Department of Obstetrics & Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People's Republic of China
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: implantation rate/IVF/luteal phase/oestradiol/pregnancy rate
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of progesterone in the luteal phase is well established. After removal of the corpus luteum at seven weeks of pregnancy, normal pregnancy was sustained when progesterone (200 mg/day) was given i.m. whereas pregnancy loss could not be prevented if these patients were instead treated with 1 mg oestradiol per day (Csapo et al., 1973a,b
). The role of oestradiol in the luteal phase is, however, unclear and it is suggested that it only plays a permissive rather than an obligatory role (de Ziegler, 1995
; Ghosh and Sengupta, 1995
).
Ovarian stimulation has been extensively used in the majority of assisted reproduction units in order to improve the success rate by increasing the number of oocytes and thus the number of embryos to be replaced. The resulting supraphysiological concentrations of oestradiol have significant implications to the outcomes of IVFembryo transfer treatment. Significantly lower implantation and pregnancy rates were shown in cycles with high serum oestradiol concentrations on the ovulatory human chorionic gonadotrophin (HCG) day (Forman et al., 1988; Simón et al., 1995
; Ng et al., 2000
).
A recent report (Sharara and McClamrock, 1999) found that the ratio of day-of-HCG oestradiol to mid-luteal oestradiol was highly predictive of successful outcome in IVFembryo transfer. The ongoing pregnancy rate and implantation rate were significantly impaired if the above ratio was >5. It was postulated that the endometrial integrity might become compromised when there was a dramatic decline in oestradiol concentrations around the mid-luteal period as reflected by a high ratio.
There are very few studies in the literature to clarify the importance of rapid decline of oestradiol around the mid-luteal phase in IVF treatment, especially with regard to cycles with high oestradiol concentrations on the day of ovulatory dose of HCG. The purpose of this study was to examine the effects of the decline of serum oestradiol concentrations around the mid-luteal phase on pregnancy rate and implantation rate in an IVFembryo transfer programme.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Couples had to fulfil the following criteria before they were included in this study in order to control for all confounding factors as far as possible. (i) Only the first treatment cycle was considered. (ii) The age of the women should be <40 years. (iii) A maximum of three embryos was replaced in the cycles.
IVF or ICSI cycles
The details of the long protocol of ovarian stimulation regimen used at our centre had been previously published (Ng et al., 1997, 2000
). In short, women were all pre-treated with a gonadotrophin-releasing hormone (GnRH) analogue, buserelin (Suprecur; Hoechst, Frankfurt, Germany) nasal spray 150 µg four times a day from the mid-luteal phase of the cycle preceding the treatment cycle. Pituitary down-regulation was confirmed by both transvaginal scanning and serum oestradiol performed on the second day of the treatment cycle. Human menopausal gonadotrophin (HMG: 75 IU FSH and LH; Pergonal, Serono, Switzerland; or Humegon; Organon, The Netherlands) injections were then started. The ovarian response was monitored by serial transvaginal scanning and serum oestradiol concentrations. HCG (Profasi, Serono, Switzerland) 10 000 IU was given i.m. when the leading follicle reached 18 mm in diameter and there were at least three follicles >15 mm in diameter. The day of this HCG injection was designated as day 0. Transvaginal ultrasound-guided oocyte retrieval was scheduled 3638 h after the HCG injection. All follicles >10 mm on scanning were aspirated and flushed once with culture medium to improve the recovery of eggs.
A maximum of three normally cleaved embryos were replaced into the uterine cavity 48 h after the retrieval. Excess good quality embryos were frozen for subsequent transfer if the woman did not conceive in that cycle. Luteal phase was supported by 1500 IU HCG injections on the day of embryo transfer and 6 days later. When serum oestradiol concentration on the day of ovulatory HCG was >18 000 pmol/l or features suggestive of ovarian hyperstimulation syndrome (OHSS) were present, progesterone injections (50 mg i.m. daily, Weimer Pharma, Rastatt, Germany) or vaginal pessaries (Cyclogest 400 mg twice daily; Cox Pharmaceuticals, Barnstaple, UK) were used instead from the day of embryo transfer for 10 days. Serum oestradiol and progesterone concentrations were measured 6 days after embryo transfer (day 10, mid-luteal phase). A urine pregnancy test was done 16 days after embryo transfer. If it was positive, ultrasound examination was performed 1014 days later to confirm intrauterine pregnancy and to determine the number of gestation sacs present.
