Different implantation rates after transfers of cryopreserved embryos originating from donated oocytes or from regular in vitro fertilization*

Transcription

1 FERTILITY AND STERILITY Vol. 54, No. 4, October 1990 Copyright co 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Different implantation rates after transfers of cryopreserved embryos originating from donated oocytes or from regular in vitro fertilization* Dominique de Ziegler, M.D.t Rene Frydman, M.D. University of Paris Sud (Bicetre), Hopital Antoine Beclere, Clamart, France Most oocyte donation programs have experienced higher pregnancy rates than usually seen in regular in vitro fertilization (IVF), suggesting that the quality of either the oocytes or the endometrium is superior. To clarify this issue we analyzed the results of transfers of 136 cryopreserved embryos originating either from donated oocytes ( 18 transfers) or from regular IVF ( 118 transfers). Transfers of embryos originating from donated oocytes took place after administrating oral estradiol (E 2) valerate and vaginal micronized progesterone (P) following a regimen designed to mimic the serum levels of E 2 and P observed during the menstrual cycle. Transfers of embryos originating from regular IVF took place either in the natural cycle (53 transfers) or after suppressing ovarian function with a single injection of a gonadotropin-releasing hormone agonist ( GnRH -a), Decapeptyl-Retard 3.75 mg, and administering the same hormone replacement regimen (E2/P) used in oocyte donation (65 transfers). Eighteen transfers involving 24 embryos originating from donated oocytes were affected, resulting in six pregnancies (4 ongoing). The ongoing pregnancy rate per transfer was 22%. Seventy-nine embryos originating from regular IVF were transferred (53 transfers) in the natural cycle resulting in six pregnancies (2 ongoing). One hundred three other embryos originating from regular IVF were transferred ( 65 transfers) after administration of GnRH -a and E 2/P resulting in four pregnancies. The pregnancy rate after transfers of embryos originating from regular IVF was 9% per transfer. This was significantly less than the pregnancy rate of 33% per transfer seen after the transfers of embryos originating from donated oocytes. Our results indicated that a difference in embryo implantation persisted between oocyte donation and regular IVF even in the absence of ovarian hyperstimulation. This suggests, therefore, that either the embryos originating from donated oocytes are of higher quality than those originating from regular IVF, or that the endometrium is less receptive in IVF patients. Fertil Steril54:682, 1990 Mastering in vitro fertilization (IVF) has permitted the development of oocyte donation programs that enable young women without functioning ovaries, or unable to use their own oocytes, to become pregnant through IVF of donated oocytes. Since the original report of Lutjen et al., 1 we 2 and others 3-6 have observed excellent pregnancy rates Received October 16, 1989; revised and accepted June 25, * Presented at the VIth in vitro fertilization Meeting in Jerusalem, Israel, April2 to 7, t Reprint requests: Dominique de Ziegler, M.D., Hopital Antoine Beclere, Maternite, 157 rue de laporte de Trivaux, Clamart, France. in oocyte donation. Despite the relatively small number of cases presented in these reports, results from oocyte donation tend to exceed those seen in regular IVF. 2-6 The higher pregnancy rates observed in oocyte donation have suggested a difference in quality of either the oocytes or the endometrium, between oocyte donation and regular IVF. Ovarian hyperstimulation required for IVF cycles is regarded as the factor responsible for altering endometrium receptivity. 7-9 An effect of ovarian hyperstimulation on endometrium could, indeed, explain the lower pregnancy rates seen in IVF as compared with oocyte donation. We studied the results of transfers of cryopreserved embryos originating from donated oocytes and from regular IVF 682 de Ziegler and Frydman Implantation of cryopreserved embryos Fertility and Sterility

