Nov 27, 1995 - and Embryo Transfer (IVFET). DAN TULCHINSKY, I'~'4 AMIR TULCHINSKY, -~ VIRGINIA PAOLETTI-FALCONE, l HOLLY NASH, t. STEPHEN ...
Journal of Assisted Reproduction and Genetics, Vol. 13, No. 7, 1996
CLINICAL ASSISTED REPRODUCTION
CLINICAL ASSISTED REPRODUCTION Delayed Embryo Implantation Following in Vitro Fertilization and Embryo Transfer (IVFET) DAN TULCHINSKY, I'~'4 AMIR TULCHINSKY, -~ VIRGINIA PAOLETTI-FALCONE, l HOLLY NASH, t STEPHEN PAZDZIORKO, 1 and KENNETH BROWN t
Submitted: November 27, 1995 Accepted: Februao' 14, 1996
It was implied that the use of GnRH-a caused a widening in the implantation window. We have undertaken a study designed to compare hCG levels in pregnancies achieved following IVFET where GnRH-a was used for pituitary down regulation with those achieved following spontaneous pregnancy achieved with and without ovarian stimulation. Because hCG levels depend largely on the normalcy of pregnancy and the number of embryos, only normal singleton pregnancies were included.
Purpose: Our goal was to compare serum human chorionic gonadotropin (hCG) levels in singleton pregnancies achieved following IVFET with those achieved following spontaneous conception. Results: The mean serum hCG level of patients who became pregnant following IVFET lagged 1.5 days behind that of patients who became pregnant spontaneously. Conclusions: The use of gonadotropin releasing hormone attalogue as part of the stimulation protocol leading to egg retrieval and IVFET results in a delay in embryo implantation.
MATERIALS AND METHODS
KEY WORDS: in vitro fertilization; gonadotropin releasing hormone analogue; pregnancy; human chorionic gonadotropin.
Twenty-five women who had become pregnant following IVFET and 48 women who became pregnant either spontaneously or following ovarian stimulation with clomiphene citrate or human menopausal gonadotropin were studied. Only women who had a singleton pregnancy, as determined by ultrasound assessment at 6 weeks of pregnancy, and had a live birth at term were included in this study. The method of ovarian stimulation prior to egg retrieval for IVFET included initial pituitary GnRH-a suppression followed by ovarian stimulation as described by Meldrum et al. (2). In brief, leuprolide acetate (Lupron; TAP Pharmaceuticals, North Chicago, IL) was given at the midluteal phase until ovarian suppression was achieved. Ovarian stimulation with 150 IU of urofollitropin (Metrodin) and 150 IU of human menopausal gonadotropin (hMG; Pergonal Serono Lab) was then started while continuing Lupron administration. After 2 days the Metrodin was discontinued and the dose of Pergonal individually adjusted. Human chorionic gonadotro-
INTRODUCTION It has been reported previously that in pregnancies which resulted from IVFET, the use of gonadotropin releasing hormone analogue (GnRH-a) as part of the protocol for ovarian stimulation resulted in a 1.5-day delay in the rise of human chorionic gonadotropin (hCG) compared to IVF pregnancies achieved after gonadotropin stimulation alone (1). The delay in hCG rise was attributed to a delay in embryo implantation.
Reproductive Endocrinology and Infertility Center, 100 Hospital Road, The Malden Hospital, Malden, Massachusetts 02148. 2 Boston University School of Medicine, Boston, Massachusetts. 3 The University of Connecticut Health Center, Farmington, Connecticut. 4 To whom correspondence should be addressed.
