Pediatrics, Obstetrics and Gynecology, University School of Medicine, Valencia University, Valencia, Spain

Delaying the initiation of progesterone supplementation until the day of fertilization does not compromise cycle outcome in patients receiving donated oocytes: a randomized study María-José Escribá, Ph.D., a José Bellver, M.D., a Ernesto Bosch, M.D., a María Sánchez, M.D., a,b Antonio Pellicer, M.D., a,b,c and José Remohí, M.D. a,c a Universitary Institute IVI Valencia; b Department of Obstetrics and Gynaecology. Hospital Dr. Peset; and c Department of Pediatrics, Obstetrics and Gynecology, University School of Medicine, Valencia University, Valencia, Spain Objective: To determine whether the initiation of P supplementation as artificial luteal phase support (day 1, day 0, or day 1 of egg donation) in extensive programs of ovum donation influences cycle cancellation, pregnancy outcome, and implantation rate in day 3 embryo transfers. Design: Prospective randomized trial. Setting: Oocyte donation program at the Instituto Valenciano de Infertilidad, Valencia, Spain. Patient(s): Three hundred recipients with normal ovarian function, absence of uterine anomalies, and undergoing their first egg donation were recruited between September 2003 and September 2004. Intervention(s): A computer-based randomization divided the recipients into three groups when hcg was administered to their matched donors. The first group (group A) started P supplementation the day before oocyte retrieval; the second group (group B) started P supplementation on the day of the oocyte retrieval; and the third group (group C) started P supplementation 1 day after the egg retrieval once fertilization was confirmed. Main Outcome Measure(s): Implantation, pregnancy, and ongoing pregnancy rates were the primary outcome measures considered. The secondary outcome measure was the cancellation rate, especially due to fertilization failure. Result(s): Global cancellation rate and cancellation rate due to fertilization failure were significantly higher in group A (12.4% and 8.2%, respectively) than in group C (3.3% and 0%, respectively). Reproductive outcome was similar in all the groups except for a higher biochemical pregnancy rate in group A (12.9%) than in groups B (6.6%) and C (2.3%). Conclusion(s): Initiation of P on day 1 of embryo development decreases cancellation rates of day 3 embryo transfers in extensive programs of ovum donation without any deleterious effect on pregnancy outcome or implantation rate. (Fertil Steril 2006;86:92 7. 2006 by American Society for Reproductive Medicine.) Key Words: Oocyte donation, initiation of progesterone supplementation, embryo transfer, endometrial receptivity, implantation window In recent years ovum donation has been considered, on an international level, as the most effective assisted reproduction technology for women whose oocytes are of poor quality or do not produce oocytes. There are varying protocols for donor recipient synchronization, which are determined mainly by the ovarian function of the recipient, the availability of donors, and recipient demand. When ovarian function persists, it is viable to prepare the endometrium after a natural cycle or hormonal replacement therapy (estrogen plus P), with or without prior administration of a GnRH agonist (GnRH-a). When ovarian function is nonexistent, hormonal replacement therapy is mandatory (1 3). Received June 1, 2005; revised and accepted December 4, 2005. Reprint requests: María-José Escribá, Ph.D., Instituto Universitario IVI, Instituto Valenciano de Infertilidad, Plaza Policía Local, 3, Valencia 46015, Spain (FAX: 34-963050999; E-mail: mjescriba@ivi.es). In some egg donation programs, the availability of donors or recipient demand is low. Therefore, a perfect synchronization must be achieved between donor and recipient (one to one) with simultaneous initiation of endometrial preparation of the recipient on the one hand and controlled ovarian hyperstimulation (COH) of the donor on the other (4). In this way, a perfect synchronization in the dialogue between embryo and endometrium can be obtained, facilitating embryo transfer to the endometrium at the appropriate moment, known as the implantation window (5, 6). However, cancellation of the donor due to an inadequate ovarian response, albeit in relatively few cases, is the principal handicap of these protocols. On the other hand, in large programs with a high number of recipients on the waiting list who are receiving estrogen therapy the main disadvantage is the high cancellation rate (approximately 20%) due to the endometrial bleeding that 92 Fertility and Sterility Vol. 86, No. 1, July 2006 0015-0282/06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2005.12.048

