapiro, M.D., Ph.D., Fertility Center of Las Vegas, 8851 West Sahara Avenue, Las Vegas, NV 89117 (E-mail: bsshapiro@aol.com). Fertility and Sterility Vol. 98, No. 6, December 2012 0015-0282/$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1136 disease and other infertility diagnoses (3 5). Other hypothesized causes include the use of ovarian stimulation (6), resulting in increased uterine contractions secondary to elevated E 2 levels (7, 8), or ciliary dysfunction secondary to elevated P levels (9). Tobacco use has also been correlated with EP after IVF (10). A large study of ART registry data from US clinics in 1999 2001 found the rates of EP were reduced in fresh oocyte donation cycles, but not in cycles of frozen-thawed ET, when compared with fresh autologous cycles, and generally concluded that embryos with high implantation potential were protective against EP (5). That study also associated increased EP 1490 VOL. 98 NO. 6 / DECEMBER 2012

Fertility and Sterility incidence with diagnoses of tubal factor, uterine factor, endometriosis, diminished ovarian reserve, and unexplained infertility, when compared with a reference group with male factor diagnosis. A subsequent registry study using data from 2008 compared single ETs and associated frozen-thawed ET with decreased risk of EP when compared with fresh autologous transfer, concluding that a negative effect of ovarian stimulation on endometrial receptivity was reflected in increased EP rates in fresh autologous cycles (11). These two registry studies failed to reach consensus on whether EP incidence was reduced in frozen-thawed ET cycles. Autologous blastocysts derived from post-thaw extended culture (PTEC) of thawed bipronuclear oocytes have been found to have implantation potential comparable with fresh blastocysts transferred in oocyte donation cycles (12). Two prospective trials found implantation rates of 67% and 71% in autologous PTEC cycles (13, 14). This technique has been used for other purposes, including reducing ovarian hyperstimulation syndrome (OHSS) risk (15, 16). The current study compares the rate of EP in autologous PTEC cycles to that after fresh autologous blastocyst transfer to determine whether PTEC is associated with different EP risk than fresh autologous IVF. This might help to resolve the lack of consensus on whether EP incidence may be reduced through frozen-thawed ET. MATERIALS AND METHODS This study included all fresh autologous blastocyst transfers and all transfers of blastocysts derived from thawed bipronuclear oocytes in the 8-year study period ending December 31, 2011. Cycles using gestational carriers or donated oocytes were excluded. Patients underwent ovarian stimulation with gonadotropins (FSH or hmg, typically in combination). Pituitary suppression was routinely achieved with GnRH antagonist. When at least half of those follicles with a mean diameter R12 mm had reached 16 mm in mean diameter, the patient was given hcg (typically 2,500 10,000 IU) or GnRH agonist (GnRH-a; 4 mg leuprolide acetate [LA]), or both in combination (4 mg LA with concomitant hcg, typically 11 33 IU per kg of body weight) for final oocyte maturation, followed approximately 35 hours later by oocyte retrieval. Oocytes were inseminated only by intracytoplasmic sperm injection (ICSI), and resulting embryos were cultured to the blastocyst stage in sequential media, as described previously (13). Blastocysts were transferred by ultrasound guidance on day 5, 6, or, rarely, on day 7 of embryo development, depending on the day of blastocyst expansion. The ICSI and blastocyst transfer were standards of care for all patients at this center throughout the study period. Bipronuclear oocytes were cryopreserved and thawed using conventional slow freezing, as described previously (13). Patients were offered the option of cohort cryopreservation based on premature luteinization, history of implantation failure after fresh transfer, or as participants in randomized trials (13, 14). Before transfer, patients received E 2 supplements (oral pills [Estrace Warner Chilcott] 6.0 mg daily and patches as needed) starting 10 14 days before thaw. Daily P injections (usually 100 mg) were started the day before thaw. After thaw, embryo culture and blastocyst transfer were as described for fresh transfers. Before transfer, the diameter of each blastocyst was measured along its longest axis through an ocular micrometer. Inner cell masses (ICMs) were measured along their longest axis and along its widest perpendicular, and these two measures were multiplied to estimate the cross-sectional area. The trophectoderm was measured by counting cells along an equator in one plane of focus. Pregnancy was established through subsequent serially increasing serum titers of hcg measured to day 10 after blastocyst transfer, with subsequent measurements every 2 4 days in pregnant patients until the first ultrasound examination, and approximately weekly thereafter. Pregnant patients typically received three