Induction of artificial endometrial cycles with s.c. oestrogen implants ...

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Human Reproduction vol.12 no.10 pp.2267–2270, 1997. Induction of ... In the preparatory cycles with oral oestrogens, all failed to ... oocyte donation cycles yielded one biochemical and eight .... Thus, the uninterrupted oestrogen action.
Human Reproduction vol.12 no.10 pp.2267–2270, 1997

Induction of artificial endometrial cycles with s.c. oestrogen implants and injectable progesterone in in-vitro fertilization treatment with donated oocytes: a preliminary report I.Ben-Nun1,2 and A.Shulman1,2,3

Materials and methods

1Departments

Patients This study included 10 patients with retained ovarian function who failed to conceive after ovum retrieval and embryo transfer. The initial diagnoses for IVF treatment were: endometriosis (n 5 3), mechanical infertility (n 5 1), ovulatory dysfunction (n 5 1), unexplained infertility (n 5 3) and elevated baseline follicle stimulating hormone (FSH) concentrations (n 5 3). In all cases, the male factor was normal. Treatment by oocyte donation was offered because of the patients’ limited response to ovarian stimulation and/or repeat IVF/embryo transfer failure. Endometrial preparation was attempted several times using oral oestrogen and progesterone (Ben-Nun et al., 1989) but, because of an unwanted premature LH surge, appropriate endometrial development (as assessed by an endometrial thickness of ø5 mm on an ultrasonographic scan) and maturation (as assessed by endometrial biopsy) were not achieved. Prior to treatment, all recipients were subjected to a careful precycle medical screening to exclude those patients with preexisting diseases, such as cardiovascular problems or diabetes mellitus.

of Obstetrics and Gynecology, Sapir Medical Center, Kfar Saba and 2Chaim Sheba Medical Center, Tel Hashomer, Israel

3To

whom correspondence should be addressed at: IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer, Israel

Endometrial preparation for embryo implantation in oocyte recipients with retained ovarian function presents a special problem. In all, 10 women with preserved ovarian function received donated oocytes in an in-vitro fertilization programme. In the preparatory cycles with oral oestrogens, all failed to develop adequate secretory endometrium because of ill-timed early luteinization occurring during the proliferative phase of the cycle. Subsequently the patients were treated with s.c. 17-β oestradiol implants and injectable progesterone. The implants successfully induced complete down-regulation of the hypothalamus, pituitary axis, and prevented luteinizing hormone (LH) surge. In these preparatory cycles, all the treated patients produced adequate secretory endometrium. Clinical trials of 27 oocyte donation cycles yielded one biochemical and eight clinical pregancies. Key words: in-vitro fertilization/oestrogen implants/oestrogen replacement/oocyte donation

Introduction In addition to ovarian failure, women with preserved ovarian function, including poor responders to ovarian stimulation and those undergoing repeat in-vitro fertilization (IVF) and embryo transfer failures, have increasingly become candidates for oocyte donation programmes. In this particular group of patients, the synchronization between donor and recipient cycles presents a special problem. Unwanted, ill-timed luteinizing hormone (LH) release often induces early secretory changes of the endometrium, and deleteriously affects the embryonic endometrial interaction. The combined use of gonadotrophin-releasing hormone analogues (GnRHa) and a high dose of oestrogens prior to treatment has been suggested as a possible solution to this problem (Meldrum et al., 1989; Yaron et al., 1995). Herein, we report our preliminary experience using micronized 17-β oestradiol implants in a group of selected patients with retained ovarian function who were treated by IVF with donated oocytes. © European Society for Human Reproduction and Embryology

Oocyte donation protocol The age of the population of patients used for this study ranged from 25 to 39 years, with a mean of 35 years. Every patient on the oocyte donation programme had an evaluation cycle before oocyte donation was performed. During the preparatory cycle, the patients were evaluated by determination of serum concentrations of oestradiol, progesterone, ultrasound measurement of endometrial thickness, as well as by analysis of endometrial biopsies on days 5, 7 and 9 after exposure to progesterone. The procedures for the hormonal assays were described elsewhere (Shulman et al., 1996). Patients with repeated implantation failure, and who repeatedly fail to achieve endometrial maturation, received s.c. oestradiol implants of 75–100 mg (Organon Laboratories Ltd, Cambridge, UK). The procedure was repeated when serum oestradiol concentrations declined to ,75 pg/ml. Endometrial biopsy study cycles These were carried out as follows: 14–16 days after progesteroneinduced bleeding, transvaginal ultrasonographic measurements of endometrial thickness were performed, and blood was obtained for oestradiol and plasma progesterone assays. If the endometrial thickness was ù8 mm and plasma progesterone ,1 ng/ml, i.m. administration of progesterone 50 mg (Gestone; Paine and Byrne Ltd., Greenford, UK) twice daily was begun. As previously described (Ben-Nun et al., 1992), endometrial samplings were performed on days 5, 7, and 9 of the progesterone treatment (days 19, 21, and 23 respectively of the cycle). The biopsies were obtained from the uterine fundus with a Novak curette or Pipelle de Cornier (Laboratoire CCD, Paris, France), and processed as previously described (Ben-Nun et al., 1989). The samples were