often occurs while waiting for a donor to be found and a transfer performed (7). This could be avoided with limited E 2 replacement therapy for every recipient undergoing endometrial preparation and with embryo transfer once a healthy endometrial thickness and pattern has been confirmed (8, 9). This is possible when there is a constant pool of donors available undergoing COH, so that once a recipient is ready, she can be matched immediately with the donor that proves most compatible according to her phenotype and blood group. Following this procedure, recipient waiting time after the initiation of estrogen administration tends to be less than 40 days, a period during which endometrial bleeding is unusual (10). The primary problem with initiating P administration as an artificial luteal phase support on the day of or day before egg donation, as some investigators have described (3, 4), is the significant cancellation rate due to failure to achieve fertilization, mainly due to sperm anomalies or, to a lesser extent, low quality of the oocyte. A possible solution is to begin P supplementation on the day after egg donation, once fertilization has been confirmed. In this way, when gamete quality is inadequate or fertilization has not occurred, P administration is postponed and recipients continue with estrogen therapy until eggs from another compatible donor or partner/donor sperm sample become available, thereby affording recipients a second chance. The only foreseeable handicap of this measure is that cell-stage embryos (on day 2 or 3 of development) could be transferred to an endometrium that is not yet prepared (day 1 or 2 of P supplementation) and, therefore, outside the implantation window (11, 12). The aim of the present randomized study was to determine the effect of P administration (as an artificial luteal phase support) on the pregnancy outcome of day 3 embryo transfers with respect to day of initiation (day 1, day 0, or day 1 of egg donation) and its impact on cycle cancellation as a result of fertilization failure. MATERIALS AND METHODS This is a prospective randomized study that was approved by the Institutional Review Board and carried out at the Instituto Valenciano de Infertilidad, Valencia, Spain, between September 2003 and September 2004. Subject inclusion criteria were oocyte recipients with active ovarian function and showing absence of uterine anomalies, who underwent a day 3 embryo transfer. The following were indications for egg donation: endometriosis, failure of previous assisted reproduction technology attempts, genetic or chromosomal anomalies of maternal origin (including recurrent pregnancy losses), poor oocyte quality, low ovarian response, and advanced age. Oocyte Donors In Spain, ovum donation is voluntary and anonymous. Donors are aged between 18 and 35 years and must be healthy, with no family history of chromosomal diseases. They must undergo a complete gynecological examination, karyotype, and screening for infectious diseases such as HIV, hepatitis B and C, gonoccocia, and lues. The protocol for COH has been described elsewhere (13). In brief, pituitary desensitization with daily SC administration of 1 mg of leuprolide acetate (LA; Procrin, Abbott S.A., Madrid, Spain) or inhaled administration of 800 g of nafarelin (Synarel, SEID Laboratories, Barcelona, Spain) was initiated in the luteal phase of the menstrual cycle. This dose was continued until ovarian quiescence was confirmed by ultrasound during the subsequent menstruation, at which point the dose of the GnRH-a was halved. On