Oestradiol was measured using a commercially available radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The inter- and intra-assay variabilities on high concentration control (oestradiol: 1082 pg/ml) were 4.2 and 4.0% respectively. Progesterone was measured using a commercially available radioimmunoassay kit (Chiron Diagnostics Corporation, MA, USA) and the inter-assay and intra-assay variabilities were 9.4 and 8.4% respectively.
Statistical analysis
Fertilization rate was defined as the proportion of oocytes resulting in two pronuclei formation and only metaphase II oocytes were counted in ICSI cycles. Only clinical pregnancies were considered in this study. A clinical pregnancy is defined by the presence of one or more gestation sacs by ultrasound, including ectopic pregnancy or the demonstration of gestational product in the uterine evacuate. On-going pregnancies were those pregnancies beyond 1012 weeks of gestation, at which stage the patients were referred out for antenatal care. Mean implantation rate was the proportion of embryos transferred resulting in an intrauterine gestational sac.
As the data were not normally distributed, continuous data were expressed as median (2.5th97.5th centiles). Hormonal data were log-transformed to correct for skewness prior to statistical analysis (Lenton et al., 1982). Cycles receiving HCG and progesterone supplementation were analysed separately. Data on the type of treatment (IVF/ICSI), the age of women, the duration/dosage of HMG used, the number of oocytes obtained/fertilized, the fertilization/cleavage rates, the pregnancy/implantation rates and the pregnancy outcomes were compared between pregnant and non-pregnant cycles. Statistical comparisons were carried out by MannWhitney U-test, Student's t-test,
2-test and KruskalWallis test, where appropriate. Correlation was assessed by the Pearson method. Two-tailed P < 0.05 was taken as significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HCG as luteal support
The number of oocytes fertilized and the number of embryos cleaved/replaced/frozen were significantly higher in pregnant cycles than non-pregnant cycles (Table I). There were no differences in the type of treatment, the age of women, HMG dosage/duration, number of oocytes obtained, fertilization rate and cleavage rate between pregnant and non-pregnant cycles. Hormonal parameters were similar in pregnant and non-pregnant cycles on day 0 and day 10 (Table II
).
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When spontaneous cycles of normal fertile women were studied, significantly higher oestradiol concentrations were shown in serum (Stewart et al., 1993), in saliva (Lipson and Ellison, 1996
) and in urine (Baird et al., 1997
) during the mid-luteal phase of pregnant cycles. The significant increase in mid-luteal oestradiol and progesterone concentrations in pregnant cycles occurred 4 days before the average time of appearance of HCG in normal pregnant cycles. Stewart et al. (1993) suggested that the enhanced ovarian steroid secretion in pregnant cycles could be due to a gonadotrophic stimulus such as HCG from the preimplantation embryo. In contrast to fertile subjects, mid-luteal oestradiol and progesterone concentrations during spontaneous cycles were similar between pregnant and non-pregnant cycles of infertile patients (Laufer et al., 1982
). The explanation for this difference between fertile and infertile women was still unknown.
There is little recent information in humans with regard to the importance and necessity of luteal oestradiol during stimulated cycles in patients undergoing IVFembryo transfer. Luteal phase serum oestradiol and progesterone concentrations were not related to the outcome of IVF treatment (Muasher et al., 1984; Mettler and Michelmann, 1987
; Nylund et al., 1990
). On the other hand, Gidley-Baird et al. (1986) and Hutchinson-Williams et al. (1989) found significantly higher oestradiol concentrations during the mid-luteal period in pregnant than non-pregnant cycles. Different luteal supports including no support, HCG and progesterone were employed and this may explain the different results observed in these studies. Pituitary down-regulation was also not used in these studies. In this study, hormonal parameters were not significantly different on day 0 and day 10 between pregnant and non-pregnant groups in cycles receiving HCG and progesterone as luteal support.