2 as the basis for establishing whether the difference in pregnancy rates between oocyte donation and regular IVF results only from ovarian hyperstimulation. Studying the implantation of cryopreserved embryos rather than fresh embryos offered the advantage that in both groups, the endometrium was exposed to a similar pattern of serum estradiol (E 2) and progesterone (P): cryopreserved embryos originating from oocyte donation were transferred after administrating exogenous E 2 and P after a regimen designed to mimic the levels of E 2 and P observed during the menstrual cycle. Cryopreserved embryos originating from regular IVF were transferred either after administering the same regimen of exogenous E 2 and P in women whose ovarian function was suppressed with a gonadotropin-releasing hormone agonist (GnRH-a), or during the menstrual cycle itself. Our results indicated that cryopreserved embryos originating from donated oocytes had a significantly higher implantation rate (25%) than those originating from regular IVF (6%). MATERIALS AND METHODS Patient Characteristics Oocyte Donation Eighteen women with absent or inactive ovaries were the recipients of cryopreserved embryos obtained from IVF of donated oocytes. In these women, infertility resulted from premature ovarian failure (13 cases), gonadal dysgenesis (2 cases), status after chemotherapy and/or radiation therapy (2 cases), and status after surgical oophorectomy (1 case). Oocytes were obtained from volunteer donors selected among fertile friends or family members. Oocytes were exchanged between phenotipically matched donor-recipient pairs 2 as a means of guaranteeing the anonymous donation of gametes. Oocyte donors were years of age (mean ± SD) whereas recipients were 32.1 ± 4.8 years old. For 14 patients the studied transfer was the first embryo transfer (ET), whereas 4 patients had a previous unsuccessful attempt at achieving pregnancy by oocyte donation. Regular IVF One hundred eighteen IVF patients had cryopreserved embryos from a previous IVF cycle. Their age was 34.9 ± 3.8. In 73% of the cases infertility was due to an absolute or a relative tubal factor. In 20% of the patients, the transferred embryos were obtained during a first IVF attempt, whereas they originated from a second, a third, or a fourth IVF attempt in 26%, 23%, and 20% of patients, respectively. In 4%, embryos originated from a fifth IVF attempt, and in 6%, patients had six or more previous IVF attempts. In Vitro Fertilization, Cryopreservation, and ET Ovarian Stimulation Regimen Fourteen oocyte donors received an ovarian stimulation regimen combining a GnRH-a and human menopausal gonadotropin (hmg) as previously described. 10 Four oocyte donors received a combination of clomiphene citrate (CC) and hmgy Seventy-eight regular IVF patients received GnRH-a/hMG, whereas 40 received CC/ hmg for ovarian stimulation. Cryopreservation Thirteen embryos (17%) originating from regular IVF and 5 (22%) originating from donated oocytes were cryopreserved at the two pronuclear stage using 1.5 M, 1'2 propanediol for cryopretection as previously described. 12 The remaining embryos were cryopreserved at the cellular stage (2 to 8 cells) 24 hours later in their developmental process using the same technique. When a choice existed, embryos having an even number of blastomeres and a more regular morphological appearance were selected for cryopreservation rather than fresh transfer. In the present study, we have analyzed only the results of all the transfers performed at our institution in which there was at least one embryo with at least one regular blastomere having no more than a moderate degree of cytoplasmic vacuolation. This represented 91% of all the transfers of cryopreserved embryos performed at our institution during the same period. Embryo Transfers Embryos were transferred within 1 to 2 hours of thawing in 25 to 35 JLL of B2 culture medium (API System, La Balme les Grottes, France) using an a traumatic catheter ( CCD Laboratories, Paris, France). Vol. 54, No.4, October 1990 de Ziegler and Frydman Implantation of cryopreserued embryos 683

3 Hormone Treatment/Hormone Measurements and Timing of ET Recipients Recipients of oocyte donation received oral E 2 valerate (Shering Pharmaceuticals, Lys Les Lannoy, France) and vaginal micronized P (Besins-Iscovesco Pharmaceuticals, Paris, France) after a modification of a regimen described by Lutjen et al. 1 Hormone replacement was scheduled as follows: E 2 valerate; 2 mg/d from cycle day 1 to day 6, 4 mg from day 7 to day 9, 6 mg from day 10 to day 12, 8 mg on day 13, 4 mg from day 14 to day 16, and 6 mg from day 17 to day 28. Micronized P 300 mg/ d was administered from day 14 to day 28. Embryo transfers were timed arbitrarely between cycle day 16 and cycle