days 1 and 2 of ovarian stimulation, recombinant FSH (Gonal-F, Serono Laboratories, Madrid, Spain; or Puregon, Organon, Barcelona, Spain) or highly purified hmg (Menopur, Ferring, Madrid, Spain) were administered; the dose varying between 150 and 300 IU/day depending of the age of the donor, body mass index, ovarian pattern, menstrual cycles, basal hormones, and response to previous COH (if any). From day 3 onward, gonadotropin doses were adjusted every second day according to serum E 2 levels and ovarian appearance as detected by vaginal ultrasound. Human chorionic gonadotropin (Ovitrelle, Serono Laboratories) was administered SC when at least two leading follicles reached a mean diameter of 18 mm. Daily GnRH-a and gonadotropin administration were discontinued on the day of hcg administration. Transvaginal oocyte retrieval was scheduled 36 hours later. Donors were matched with recipients according to phenotype and blood group. Oocyte Recipients Three hundred recipients with active ovarian function and no uterine anomaly, who had not undergone previous egg donation, were admitted for the study. Patients underwent ovum donation because of the women s age (47.6%), low response to ovarian stimulation (25.6%), implantation failure (8.5%), severe endometriosis (8.5%), and other causes such as recurrent pregnancy loss, bad oocyte quality, or genetic infertility (9.8%). The protocol for steroid replacement included pituitary desensitization with a single intramuscular ampule administration of 3.75 mg of triptorelin (Decapeptyl depot 3.75, Ipsen Pharma, Madrid, Spain) in the midluteal phase of the menstrual cycle. Hormonal replacement therapy was initiated when ultrasound confirmed ovarian quiescence during the following menstruation. Two milligrams of E 2 valerate (Progynova, Fertility and Sterility 93

Schering Spain, Madrid, Spain) were administered daily for the first 8 days, 4 mg for the next 3 days, and 6 mg from thereon. After 13 days of E 2 valerate administration, endometrial thickness and pattern were tested. If a three-layer pattern was observed in a 7 mm endometrium, the aforementioned dose of E therapy was continued at least until the pregnancy test was performed. If the endometrium was not seen to be sufficiently developed, doses of E 2 valerate were increased to 8 mg/day. Serum E 2 levels were tested to detect possible E 2 bowel malabsortion when the endometrium appeared to be thin. Our egg donation program differs from others described due to the high demand of recipients and the availability of our donors. When a recipient is ready, she must undergo estrogen therapy until egg donation is performed. We aim for this period to be shorter than 40 days, as endometrial bleeding is rare during this time span, and recipient cancellation as a consequence is avoided (10). Therefore, when a donor from our continuous pool of donors undergoing COH is at the point of ovarian retrieval, we match her with a recipient whose compatibility has previously been confirmed and who is ready to receive the donation. In this way, our program permits donors and recipients to be matched on a daily basis, thereby avoiding the wait before entering into the program. In our program, daily administration of 800 mg/day of micronized intravaginal P (Progeffik, Laboratories Effik S.A., Madrid, Spain) was initiated on the day of oocyte retrieval, and continued at least until the result of the pregnancy test was known. For this study, P was initiated on different days after the randomization protocol of the study (discussed later). Embryo transfer was performed 3 days after egg retrieval by the vaginal route in all cases. Sixteen days after oocyte retrieval, a quantitative serum value of -hcg was obtained, with a result of 10 IU/L being considered positive. When a positive result was obtained, a second -hcg test and a primary transvaginal ultrasound were performed 1 week later. Scans were repeated weekly until the detection of embryo heart beat, and repeated monthly from thereon. The E 2 valerate and micronized P administration were continued at the same dosage until day 80 of the pregnancy. Biochemical pregnancy was diagnosed