1058-0468/96/08(X)-0536S{)9.50/0© 1996 Plenum Publishing Corpor'~tion
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pin (hCG), 5000 USP (Serono Lab), was administered when estradiol levels of 500 pg/ml or greater were achieved and at least two follicles reached, or exceeded, a size of 16 mm in mean diameter. Egg retrieval was initiated 36 hr after the hCG was administered. Embryo transfer was carried out approximately 48 hr after egg retrieval and 84 hr after hCG administration. Luteal-phase support was achieved by giving either progesterone in oil, 100 mg daily (10 cases), or hCG, 1500 U, on the 4th, 7th, and 10th days following egg retrieval (18 cases). Serum hCG was determined beginning 12-14 days following egg fertilization and every 3-7 days afterward. Women who became pregnant spontaneously following induction of ovulation, received either clomiphene citrate at daily doses of 50-100 mg from day 5 to 9 of the cycle, or human menopausal gonadotropin (hMG), 75-300 U daily from day 5 until the primary follicle exceeded 16 mm in diameter, at which time the patients received 5000 U of hCG. Patients who received hMG followed by hCG were also given 100 mg progesterone daily for luteal-phase support. In spontaneously ovulating women, the day of conception was considered the day after the LH surge. In women in whom ovulation was induced using hCG, the day of insemination, which took place 36-44 hr after hCG was given, was considered the day of conception. The mean age of patients who conceived following IVFET was 32.5 _+ 2.0 (SE), whereas the mean age of patients who conceived spontaneously was 34.0 __+ 2.1. Patients who conceived following IVFET received Pergonal for an average of 10.1 days, achieving a mean estradiol level of 1550 _+ 220 pg/ml. Patients who conceived spontaneously received Pergonal for an average of 8.0 days, achieving mean estradiol level of 526 ___ 130 pg/ml, hCG was measured using microparticle enzyme immunoassay manufactured by Abott Laboratories, Chicago, IL. The between-assay coefficient of the variation of hCG measurement is less than 10%. hCG values were log transformed and a linear regression analysis of the log values versus the days postconception was pertbrmed using a commercial statistical package (Stat view; Abacus Inc., CA). Separate hCG regression lines were constructed for IVFET and non-IVF resulting pregnancies and the lines were compared for their slopes using Student's t test. The protocol received approval from The Malden Hospital Human Research Committee. RESULTS hCG serum levels of 25 women who became pregnant following IVFET were compared to those of 48 Journal of Assisted Reproduction and Genetics, VoL 13. No. 7, 1996
women who became pregnant spontaneously with or without ovarian stimulation. Since hCG levels of normally ovulating patients who became pregnant spontaneously were not different from those of patients who became pregnant following ovarian stimulation, they were all combined into one group. In each group, the number of hCG determinations exceeded the number of patients since many patients had multiple measurements. The regression line of log hCG of patients who conceived following IVFET was compared with that of women who became pregnant spontaneously. They are shown in Fig. 1. The slopes of the regression lines were 0.177 and 0.165 for IVFET induced and spontaneous achieved pregnancies, respectively. The difference between the slopes was not significant (P > 0.05). However, the elevations, or the Y intercepts, were significantly higher for spontaneously conceived pregnancies than for pregnancies resulting from IVFET (P < 0.05), indicating that these two were statistically separate lines with statistically indistinguishable slopes. At 15 days postconception the mean hCG level of the IVF group was approximately equivalent to that seen in the spontaneously conceived group at 13 days postconception, whereas at 25 days values of the two groups were 1 day apart.
DISCUSSION We have found in this study that the hCG levels of patients conceiving after IVFET were significantly lower than those of patients conceiving spontaneously. Mean hCG levels of the IVFET group at 15 days postconception were equivalent to those observed in spontaneous achieved pregnancies at 13 days postconception and they continued to be lower throughout the observation period. These lower serum hCG levels in patients who became pregnant after undergoing IVFET may reflect late implantation of embryos. These observations are also in agreement with a previous report describing a delay of 1.5 days in the estimated implantation date of patients conceiving by IVFET after receiving GnRH agonist and hMG hormones compared with patients who conceived by IVFET after receiving hMG alone (1). This delay in presumed embryo implantation is not due to the use of hMG itself, since normal rise of hCG was observed in our patients who conceived spontaneously after receiving hMG. Therefore, it appears that it is most likely due to the use of GnRH agonist in the protocol leading to egg retrieval and IVFET.
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Fig. 1. Regression lines of log hCG versus days postconception in women conceiving spontaneously or after IVFET.