day 18, i.e., between luteal day 3 and luteal day 5 by a physician not aware of the comparative study. Regular IVF Patients Menstrual Cycle. In vitro fertilization patients having regular menstrual cycles were randomly assigned to have their embryos transferred either in the natural cycle or after receiving GnRH -a and exogenous E 2 and P (GEEP). In vitro fertilization patients with a history of oligoanovulation were arbitrarily attributed to the GEEP group. Transfers in the natural cycle were timed as follows: daily semiquantitative measurements of urinary luteinizing hormone (LH) in first voided urine were started from cycle day 10 using First Response ovulation predictor kits (Talco Laboratories, Joue-Les Tours, France). On the day of urinary LH rise, serum LH was measured to confirm an actual LH surge. Embryo transfers took place on the third day after the LH surge. In doubtful cases, ETs were cancelled and a new attempt was rescheduled. GEEP. Preparation for ET with exogenous hormones (GEEP) was conducted as follows: a single injection of a depot preparation of a GnRH-a, Decapeptyl-Retard 3.75 mg IM (lpsen-biotech Pharmaceuticals, Paris, France), was administered on the first day after spontaneous or induced menstruations. Hormone replacement with oral E 2 valerate and vaginal micronized P following the same regimen used in oocyte donation was started on cycle day 11 provided that serum E 2 measured on cycle day 10 was <35 pgjml. If serum E 2 on cycle day 10 exceeded 35 pg/ml, hormone replacement was withheld and serum E 2 was measured again 4 or 5 days later. In all cases, hormone therapy was Table 1 Outcome of Transfers of Cryopreserved Embryos Originating From Oocyte Donation Program or Regular IVF Regular IVF Cryopreserved embryos originating Oocyte Natural from donation cycle GEEP Total No. of transfers No. of embryos transferred No. of embryos per transfer Pregnancies (rate/ transfer) 6 (33)" 6 (11) 4 (6) 10 (9)b Ongoing pregnancies (rate/transfer) 4 (22)" 2 (4) 3 (5) 5 (4) Implantation rate(%) b a Values are no. of pregnancies with percents in parentheses. b Indicates significant difference with results of oocyte donation (P < 0.05). started only after complete ovarian suppression was achieved. Embryo transfers were timed arbitrarily between E 2/P treatment days 16 and 19, i.e., between luteal day 3 and luteal day 6. Hormone Supplementation After Establishment of Pregnancy After the establishment of pregnancy 11 to 13 days after ET, women in the oocyte donation group and those in regular IVF who received GEEP were prescribed hormone replacement until the production of E 2 and P by the placenta became manifest. During this interval, pregnant women received 6 to 8 mg of oral E 2 valerate together with vaginal (1,200 mg/24 h) and IMP (25 to 50 mg/24 h). Hormone supplementation was discontinued when serum E 2 and P started to rise despite constant supply of E 2 and P. In all cases this had occurred by the 11th week after ET. Statistical Analysis Results were compared by the x 2 analysis of population distributions where applicable. Statistical significance was defined asp < RESULTS Results of transfers of cryopreserved embryos originating from oocyte donation and from regular IVF appear in Table 1. When results of transfers of oocyte donation originating from regular IVF were regrouped (natural cycle+ GEEP), the pregnancy rate was 9% per transfer. This was significantly 684 de Ziegler and Frydman Implantation of cryopreserved embryos Fertility and Sterility

4 Table2 Impact of the Duration of Exposure to Exogenous P Luteal day No. ofets Pregnancies Cryopreserved embryos (4)a from oocyte donation Cryopreserved embryos (1) from regular IVF (2) (GEEP) a Values in parentheses represent ongoing pregnancies. lower (P < 0.05) than the pregnancy rate of 33% per transfer obtained after the transfers of cryopreserved embryos originating from donated oocytes. The implantation rates (implantations/embryos transferred) obtained after transferring embryos originating from regular IVF and oocyte donation were also significantly different at 6% and 25%, respectively (P < 0.05). The number of embryos transfered during successful cycles was 1.3 ± 0.1 and 1.6 ± 0.2 (mean ± SEM) in oocyte donation and IVF cycles. This was not significantly different from unsuccessful cycles in which 1.3 and 1.5 embryos were transferred in oocyte donation and regular IVF patients. Results were analyzed