when a previously positive pregnancy test result became negative before ultrasonographic detection of an embryonic sac. Miscarriage was defined as a pregnancy failing to reach week 20 after the detection of the gestational sac by ultrasound. Ectopic pregnancy was defined as a pregnancy sited outside the uterine cavity, demonstrated by ultrasound or laparoscopy, or highly suspected due to symptoms or the -hcg serum curve of the patient. Ongoing pregnancy was considered when prolonged more than 20 weeks. The implantation rate was defined as the percentage of gestational sacs per number of transferred embryos. Study Protocol and Randomization The present study followed the CONSORT guidelines for randomized control trials (14). A computer-based randomization divided recipients into three groups immediately after confirmation of hcg administration in a matched donor. The first group (group A) began P supplementation the day before oocyte retrieval. In the second group (group B) P was administered from the day of oocyte retrieval. The third group (group C) began P one day after retrieval, once fertilization had been confirmed. Implantation, pregnancy, and ongoing pregnancy rate (PR) were the primary outcome measures considered. The secondary outcome measure was the cancellation rate, which occurred especially due to fertilization failure. Statistical Analysis The statistical analysis was performed using the Statistical Package for Social Science version 10.0 (SPSS Inc., Chicago, IL). Categorical data were expressed as number, percentage, and 95% confidence interval, and numerical data as mean and standard deviation. One-way ANOVA and Kruskal- Wallis test were used when appropriate. The primary end points in this study were clinical pregnancy and implantation rate, expressed as a ratio with corresponding 95% confidence intervals. To detect a two-sided difference of 10% in clinical PR between the group that started P supplementation the day of oocyte retrieval, and the groups that started the day before or the day after oocyte retrieval, during a control rate of 50%, 84 patients were needed per study arm. We included 300 patients, taking into account a withdrawal of 15%. The study was designed with a power of 85% and an error of 0.05. Significance was assumed at P.05. RESULTS Of the initial 300 patients, some were excluded when embryo transfer did not take place on day 3 of embryo development: three patients from group A, six from group B, and nine from group C. This was due to postponement of transfer until the blastocyst stage because these patients were unwilling to have the embryos frozen, or due to poor quality of the embryo on day 3. Therefore, groups A, B, and C consisted of 97, 94, and 91 patients, respectively. No difference was detected between the three groups in the following parameters: age of the recipient, body mass index, days undergoing estrogen therapy, endometrial thickness achieved, and incidence of severe sperm pathology, the latter defined as severe teratozoospermia ( 5% normal forms), severe oligozoospermia ( 5 million fresh spermatozoids/mm 3 ), or nonobstructive azoospermia (Table 1). 94 Escribá et al. Progesterone onset in egg donation Vol. 86, No. 1, July 2006

TABLE 1 Descriptive characteristics of the study groups (n 282). Group A ( 1) (n 97) Group B (0) (n 94) Group C ( 1) (n 91) P value Age of recipient (y) 39.4 5.1 39.3 4.7 39.5 4.5 NS Recipients range age (y) 25 47 27 47 29 48 Body mass index (kg/m 2 ) 22.0 2.9 23.4 4.6 22.7 4.0 NS Days under E 2 therapy 29.7 9.3 26.4 9.2 26.5 9.7 NS Endometrial thickness 8.9 2.0 9.1 2.0 9.2 2.1 NS Severe sperm pathology (%) 18 (18.6) 23 (24.4) 14 (15.3) NS No. of donated oocytes 10.8 3.4 10.8 3.5 10.9 4.0 NS No. transfer (n/%) 85 (87.6) a 90 (95.7) 88 (96.7) b.04 a,b Fertilization failure (n/%) 8 (8.2) a 1 (1.1) b 0 c.04 a,b.01 a,c E 2 serum level at ET (pg/ml) 204.6 90.2 199.7 69.6 202.9 79.2 NS P serum level at ET (ng/ml) 19.5 19.5 16.7 19.0 18.8 16.0 NS No. of transferred embryos 2.1 0.3 2.0 0.2 2.0 0.3 NS No. of frozen embryos 1.6 2.2 1.4 1.9 1.6 2.1 NS Note: Unless otherwise indicated, values are means SD. NS not significant; ET embryo