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Table I. Histological findings from endometrial biopsies obtained during the preparatory cycles, after 4 (5 day 19), 6 (5 day 21) and 8 (5 day 23) days of progesterone exposure Patient no. 1 2 3 4 5 6 7 8 9 10

Day 19

Day 21

Day 23

Glands

Stroma

Glands

Stroma

Glands

Stroma

17 17 16 18 16 17 16 16 16 15

17 17 16–22 18 16 17 16 16 16–22 22

19 21 22 20 18 N.A. N.A. 22 22 22

Decidua 17 – 20 18 N.A N.A. 22 22 22

22 24 22 22–23 FOC. 23 21 22 23 25 25

25 24 22 22–23 25 23 22–25 23 25 25

N.A.5 not available; FOC. 5 focal.

fixed in Bouin’s fluid and embedded in paraffin. Serial sections of 5 µm thickness were stained with haematoxylin and eosin. The dating was established by light microscopy, based on the criteria of Noyes et al. (1950). Embryo transfer cycles The patients were prepared as for the study cycles, and administration of twice daily i.m. progesterone 50 mg commenced between 8 and 60 days from the last vaginal bleeding (the proliferative phase length varied between 8 and 60 days). The embryo transfer was performed on the fifth day of progesterone supplementation. In the successful cycles (in which pregnancy was achieved), progesterone supplementation was discontinued after 8–9 weeks (Ben-Nun et al., 1989).

Table II. Clinical results Patient no.

Days since withdrawal bleeding

No. of replaced embryos

Outcome

1 2 4 5 5 6 7 7 7

N.A. 21 24 60 29 17 17 16 29

3 3 6 2 5 3 2 4 3

EUP Delivery Delivery Biochemical pregnancy Delivery Delivery First trimester abortion (twins) First trimester abortion Delivery

N.A. 5 not available; EUP 5 extra-uterine pregnancy.

Source of the donated oocytes According to the regulations of the Israeli Ministry of Health, oocytes only may be donated anonymously by patients undergoing IVF themselves. Therefore, our oocyte reservoir is from young (aged ,34 years), healthy women undergoing IVF, who have willingly consented to donate excess oocytes. Our own policy is to encourage them to donate a limited number of oocytes if more than 15 oocytes are retrieved. Usually up to three embryos are transferred; however, if extra embryos were available which were morphologically unsuitable for cryopreservation, then up to six embryos were transferred (Shulman et al., 1993).

Results The oestradiol concentrations varied between 75 and 240 pg/ml in the biopsy and oocyte donation treatment cycles. The plasma progesterone concentrations were ,1 ng/ml before exogenous progesterone administration and were .30 ng/ml thereafter. The endometrial thickness was ù10 mm. The results of the histological studies are presented in Table I. There were 27 embryo transfer cycles in which a total of 76 embryos were replaced (an average of 2.8 embryos/embryo transfer cycle). In nine cycles from six women, positive β-human chorionic gonadotrophin (HCG) values were obtained 12 days after embryo transfer. Of these, one was a biochemical pregnancy, one an extrauterine pregnancy, two ended as abortions and five were delivered. This resulted in a rate of 33.3% pregnancies per treatment cycle with an implanta2268

tion rate of 10.5%. Relevant data on the conception cycles are presented in Table II. Discussion The accumulated experience with the induction of artificial endometrial cycles in women with ovarian failure has demonstrated beyond a doubt that the human endometrium is highly conducive to hormonal manipulations (Ben-Nun et. al., 1992). Borini et al. (1995) found that menopausal women achieved higher implantation and pregnancy rates than cyclic women, although donors in both groups had equivalent pregnancy rates. Recently, Khastgir et al. (1997) achieved a pregnancy rate as high as 41.2% per treatment cycle in agonadal women with Turner’s syndrome. In contrast with agonadal women, patients with retained ovarian functions may begin monthly follicular development. As a consequence of feedback mechanisms, premature luteinization due to an uncontrolled LH surge may expose the endometrium to untimed progesterone secretion. In low responder patients prepared with oral oestrogens for oocyte donation, Remohi et al. (1993) demonstrated the presence of secretory endometrium in 36.4% of the biopsies obtained during the evaluation cycles prior to progesterone administration. Hoffman et al. (1993) and Shulman et al. (1996) have demonstrated decreased pregnancy rates in patients with rising progesterone secretion during the follicular phase