according to the duration of exposure of the endometrium to exogenous P at the time of transfer. As seen in Table 2, irrespective of the origin of the embryo, pregnancies occurred only when transfers were performed on or before luteal day 4. A larger number of embryos were transferred on luteal day 5 or later in the IVF / GEEP group (22) than in the oocyte donation group (2). Although this factor could be responsible for a slightly lower pregnancy rate in the IVF I GEEP group, it cannot explain the lower pregnancy and implantation rate encountered in the IVF /natural cycle group. The possible impact of using a GnRH -a during ovarian stimulation on the subsequent ability for cryopreserved embryos to implant was studied. As shown in Table 3, exposure to GnRH -a during ovarian stimulation did not appear to influence the implantation of embryos that survived thawing and had an acceptable morphological appearance at the time of transfer. One hundred forty-one embryos obtained from GnRH-a cycles showed an implantation rate of 7.1 %. A similar implantation rate of 7.7% was achieved by 65 embryos originating from CC/hMG cycles. The embryo development stage at the time of freezing and the number of intact blastomeres at the time of transfer were compared between oocyte donation and regular IVF groups. Seventeen percent of embryos originating from regular IVF and 22% of embryos originating from oocyte donation were cryopreserved at the two pronuclear stage, a difference that was not statistically significant. Likewise, the fractions of embryos having 1 to 2, 3 to 4, and 5 or more intact blastomeres at the time of transfer were similar at 54%, 40%, and 6% for embryos originating from donated oocytes and at 57%, 38%, and 5%, respectively, for regular IVF embryos. DISCUSSION The aim of our study was to compare embryo implantation in oocyte donation and regular IVF. By focusing our comparison only on the outcomes of transfers of cryopreserved embryos, our study design excluded any possible effect of ovarian hyperstimulation on the endometrium. Moreover, we also restricted our study to the implantations of embryos showing an acceptable morphological appearance after thawing. This precaution was motivated by our desire to compare embryo implantation in oocyte donation and regular IVF, rather than analyzing possible differences in freezingthawing efficiency that may exist between these two groups. In the context of our study design, the observation of different implantation rates for cryopreserved embryos originating from donated oocytes and regular IVF was unexpected. In both groups, Table 3 Impact of Using a GnRH -a During the Ovarian Stimulation Regimen Cryopreserved from oocyte donation GnRH-a/hMG CC/hMG Cryopreserved from regular IVF (natural cycle) GnRH-a/hMG CC/hMG Cryopreserved from regular IVF (GEEP) GnRH-a/hMG CC/hMG Number of Number of Number of stimulation embryos embryos cycles transferred implantatinga a Values in parentheses are percents (17) 6 2 (33) 53 4 (8) 26 2 (8) 70 3 (4) 33 1 (3) Vol. 54, No.4, October 1990 de Ziegler and Frydman Implantation of cryopreserved embryos 685

5 embryos were obtained from oocytes retrieved by similar techniques after similar ovarian stimulation regimens. Embryo freezing techniques were also identical. Moreover, before ET, patients from both groups were exposed to similar patterns of serum E 2 and P: oocyte donation patients received exogenous E 2 and P following a regimen designed to mimic the pattern of serum E 2 and P seen in the menstrual cycle. 1 Regular IVF patients were divided into two groups: 53 had their ET in the natural cycle, whereas 65 others received the same regimen of exogenous E 2 and P as administered in oocyte donation patients. This latter group with active ovaries differed slightly from oocyte donation patients in that GnRH -a was administered to suppress their ovarian function before the administration of exogenous E 2 and P. An abortive effect of GnRH -a has been described in animals but the mechanism involved, luteolysis, could not apply here. 13 Furthermore, there is no data to support a direct action of GnRH-a on the endometrium. Transfers of cryopreserved embryos originating from regular IVF were timed according to the number of days after the LH surge or according to the number of days of exposure to exogenous P. We previously studied the correlation existing between the number of days after LH surge and the number of