Sharara and McClamrock (1999) recently found an adverse outcome in 106 IVFembryo transfer cycles using progesterone supplementation when the ratio of day-of-HCG oestradiol to mid-luteal oestradiol was >5. Impaired endometrial receptivity was suggested to be caused by a rapid decline in oestradiol concentrations in the luteal phase, i.e. a high oestradiol ratio. In our programme, HCG was usually given on the day of embryo transfer and 6 days after embryo transfer for the luteal support in cycles with oestradiol on the ovulatory HCG day <18 000 pmol/l. Our data showed that the number of embryos replaced in the pregnant cycles (median 3; 2.5th97.5th centiles: 23) was significantly higher than the non-pregnant group (median 3; 2.5th97.5th centiles: 13). Our results, however, could not confirm an adverse effect of high oestradiol ratio around the mid-luteal phase on the outcomes in cycles receiving HCG supplementation because similar outcomes were observed in cycles with different oestradiol ratios.
The results of our study were different from those of Sharara and McClamrock (1999). Their patients received either long or flare-up protocol of GnRH analogue and flushing of follicles was not performed after egg collection. Luteal phase was supported by i.m. progesterone (50100 mg per day) only and oestradiol and progesterone concentrations were measured on day 8 after the ovulatory HCG injection. All our patients were down-regulated by taking Buserelin nasal spray and each follicle was flushed once after aspiration. Cycles supported with HCG and progesterone in the luteal phase were separately analysed. We checked serum oestradiol and progesterone concentrations 10 days after the HCG injection. It remains uncertain whether the discrepancy of the results could be due to the different protocols of two programmes.
Menopausal or functionally agonadal women could achieve donor oocyte pregnancies without support of oestradiol in the luteal phase (Kapetanakis and Pantos, 1990; Stassart et al., 1995
; Zegers-Hochschild and Altieri, 1995
). The permissive function of luteal oestradiol in humans was also suggested by the examination of endometrial development. Mid-luteal endometrial morphology of glandular and stromal elements was not significantly affected by the cessation of oestradiol treatment and the reduction of oestradiol concentration to hypogonadic levels in the luteal phase after an adequate follicular priming (de Ziegler et al., 1992
; Younis et al., 1994
). Endometrial maturation assessed by oestrogen and progesterone receptors showed the typical distribution of the normal mid-luteal endometrium in those patients with and without oestradiol supplementation in the luteal phase (de Ziegler et al., 1992
). Our data on cycles receiving HCG supplementation may add further support to a permissive rather than obligatory role of luteal oestradiol during the peri-implantation period in humans (Ghosh and Sengupta, 1995
).
The suggestion that luteal oestradiol is not essential for normal endometrial development has not been examined in the hyperstimulated cycles. We have recently shown an association between serum oestradiol >20 000 pmol/l on the day of HCG injection and impaired implantation and pregnancy rates in fresh IVFembryo transfer cycles (Ng et al., 2000). Excess embryos from cycles with different oestradiol concentrations, however, had similar outcomes in frozenthawed transfer cycles. These data suggested that impairment in implantation was likely to be related to an adverse environment in the endometrium resulting from high serum oestradiol concentrations. To the best of our knowledge, this is the first study examining the role of oestradiol and the decline of oestradiol concentrations during the mid-luteal phase on implantation rate and pregnancy rates of these hyperstimulated cycles.
In our programme, patients whose oestradiol concentrations on the day of HCG injection were >18 000 pmol/l or had features suggestive of OHSS would receive progesterone supplementation only in order to reduce the risk of OHSS. Hormonal parameters on day 0 and day 10 were again not found to be significantly different between pregnant and non-pregnant cycles. The pregnancy rates were 16.7% and 11.4% per cycle respectively when oestradiol ratio (day 0/day 10) was 5.0 and >5.0. There seems to be a trend of a higher pregnancy rate in those cycles with lower oestradiol ratio (day 0/day 10) or less rapid decline of oestradiol concentrations in the mid-luteal phase. De Ziegler et al. (1992) and Younis et al. (1994) only examined the endometrial biopsies taken from cycles with oestradiol concentrations <10 000 pmol/l. Moreover, de Ziegler et al. (1992) showed that endometrial morphology in those women not taking oestradiol in the luteal phase had smaller subnuclear vacuoles and these vacuoles were present in <50% of glandular cells. It will be important to examine the endometrium from cycles with oestradiol concentrations >20 000 pmol/l and to compare the differences between cycles with low and high oestradiol ratios. Moreover, morphometry rather than traditional dating should be used to examine the endometrium in these cycles in order to detect subtle differences in the morphology (Li et al., 1988
).