to four serial ultrasound examinations commencing at 5 6 weeks' gestation to confirm intrauterine gestation, examine the adnexa, and follow subsequent development. Those with abnormally increasing hcg serum titers were considered suggestive of potential EP. These received more frequent ultrasound examinations in the 5 6 week time frame, often at 48-hour intervals, until the location of the pregnancy was visualized or hcg titers began to resolve. Luteal support in all cycles included E 2 and P supplements sufficient to sustain serum levels of 200 pg/ml and 15 ng/ml, respectively, as previously described (13). Visualized EP (VEP) was defined by a pregnancy accompanied by sonographic visualization of an extrauterine gestational sac (including any heterotopic gestations). Treated persistent pregnancy of unknown location (TPPUL) was defined by persistent abnormally increasing hcg titers without sonographic visualization, which were treated with methotrexate (17). Clinical pregnancy was defined based on observed fetal heart motion during sonographic examination and multiple pregnancy was based on the maximum number of fetal hearts. Ongoing pregnancy was defined as viable fetal heart motion at approximately 10 weeks' gestation, whereas spontaneous abortions were clinical pregnancies that did not achieve ongoing pregnancy. Fisher's exact test was used to compare nominal variables. After applying the Shapiro-Wilk test for normality, the Wilcoxon test was used to compare numeric variables if significant deviation from the normal distribution was observed. P values less than.05 were considered significant. The JMP version 5.01 (SAS Institute, Inc.) was used for these statistical analyses. The JMP 7.02 (SAS Institute, Inc.) was used to examine risk ratios. Institutional Review Board approval was obtained for this retrospective study. RESULTS There were 1,460 fresh autologous blastocyst transfers and 690 autologous transfers of blastocysts derived from thawed bipronuclear oocytes in the study period. The characteristics of these cycles are described in Table 1. When compared with those in the fresh transfer group, women in the PTEC group were significantly older at ET, older at oocyte retrieval, and more likely to be diagnosed with diminished ovarian reserve. No significant differences were observed in body mass index (BMI), the incidence of tubal factor, VOL. 98 NO. 6 / DECEMBER 2012 1491

ORIGINAL ARTICLE: EARLY PREGNANCY TABLE 1 Demographics and potential confounders in the two study groups. Fresh transfer PTEC P value Blastocyst transfers 1,460 690 Age at transfer (y) 33.3 4.8 a 34.1 4.7 a.0001 Age at retrieval (y) 33.3 4.8 a 33.8 4.7 a.0087 Age range at retrieval (y) 20 46 22 45 Body mass index (kg/m 2 ) 25.5 5.9 a 25.3 5.6 a.7447 Tubal factor diagnosis (%) 320 (21.9) 161 (23.3).4714 Endometriosis (%) 74 (5.1) 42 (6.1).3575 Diminished ovarian reserve (%) 443 (30.3) 275 (39.9) <.0001 Unexplained infertility (%) 89 (6.1) 56 (8.1).0969 Tobacco use (%) 240 (16.9) 108 (16.7) 1.0000 Blastocysts transferred 1.88 0.51 a 1.87 0.50 a.5822 Note: Values are mean SD, range, or frequency (percentage). PTEC ¼ post-thaw extended culture. a Numeric variable not normally distributed per Shapiro-Wilk test (P<.0001). unexplained infertility, tobacco use, or the number of blastocysts transferred. Embryo morphology is compared in Table 2. An overall c 2 test found that ET day was not independent of the type of cycle (fresh vs. PTEC) (P<.0001). Compared with fresh transfers, the PTEC transfers were more likely to occur on day 6 or day 7 and less likely on day 5. Significantly larger mean ICM was observed for the fresh transfers on day 5 and day 6. Significantly greater mean embryo diameter and mean trophectoderm cell count were observed for the fresh transfers on day 5. All significant differences in blastocyst morphology favored the fresh transfer group. Outcomes are described in Table 3. There were significant differences in the rates of pregnancy, implantation, ongoing pregnancy, TPPUL, and VEP, but not in the rates of multiple pregnancy or spontaneous abortion. Pregnancies resulting from fresh autologous blastocyst transfers were significantly more likely to result in TPPUL or VEP when compared with those resulting from autologous PTEC. Among pregnant patients without tubal factor diagnosis, the incidence of TPPUL was 2.5% (18/725) in fresh cycles and 0.2% (1/433) in the PTEC group (P¼.0029). Among pregnant patients with tubal factor diagnosis, these rates were 2.7% (5/ 184) and 0.7% (1/147), respectively (P¼.2321). Among pregnant patients without tubal factor diagnosis, the incidence of VEP was 1.9% (14/725) in fresh autologous cycles, and 0 (0/433) in PTEC cycles (P¼.0016). Among pregnant patients with tubal factor diagnosis, there were no VEPs. DISCUSSION These findings corroborate the conclusion of Clayton et al. (5) that high implantation potential is protective against EP. Autologous PTEC cycles have had reported implantation rates of 67% and 