days of exposure to P. 14 In our hands this correlation did not exist for all patients. In 58% of the menstrual cycles that were monitored, the first day that serum P exceeded 1.2 ng/ml was after LH day 1. In the rest of the patients, serum P exceeded 1.2 ng/ml either a day before (19%) or a day later (23%). Therefore, in the present study we analyzed ET outcomes in relation to the duration of exposure to P only in patients receiving exogenous P (Table 2). One possible explanation for the different implantation rates seen after transfers performed in oocyte donation and regular IVF may be the difference in oocyte origin between the two groups. In oocyte donation, embryos were obtained from oocytes originating from fertile donors who were slightly younger than our infertile IVF candidates. The age difference between oocyte donation (donors) and IVF patients, 29.1 ± 4.9 and 34.9 ± 3.8 years old (mean± SD), respectively, was not statistically significant. It is therefore doubtful that the age difference alone could explain the different implantation rates of cryopreserved embryos originating from donated oocytes and regular IVF. To further substantiate this point, we divided our regular IVF population into comparable age groups. Four hundred sixty-eight consecutive regular IVF oocyte retrievals were performed at our institution during the observation period of the present study (15 months). During this period, 212 of the retrievals were performed in women aged 28 to 32, and 207 in women aged 33 to 3 7. Pregnancy rates after fresh ET were similar at 26% and 27% per transfer in the older and younger group, respectively. Furthermore, no difference in embryo quality assessed by their morphological appearance could be noted between oocyte donation and IVF. Another source of difference between regular IVF and oocyte donation patients is the difference in the number of previous IVF attempts between these two groups. Regular IVF patients had more unsuccessful IVF attempts before the cycle that yielded the cryopreserved embryos than did oocyte donation patients. For 70% of regular IVF patients, however, the number of previous IVF attempts was :::;2 and for 94% of them it was :::;4. As the chances of pregnancy remain unchanged during at least the first four IVF attempts, 15 it is unlikely that the difference in the number of previous IVF attempts represents the cause for the difference in pregnancy rates observed after the transfers of cryopreserved embryos originating from regular IVF or oocyte donation. Moreover, the freezing-thawing interval differed between oocyte donation and regular IVF. Embryos originating from donated oocytes remained frozen for 0.9 ± 0.2 months (mean ± SEM), whereas those originating from regular IVF stayed frozen for 15 ± 1.3 months. Whereas a higher postthawing survival rate of blastomeres has been reported when thawing occurred <1 month after freezing as compared with >2 months after freezing, no difference in embryo implantation rate was observed in relation to the duration of the freezingthawing interval. 12 Therefore, it is unlikely that the difference in duration of the freezing-thawing interval was responsible for the different implantation rates observed in oocyte donation and regular IVF. Still another possible explanation for the difference in pregnancy rates seen between oocyte donation and regular IVF is a possible difference in endometrium receptivity. This hypothesis implies a non-e 2/P mediated factor acting on the endometrium of IVF patients and having a detrimental influence on embryo implantation. The high incidence of tubal factors in our IVF population (73%) suggests that this hypothetical factor impairing implantation in IVF might also represent a sequela 686 de Ziegler and Frydman Implantation of cryopreserved embryos Fertility and Sterility