The reasons for a higher oestradiol ratio in some of the cycles indicating a more rapid decline in oestradiol concentrations around the mid-luteal phase were unknown. Oestradiol ratio (day 0/day 10) was not correlated with oestradiol concentrations on the day of HCG in cycles receiving HCG or progesterone supplementation whereas it was negatively correlated with the total number of oocytes obtained in cycles receiving HCG supplementation. The routine supplementation with oestradiol in addition to intravaginal progesterone luteal support was, however, not shown to affect the pregnancy rates of down-regulated IVF cycles in a prospective randomized study (Smitz et al., 1993).
In conclusion, our results could not find an adverse outcome in the cycles showing a rapid decline in oestradiol concentrations during the luteal phase whether HCG or progesterone supplementation was given. The endometrium from these hyperstimulated cycles should be examined by morphometry to detect any subtle difference when there is a rapid decline of oestradiol concentrations around the mid-luteal phase.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bosu, W.T.K. and Johansson, E.D.B. (1975) Implantation and maintenance of pregnancy in mated rhesus monkeys following bilateral oophorectomy or lutectomy with and without progesterone replacement. Acta Endocrinol., 79, 598609.[ISI][Medline]
Csapo, A.I., Pulkkinen, M.O. and Wiest, W.G. (1973a) Effects of lutectomy and progesterone replacement therapy in early pregnant patients. Am. J. Obstet. Gynecol., 115, 759765.[ISI][Medline]
Csapo, A.I., Pulkkinen, M.O. and Kaihola, H.L. (1973b) The effect of estradiol replacement therapy on early pregnant luteectomized patients. Am. J. Obstet. Gynecol., 117, 987990.[ISI][Medline]
de Ziegler, D. (1995) Hormonal control of endometrial receptivity. Hum. Reprod., 10, 47.[ISI][Medline]
de Ziegler, D., Bergeron, C., Cornel, C. et al. (1992) Effects of luteal oestradiol on the secretory transformation of human endometrium and plasma gonadotrophins. J. Clin. Endocrinol. Metab., 74, 322331.[Abstract]
Finn, C.A. (1977) The implantation reaction. In Wynn, R.M. (ed.), Biology of the Uterus. Plenum Press, New York, pp. 245308.
Forman, R., Fries, N., Testart, J. et al. (1988) Evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation. Fertil. Steril., 49, 118122.[ISI][Medline]
Ghosh, D. and Sengupta, J. (1995) Another look at the issue of peri-implantation oestrogen. Hum. Reprod., 10, 12.[ISI][Medline]
Ghosh, D., De, P. and Sengupta, J. (1994) Luteal phase ovarian oestrogen is not essential for implantation and maintenance of pregnancy from surrogate embryo transfer in the rhesus monkey. Hum. Reprod., 9, 629637.[Abstract]
Gidley-Baird, A.A., O'Neill, C., Sinosich, M.J. et al. (1986) Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in humans and mice. Fertil. Steril., 45, 6974.[ISI][Medline]
Hutchinson-Williams, K., Lunenfeld, B., Diamond, M.P. et al. (1989) Human chorionic gonadotropin, estradiol, and progesterone profiles in conception and nonconeption cycles in an in vitro fertilization program. Fertil. Steril., 52, 441445.[ISI][Medline]
Kapetanakis, E. and Pantos, K.J. (1990) Continuation of a donor oocyte pregnancy in menopause without early pregnancy support. Fertil. Steril., 54, 11711173.[ISI][Medline]
Laufer, N., Navot, D. and Schenker, J.G. (1982) The pattern of luteal phase plasma progesterone and estradiol in fertile cycles. Am. J. Obstet. Gynecol., 143, 808813.[ISI][Medline]
Lenton, E.A., Sulaiman, R., Sobowale, O. et al. (1982) The human menstrual cycle: plasma concentrations of prolactin, LH, FSH, oestradiol and progesterone in conceiving and non-conceiving women. J. Reprod. Fertil., 65, 131139.[Abstract]
Li, T.C., Rogers, A.W., Dockery, P. et al. (1988) A new method of histologic dating of human endometrium in the luteal phase. Fertil. Steril., 50, 5260.[ISI][Medline]
Lipson, S.F. and Ellison, P.T. (1996) Comparison of salivary steroid profiles in naturally occurring conception and non-conception cycles. Hum. Reprod., 11, 20902096.[Abstract]
McLaren, A. (1973) Blastocyst activation. In Segal, S.J., Crozier, R., Corfman, P.A. and Condliffe, P. (ed.), Regulation of Mammalian Reproduction. Charles C.Thomas, Springfield, pp. 321328.