transfer. Severe sperm pathology was defined as severe teratozoospermia ( 5% normal forms), severe oligozoospermia ( 5 million of fresh spermatozoids/mm 3 ), and nonobstructive azoospermia. a,b,c Different superscripts within the same line means statistically significant differences. Escribá. Progesterone onset in egg donation. Fertil Steril 2006. The mean numbers of donated oocytes in each group were similar, but the global cancellation rate and cancellation rate due to fertilization failure was significantly higher in group A. There were 12 transfer cancellations in group A: 8 because of fertilization failure, 3 due to poor embryo quality on day 2 of embryo cleavage, and 1 due to endometrial bleeding on day 3 of embryo cleavage. In group B, 4 patients were cancelled before transfer: 3 because of poor embryo quality on day 2 of embryo development and 1 due to fertilization failure. In group C, 3 patients were cancelled, all as a consequence of poor embryo quality on day 2 and none as a result of fertilization failure (Table 1). In the three groups, the percentage of cycles enrolled to IVF, intracytoplasmic sperm injection (ICSI), or IVF/ICSI fertilization procedure was similar (P not significant [NS]; Table 2). On day 3 of development, the mean embryo quality in the three groups was similar, without differences in the number of cells or the fragmentation rate (P NS; Table 2). Serum E 2 and P levels on the day of embryo transfer were similar in the three groups, as were the numbers of transferred and cryopreserved embryos (Table 1). Regarding cycle outcome, PR and implantation rates did not differ among the groups (Table 3). However, biochemical TABLE 2 Distribution of IVF, ICSI, and IVF/ICSI procedures and averaged number of cells and fragmentation degree of day 3-derived embryos among experimental groups (n 282). Group A Group B Group C Fertilization procedure IVF (n/%) 28 (28.9) 22 (23.4) 25 (27.5) ICSI (n/%) 62 (63.9) 67 (71.3) 63 (69.2) IVF/ICSI (n/%) 7 (7.2) 5 (5.3) 3 (3.3) No. of transferred embryos (n) 176 183 177 Number of cells 7.2 1.2 7.5 1.0 7.5 1.0 Percentage of fragmentation 10.0 7.5 9.0 6.9 9.1 7.6 Note: Unless otherwise indicated, values are means SD. There were no significant differences for any entry (P.05). Escribá. Progesterone onset in egg donation. Fertil Steril 2006. Fertility and Sterility 95

TABLE 3 Clinical outcome according to initiation of P supplement. Group A ( 1) Group B (0) Group C ( 1) P value Patients with embryo transfer 85/97 90/94 88/91 Pregnancy: n; % (95% CI) 48/85; 56.5% (45.7 67.2) 57/90; 63.3 (53.2 73.5) 50/88; 56.8% (46.3 67.4) NS Implantation: n; % (95% CI) 51/176; 28.9% (22.1 35.6) 71/83; 38.8% (31.7 45.9) 63/177; 35.6% (28.6 42.9) NS Biochemical pregnancy: n; % (95% CI) 11/85; 12.9% (5.7 20.2) a 6/90; 6.6% (1.4 11.9) 2/88; 2.3% (0 5.4) a.02 Clinical pregnancy: n; % (95% CI) 37/85; 43.5% (32.8 54.3) 51/90; 56.6% (46.2 67.1) 48/88; 54.5% (43.9 65.2) NS Miscarriage: n; % (95% CI) 6/37; 16.2% (3.8 28.7) 12/51; 23.5% (11.5 35.6) 5/48; 10.4% (1.5 19.6) NS Ectopic pregnancy: n; % (95% CI) 0 0 2/48; 4.2% (0 10.0) NS Twin pregnancy rate: n; % (95% CI) 14/37; 37.8% (21.4 54.2) 20/51; 39.2% (25.3 53.1) 15/48; 31.2% (17.6 44.9) NS Ongoing PR per transfer: n; % (95% CI) 31/85; 36.5% (26.0 46.9) 39/90; 43.3% (32.9 53.8) 41/88; 46.6% (36.0 57.2) NS Ongoing PR per egg donation: n; % (95% CI) 31/97; 31.9% (22.5 41.4) 39/94; 41.5% (31.3 51.6) 41/91; 45.0% (34.6 55.5) NS Note: NS not significant; PR pregnancy rate. a Significant differences were observed between groups A and C. Escribá. Progesterone onset in egg donation. Fertil Steril 2006. PRs were lower when P supplementation was begun on the day of egg retrieval (group B) or on the day of fertilization (group C) than when initiated the day before egg donation (group A). With respect to this, significant differences appeared between groups A and C (P.02), but, although we noted a slightly lower incidence in group C, the difference with group B was not significant. Differences were not detected among the groups in clinical PRs, miscarriage rates, or ectopic PRs. The twin PR was also similar, and no triplet pregnancies were