6 of a previous infectious process of the genital tract. We previously observed, by scanning electron microscopy in endometrium samples collected 6 days after hcg in IVF patients who did not have an ET, that apical protusions (pinopodes) normally present at the corresponding time of the menstrual cycle were missing in 11 of the 13 IVF patients. 16 We originally believed that this finding was related to ovarian hyperstimulation. Today we are extending these studies to determine if the abnormal morphological findings made in the endometrium of IVF patients (absence ofpinopodes) could not be linked to a previous infectious process of the genital tract rather than to ovarian hyperstimulation. Endometrial biopsies were, however, not obtained in regular IVF patients who had embryos transfered in their natural cycle or with GEEP. The recent report of unsuspected hcg/lh receptors in porcine 17 and human uteri 18 led us to consider also a totally different mechanism that could explain a non-e 2/P mediated difference in endometrium receptivity between oocyte donation and IVF patients. Women with inactive or absent ovaries have higher serum gonadotropin levels than seen during the menstrual cycle. 19 Therefore, as LH receptors have been documented in animal 17 and human 18 endometrium, it is possible that the higher level of serum gonadotropins present in oocyte donation patients might positively influence endometrium receptivity. Evidently this latter possibility needs to be investigatedfurther. If it is confirmed, however, fascinating new avenues could open for attempting to improve embryo implantation in IVF with exogenous gonadotropins. Ideally, to compare endometrium receptivity between regular IVF and oocyte donation patients, a unitary source of oocytes should be preferred. A unitary source of oocyte would exist if donated oocytes were obtained from regular IVF patients. This approach would, however, not be free of bias either, as oocytes donated by regular IVF patients are usually obtained from a subgroup of IVF patients who have a stronger than average ovarian response to hmg. Moreover, this study was not feasible at our institution because our oocyte donation program uses exclusively oocytes obtained from volunteer donors selected among friends or family members and anonymously exchanged between donor recipient pairs as previously described. 2 Our observation that pregnancies occurred exclusively when ET occurred on luteal days 3 and 4 confirms the previous data on the end of the period or endometrium receptivity or window oftransfer. 3 The absence of transfers performed before luteal day 3 does not allow us to comment on the onset of endometrium receptivity. If we consider the 33 embryos originating from regular IVF that were transferred on luteal day 3 or 4 in the GEEP group, the implantation rate of 12% per embryo transferred was similar to that of 11% observed for all cryopreserved embryos transferred in the natural cycle at our institution. 20 This indicates, therefore, that GEEP is a valuable alternative for timing transfers of cryopreserved embryos in individuals who do not have regular menstrual cycles. Our observation that GnRH-a exposure during ovarian stimulation did not appear to impact on implantation of cryopreserved embryos is evidently restricted by the limits of our study design. Results were presented only for the purpose of illustrating that the difference in implantation rate observed between embryos originating from oocyte donation and regular IVF did not result from different rates of exposure to GnRH -a during ovarian stimulation. The numbers reported in this study are insufficient to draw general conclusions on the effect of exposure to GnRH -a during ovarian. stimulation on the quality of cryopreserved embryos. Furthermore, a complete analysis of the effects of GnRH-a on cryopreservation would require studying the global impact of GnRH -a on the efficiency of the freezing-thawing process. Therefore, the results reported here do not contradict our observation that exposure to GnRH -a during ovarian stimulation decreased the overall efficiency of the freezing-thawing process. 21 In conclusion, our observation of a significantly higher implantation rate after transfers of cryopreserved embryos originating from oocyte donation rather than regular IVF suggests that a factor not mediated by E 2 andp might decrease endometrium receptivity in IVF patients. The high incidence of previous pelvic infections in IVF candidates led us to hypothesize that this detrimental factor for embryo implantation might represent a sequela of a previous pelvic infection. We are currently undertaking morphological studies to identify possible infectious stigmata in the endometrium of IVF patients. REFERENCES 1. Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P: The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature 307:174, 1984 Vol. 54, No.4, October 1990 de Ziegler and Frydman Implantation of cryopreserved embryos 687

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