Mettler, L. and Michelmann, H.W. (1987) Estradiol values under gonadotropin stimulation in relation to the outcome of pregnancies in in vitro fertilization and embryo transfer. J. In Vitro Fertil. Embryo Transfer, 4, 303306.[ISI][Medline]
Meyer, R.K., Wolf, R.C. and Arslan, M. (1969) Implantation and maintenance in progesterone treated ovariectomized monkeys (Macaca mulatta). In Hoffer, S. (ed.), Recent Advances in Primatology. Karger, Basel, pp. 3035.
Muasher, S., Acosta, A.A., Garcia, J.E. et al. (1984) Luteal phase serum estradiol and progesterone in in vitro fertilization. Fertil. Steril., 41, 838843.[ISI][Medline]
Ng, E.H.Y., Yeung, W.S.B. and Ho, P.C. (1997) The presence of hydrosalpinx may not adversely affect the implantation and pregnancy rates in in vitro fertilization treatment. J. Assist. Reprod. Genet., 14, 508512.[ISI][Medline]
Ng, E.H.Y., Yeung, W.S.B., Lau, E.Y.L. et al. (2000) High serum oestradiol levels in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent FET cycles. Hum. Reprod., 15, 250255.
Nylund, L., Beskow, C., Carlström, K. et al. (1990) The early luteal phase in successful and unsuccessful implantation after IVFET. Hum. Reprod., 5, 4042.[Abstract]
Sharara, F.I. and McClamrock, H.D. (1999) Ratio of oestradiol concentration on the day of human chronic gonadotrophin administration to mid-luteal oestradiol concentration is predictive of in-vitro fertilisation outcome. Hum. Reprod., 14, 27772782.
Simón, C., Cano, F., Valbuena, D. et al. (1995) Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients. Hum. Reprod., 10, 24322437.[Abstract]
Smitz, J., Bourgain, C., Van Waesberghe, L. et al. (1993) A prospective randomized study on oestradiol valerate supplementation in addition to intravaginal micronized progesterone in buserelin and HMG induced superovulation. Hum. Reprod., 8, 4045.[Abstract]
Soliman, S., Daya, S., Gollins, J. et al. (1994) The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. Fertil. Steril., 61, 10681076.[ISI][Medline]
Stassart, J.P., Corfman, R.S. and Ball, G.D. (1995) Continuation of a donor oocyte pregnancy in a functionally agonadal patient without early oestrogen support. Hum. Reprod., 10, 30613063.[Abstract]
Stewart, D.R., Overstreet, J.W., Nakajima, S.T. et al. (1993) Enhanced ovarian steroid secretion before implantation in early human pregnancy. J. Clin. Endocrinol. Metab., 76, 14701476.[Abstract]
Younis, J.S., Ezra, Y., Sherman, Y. et al. (1994) The effect of estradiol depletion during the luteal phase on endometrial development. Fertil. Steril., 62, 103107.[ISI][Medline]
Zegers-Hochschild, F. and Altieri, E. (1995) Luteal estrogen is not required for the establishment of pregnancy in the human. J. Assist. Reprod. Genet., 12, 224228.[ISI][Medline]
Submitted on February 7, 2000; accepted on June 2, 2000.