Induction of endometrial cycles with oestrogen implants

(subtle premature luteinization), despite similar oocyte and embryo quality. The sole explanation for the lower pregnancy rate was attributed to the possible deleterious effect of the progesterone on endometrial receptivity. Thus, the unexpected secretory changes of the endometrium caused by early LH rises in patients with ovarian function may deleteriously affect the embryonic endometrial interaction. There are some reports that very high doses of oral oestrogen (Yaron et al., 1995) may prevent this LH surge. Another modality is the continuous suppression of ovarian function by controlled release GnRHa with oral oestrogens and parenteral progesterone (Remohi et al., 1995). Borini et al. (1995) showed that, after GnRHa treatment, implantation and pregnancy rates were similar to those in menopausal women. The main limitation with the former is the risk in prolonged exposure of the patients to high doses of oestrogen, which may also require intra-treatment interruption intervals. Although Remohi et al. (1995) showed that prolonged oestrogen treatment was not deleterious to implantation, Yaron et al. (1995) found a drop in pregnancy rates with oestrogen administration prolonged beyond 35 days. Alternatively, the use of long-acting GnRHa in conjunction with oestrogen/progesterone substitution increases the treatment cost. The continuous oestrogen treatment achieved by the implants may have had a special impact in these cases. The maintenance of a constant plasma oestrogen concentration (Suhonem et al., 1993) resulted in continuous pituitary suppression (Nezhat et al., 1980). This condition would have prevented a resumption of LH secretion which conceivably might have occurred during the drug-free interval between the monthly cycles of hormone replacement therapy. Thus, the uninterrupted oestrogen action ensured a continuously oestrogen-rich endometrial environment and prevented deleterious untimed progesterone secretion. Non-oral oestrogen, e.g. transdermal oestradiol combined with progesterone, was found to be highly satisfactory for establishing a physiological endometrium in women with premature ovarian failure (Hung et al., 1989; Critchley et al., 1990; Massai et al., 1993). These studies showed that transdermal oestradiol patches, while being equally as effective as synthetic steroids, in inducing endometrial development in post-menopausal patients can also provide a more physiological approach that may conveniently and safely be extended into the second trimester of pregnancy. Oestradiol implants lower the sex hormone-binding globulin (SHBG) concentrations (Ben-Nun et al., 1995) and, as a consequence, lower concentrations of circulating oestradiol are sufficient for adequate endometrial response. After embryo implantation, oestrogen concentrations supplied by the implants provide sufficient long-term decidual support until the placental shift. If pregnancy is not achieved and progesterone is withdrawn, the endometrium may be ready again as soon as 8–10 days from menstruation. This is due to the continuous oestrogen milieu supported by the implants even during endometrial shedding . The exact time recommended for renewal of the implants is not known. When used for post-menopausal replacement therapy, Suhonen et al. (1993) found that a single application could give sufficient oestrogen substitution for .1 year.

However, the rate of decline of the serum oestradiol concentrations may vary (Gangar et al., 1989). For this reason we chose to renew them whenever the plasma oestradiol values fell to ,75 pg/ml. The results of the histological study clearly demonstrate the ability of the oestradiol implants and injectable progesterone to produce endometrial maturation compatible with embryo implantation and development. Furthermore, in cases where the oral oestrogens fail to induce endometrial maturation, the implants show progression of endometrial development paralleling the number of days of progesterone administration (Table II). In conclusion, endometrial preparation in women with retained ovarian function can be achieved with s.c. oestradiol and progesterone. The use of s.c. implants of oestrogen induces a down-regulation of the hypothalamus–pituitary–ovarian axis and provides windows of endometrial receptivity for embryo implantation in an oocyte donation programme. Although the data is derived from a small population of patients, our method constitutes a novel alternative which should be considered in oocyte donation programmes because of its simplicity and effectiveness.

Acknowledgments The authors thank Sally Esakov for her editorial remarks.

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I.Ben-Nun and A.Shulman Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25. Remohi, J., Vidal, A. and Pellicer, A. (1993) Oocyte donation in low responders to conventional ovarian stimulation for in vitro fertilization. Fertil. Steril., 59, 1208–1215. Remohi, J., Gallardo, E., Guanes, P.P. et al. (1995) Donor–recipient synchronization and the use of gonadotrophin-releasing hormone agonists to avoid the premature luteinizing hormone surge in oocyte donation. Hum. Reprod., 10 (Suppl. 2), 84–90. Shulman, A., Ben-Nun, I., Ghetler, Y. et al. (1993) Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertil. Steril., 60, 123–126. Shulman, A., Ghetler, Y., Beyth, Y. and Ben-Nun, I. (1996) The significance of an early (premature) rise of plasma progesterone in in vitro fertilization cycles induced by a ‘long protocol’ of gonadotropin releasing hormone analogue and human menopausal gonadotropins. J. Assist. Reprod. Genet., 13, 207–211. Suhonen, S.P., Allonen, H.O. and Lahteenmaki, P. (1993) Sustained release subdermal estradiol implants, a new alternative in estrogen replacement therapy. Am. J. Obstet. Gynecol., 169, 1248–1254. Yaron, Y., Amit, A., Mani, A. et al. (1995) Uterine preparation with estrogen for oocyte donation: assessing the effect of treatment duration on pregnancy rates. Fertil. Steril., 63, 1284–1286. Received on January 3, 1997; accepted on July 20, 1997

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