71% in prospective studies (13, 14) in first-time IVF patients <41 years of age and the implantation rate after autologous PTEC is comparable to that of fresh oocyte donation cycles when both methods use oocytes from young patients (12). In the present study, which was not restricted on patient age or history, the implantation rate in PTEC cycles was 51.2%, which significantly exceeded the 31.8% implantation rate in the fresh cycles, despite numerous significantly superior morphological embryo parameters in the fresh transfer group. The PTEC allows 4 5 days to confirm resumed TABLE 2 Embryology parameters according to the day of blastocyst transfer. Fresh transfer PTEC P value Day 5 transfers (%) 785 (53.8) 219 (31.7) <.0001 Mean ICM area, day 5 (mm 2 ) 4,582 1,228 4,309 1,280.0038 Mean embryo diameter, day 5 (mm) 186.0 14.7 183.7 17.9.0002 Mean trophectoderm cells, day 5 14.3 3.3 13.4 2.9.0002 Day 6 transfers 663 (45.4) 433 (62.8) <.0001 Mean ICM area, day 6 (mm 2 ) 4,221 1,510 3,982 1,411.0148 Mean embryo diameter, day 6 (mm) 197.4 24.7 195.8 22.2.1997 Mean trophectoderm cells, day 6 15.1 4.5 14.8 4.7.3252 Day 7 transfers 12 (0.8) 38 (5.5) <.0001 Mean ICM area, day 7 (mm 2 ) 3,441 1,173 3,247 1,503.5477 Mean embryo diameter, day 7 (mm) 192.7 13.8 206.3 26.4.1833 Mean trophectoderm cells, day 7 14.5 4.0 15.6 5.5.6671 Note: Values are mean SD or frequency (percentage). ICM ¼ inner cell mass; PTEC ¼ post-thaw extended culture. 1492 VOL. 98 NO. 6 / DECEMBER 2012

Fertility and Sterility TABLE 3 Outcomes in the two study groups. Fresh transfer PTEC P value Relative risk (95% CI) Pregnancies 909 580 Pregnancy rate (%) 62.3 84.1 <.0001 TPPULs 23 2 TPPUL/pregnancy (%) 2.5 0.3.0007 7.3 (1.7 31.0) Visualized ectopic pregnancies (VEP) 14 0 VEP/pregnancy (%) 1.5 0.0014 Undefined Clinical pregnancies 627 465 Fetal hearts 876 662 Implantation rate 31.8% 51.2%.0001 Multiple pregnancies 239 188 Multiple pregnancy rate 38.1% 40.4%.4521 Spontaneous abortions 45 40 Spontaneous abortion rate 7.2% 8.6%.4244 Ongoing pregnancies 582 425 Ongoing pregnancy rate 39.9% 61.6%.0001 Note: PTEC ¼ post-thaw extended culture; TPPUL ¼ treated persistent pregnancy of unknown location. development to the blastocyst stage. This confirmed development is a more stringent criterion than the usual practice of using only immediate post-thaw survival alone, because many embryos that appear to survive thaw cease development afterward (18, 19). By transferring a developing embryo into a uterine environment that is not compromised by ovarian stimulation, PTEC achieves conditions that approximate the fresh oocyte donation cycle. These findings also support the conclusions of Ishihara et al. (11) who compared fresh and frozen-thawed ET cycles in a national registry study and concluded a negative effect of ovarian stimulation on endometrial receptivity was reflected by increased EP rates. However, another study (20) found no significant difference in ectopic rates between fresh and thawed blastocyst transfer. There were some potential confounders in this retrospective cohort study. Despite the fresh transfer group being associated with some factors that would suggest greater implantation potential (slightly younger, less likely to be diagnosed with diminished ovarian reserve, more day 5 blastocysts, and superior embryo morphology on day 5 and day 6), that group was still associated with greater rates of VEP and TPPUL and lower rates of pregnancy, implantation, and ongoing pregnancy. The greater rates of pregnancy, implantation, and ongoing pregnancy in the PTEC group have been previously attributed to superior endometrial receptivity, absent the effects of ovarian stimulation (13). The incidence of tubal factor diagnosis did not differ significantly between the two groups, and little evidence was found of a relationship between tubal factor diagnosis and EP in these results. Limitations of this study include its retrospective nature, its modest size relative to registry studies, and its use of less common methodologies. The degree of reduction in EP risk through frozen-thawed ET relative to fresh transfer may be protocol-specific. The methodology in the present study, involving post-thaw extended culture after routine use of GnRH antagonist cycles, ICSI insemination, and alternative triggering is not the current routine at most centers, and the rates of EP observed here might have been influenced by these protocol choices. The high implantation potential that is protective against EP (5) requires favorable endometrial receptivity and endometrial receptivity can be compromised by ovarian stimulation (13). Previous reports have associated frozen-thawed ET with reduced risk of EP when compared to fresh ET (11, 21). The rates of EP with frozen-thawed ET in the present study were nominally lower than in those prior studies (0.3% for TPPULs and 0 for VEPs in the present study, compared to 0.8% [11] and 1.2% [21], each for EP ). 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