registered in any of the studied groups (Table 3). The ongoing PR per randomized patient did not vary between the three groups and the overall ongoing PR per randomized patient was estimated at 39% (Table 3). DISCUSSION Cancellation of an egg donation cycle due to poor oocyte quality or fertilization failure is a frustrating obstacle in the assisted reproduction process. A second attempt within the same cycle is possible if E 2 therapy is continued in the recipient until another compatible donor is found. However, once P supplementation has been initiated, the time in which embryo transfer can be carried out is limited, therefore if another donor is not immediately available, cancellation is inevitable. This race against time would be avoided if oocyte quality and fertilization were confirmed first and P started after, when the embryos were available. In this way, P supplementation would be initiated the day after egg donation (day 1), whereas embryo transfer would be performed only after 2 days of supplementation, very close to the opening of the implantation window. This has been described as a shortcoming, with a reduced possibility of achieving pregnancy (11). The aim of the present randomized study was to determine whether initiating P supplementation on the day on which fertilization is confirmed (day 1 after oocyte retrieval) reduces the cancellation rate in recipients without affecting pregnancy outcome. In these cases the recipient received P supplementation for only 2 days, as embryo transfers were performed on day 3 of embryo development. Some previous studies dealing with ovum donation have performed transfers of 2- or 3-day-old embryos on the second, third, fourth, and fifth days of P supplementation, or even later (12). Several investigators have reported improved results with 2- or 3-day embryos after at least 3 days of P supplementation (15, 16). Other researchers reported a lack of pregnancies after 2 days of P supplementation and embryo transfers on day 2 of development (11), suggesting that embryo transfers were performed before the implantation window, which seems to begin approximately 48 hours after P supplementation and last for 4 days (12). There is an obvious lack of references in the literature regarding transfer of day 3 embryos after 2 days of P supplementation. However, extended P administration before transfer has been recommended at this stage of embryo development (12). 96 Escribá et al. Progesterone onset in egg donation Vol. 86, No. 1, July 2006

In our study, pregnancy outcomes were similar in each of the three groups, demonstrating that the duration of previous P preparation had no influence, and that, after 48 hours of P, the window of implantation is open for day 3 embryos. Curiously, the biochemical PR was higher and the implantation rate somewhat lower when P supplementation was begun earlier. These findings reflect the results of a study by Troncoso et al. (17), in which the incidence of biochemical pregnancies did not decrease after preimplantation genetic diagnosis, suggesting that biochemical pregnancies had a nonchromosomal origin, such as deficient endometrial receptivity. In this way, we could hypothesize that an earlier administration of P affects embryo implantation through endometrial disturbances, perhaps related to the histological advancement previously described by Kolibianakis et al. (18). On the other hand, the avoidance of cancellation due to poor oocyte quality and especially due to fertilization failure is an advantage in centers with extensive programs of ovum donation. In this study, the cancellation rate due to fertilization failure was reduced from 4.7% (9/191: groups A B) to 0% (group C), achieved by delaying the initiation of P supplementation until day 1. In our center, during the 1 year of study period, 1,371 cycles of egg donation were performed. In this way, delaying the artificial luteal display just 1 day, the cancellation of 64 cycles (4.7%) could be avoided. In centers in which donors and recipients are synchronized one to one, this strategy may be of little clinical impact. 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