Although the mechanism for this delay in embryo implantation remains unknown, there could be several explanations. Although several effects of GnRH-a use on oocytes maturation have been described (3), no adverse effects on embryo qualities have been reported and pregnancy rates appear to be similar or better when GnRH-a is added to the simulation regimen (4). It is more likely that a different endometrial environment is created when GnRH agonist is added to the induction regimen. First, we have found three times higher estradiol levels in patients who received GnRH agonist plus hMG, compared to hMG alone, indicating greater ovarian stimulation in the IVFET cycles. There is evidence that in hypogonadal women receiving estradiol, the absolute blood estradiol levels achieved do not correlate with uterine receptivity (5). However, in normally ovulating women, in addition to estradiot, the stimulated ovaries are also likely to produce nonsteroidal ovarian substances (6), which if secreted in excess, may create a different endometrial environment. Second, the initial use of GnRH-a brings about a period of amenorrhea, which may lead to a different endometrial environment once hMG is begun. Histopathologic studies of endometrial biopsies have been limited in the scope and are difficult to interpret (7,8). However, morphological results suggest that endometrial glandu-
lar volume is better when GnRH agonist plus hMG is used as compared to clomiphene citrate and hMG or hMG alone (9) and that there is less asynchrony between cumulus-coronal morphology and nuclear maturity (3). The presumed effect of GnRH-a on uterine receptivity may be modulated via its ability to alter hormone secretion by the pituitary or ovary or via direct effect at the uterine levels. GnRH-a, in addition to its wellknown effect on pituitary LH and FSH secretions, may also have an effect, at least in the rat, on release of pituitary hormones other than LH and FSH, such as growth hormone (10). In addition, GnRH-a can exert extra pituitary effects ( I 1) including altering specific tissue receptors in certain tumors and cancers (12). Moreover, low-affinity high-capacity binding sites have been described previously for GnRH in human uterine tissue and GnRH agonist can directly affect the production of growth factors expressed by these cells (12). Finally, uterine receptivity may be related to the appearance of the endometrial cell adhesion molecule integrin (13). The appearance of integrin on the epithelial cells of human endometrium, at precisely the time where implantation is thought to occur, suggests its importance in enhancing implantation (14). It would Journal of Assisted Reproduction and Genetics, Vol. 13, No. 7, 1996
EMBRYO IMPLANTATION FOLLOWING IVFET
be of interest to determine if the use of GnRH-a alters the presence of the cell adhesion molecule integrin, which are considered indicators of endometrial maturation.
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6. Speroff L, Glass RH, Kase NG, Regulation of the menstrual cycle. In Clinical Gynecologic Endocrinology and Infertility. Baltimore, Williams and Wilkins, 1994, pp 183-230 7. Self MW, Pearson JM, lbrahim ZHZ, Buckley CH, Aplin JD, Buck P, Matson PL, Lieberman BA: Endometrium in in-vitro fertilization cycles: morphological and functional differentiation in the implantation phase. Hum Reprod 1992;7:6-11 8. Yoshinaga K: Endocrinology of implantation. In Maternal-Fetal Endocrinology, D Tulchinsky, BA Little (eds). Philadelphia, WB Saunders, 1994, pp 336-349 9. Hosie MJ, Murphy CR, Rogers PAW, Dwarte DM: Morphometric comparison of uterine glandular epithelium in the early secretory phase from patients treated with different superovulatory drugs on an in-vitro fertilization programme. Acta Anat 1991;142:174-178 10. Penzias AS, Goodman SR, Rossi G, Shamma FN, Aten RF, Behrman HR, Jones EE: Preliminary evidence that GnRH has the properties of a growth hormone-releasing factor in-vivo in the Rat. J Soc Gynecol Invest 1995;2:623--629 I I. Hsueh AJW, Erickson GF: Extrapituitary actions of gonadotropin-releasing hormone: Direct inhibition of ovarian steroidogenesis. Science 1979;204:854-855 12. Imai A, Furui T, Tamaya T: Is extrapituitary action of gonadotrophin-releasing hormone biologically significant? Ann Clin Biochem 1992;29:477-480 13. Lessy BA, Damjanovich L, Coutifaris C, Castelbaum A, AlbeIda SM, Buck CA: Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992;90:188-195 14. Lessy BA: The use of lntegrins for the assessment of uterine receptivity. Fertil Steril 1990;61:812-813
