A rapid decline in serum oestradiol concentrations around the mid-luteal phase had no adverse effect on outcome in 763 assisted reproduction cycles

Ernest Hung Yu Ng1, William Shu Biu Yeung, Estella Yee Lan Lau, William Wai Ki So and Pak Chung Ho

Department of Obstetrics & Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People's Republic of China


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Progesterone is essential in the luteal phase whereas luteal oestradiol may play only a permissive role on the endometrium. However, a rapid decline in oestradiol concentrations around the mid-luteal period may compromise the endometrial integrity leading to poor IVF outcomes. A retrospective analysis of 763 women aged <40 years undergoing their first IVF cycle and having <=3 embryos replaced was undertaken. In cycles receiving human chorionic gonadotrophin (HCG) for luteal support, 25th, 50th and 75th centiles of the ratio of day-of-HCG oestradiol to mid-luteal oestradiol (oestradiol ratio) were 1.8, 2.8 and 5.0 respectively. Hormonal parameters were not different between pregnant and non-pregnant cycles. The outcomes were similar irrespective of the oestradiol ratio. Progesterone supplementation was used instead when the HCG oestradiol was >18 000 pmol/l or there were features of ovarian hyperstimulation syndrome. Pregnancy rates of these hyperstimulated cycles were 16.7 and 11.4% per cycle respectively when oestradiol ratio was <=5.0 and >5.0. This difference did not reach statistical significance. Our results could not find an adverse outcome in cycles showing a rapid decline in oestradiol during the mid-luteal phase.

Key words: implantation rate/IVF/luteal phase/oestradiol/pregnancy rate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The success of implantation depends upon a perfect dialogue between good quality embryos and a receptive endometrium. The endometrium in the follicular phase undergoes a progressive increase in thickness due to hyperplasia of both glandular and stromal elements under the stimulation of rising serum oestradiol concentration as a result of follicular development. In addition, oestradiol induces the synthesis of specific proteins and growth factors. Progesterone is essential in allowing implantation in the endometrium. Under the influence of progesterone, the endometrium undergoes sequential events including maturation, secretory transformation of the glandular elements, oedema formation, vascular proliferation and decidualization of the stroma in the luteal phase.

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., 1973aGo,bGo). 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, 1995Go; Ghosh and Sengupta, 1995Go).

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 IVF–embryo 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., 1988Go; Simón et al., 1995Go; Ng et al., 2000Go).

A recent report (Sharara and McClamrock, 1999Go) found that the ratio of day-of-HCG oestradiol to mid-luteal oestradiol was highly predictive of successful outcome in IVF–embryo 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 IVF–embryo transfer programme.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A retrospective study of infertile patients attending the Assisted Reproduction Unit at the Department of Obstetrics and Gynaecology, The University of Hong Kong, for conventional IVF or intracytoplasmic sperm injection (ICSI) treatment from early 1993 to December 1998 was undertaken. Ethical approval was not required for this retrospective analysis. The indications for conventional IVF (n = 637) included tubal (n = 268), male (n = 99), endometriosis (n = 90), unexplained (n = 112) and mixed factors (n = 68). ICSI (n = 126) was performed for couples with severe semen abnormalities where <106 motile spermatozoa were recovered after sperm preparation. In cases of obstructive azoospermia, surgically retrieved spermatozoa from epididymis or testis were used for ICSI.

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., 1997Go, 2000Go). 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 36–38 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 10–14 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 10–12 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 (2.5th–97.5th centiles). Hormonal data were log-transformed to correct for skewness prior to statistical analysis (Lenton et al., 1982Go). 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 Mann–Whitney U-test, Student's t-test, {chi}2-test and Kruskal–Wallis test, where appropriate. Correlation was assessed by the Pearson method. Two-tailed P < 0.05 was taken as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In all, 763 cycles were included in the analysis: 637 conventional IVF cycles and 126 ICSI cycles. Women were of median age 34.0 years (2.5th–97.5th centiles: 28–37 years). The serum oestradiol concentrations on the day of ovulatory dose of HCG was 8207 ± 181 pmol/l (geometric mean ± SEM) and the median number of oocytes obtained was 8.0 (2.5th–97.5th centiles: 3–21). The median fertilization and cleavage rates were 82.6% (2.5th–97.5th centiles: 21.4–100%) and 100% (2.5th–97.5th centiles: 80.0–100%) respectively. The pregnancy rate was 128/763 (16.8%) per transfer cycle.

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 IGo). 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 IIGo).


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Table I. Comparison of parameters of pregnant and non-pregnant cycles in patients receiving human chorionic gonadotrophin supplementation
 

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Table II. Comparison of hormonal profile of pregnant and non-pregnant cycles in patients receiving human chorionic gonadotrophin (HCG) supplementation
 
The 25th, 50th and 75th centiles of the ratio of day-of-HCG oestradiol to mid-luteal oestradiol, i.e. oestradiol ratio (day 0/ day 10) were 1.8, 2.8 and 5.0 respectively. The outcomes were categorized into four groups according to the quartiles of the above oestradiol ratio: group A <1.8; group B 1.8–2.79; group C 2.8–5.0; and group D >5.0. Pregnancy rate, implantation rate, multiple pregnancy rate and outcome of pregnancy were not significantly different among these four groups (Table IIIGo). The oestradiol ratio (day 0/day 10) was significantly correlated with the total number of eggs obtained (r = –0.093, P = 0.015) but not correlated with day-of-HCG oestradiol concentrations, after log-transformation of the data.


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Table III. Comparison of IVF outcomes with different oestradiol ratio (day 0/day 10) in patients receiving human chorionic gonadotrophin supplementation
 
Progesterone as luteal support
No differences were observed in all demographic and ovarian response parameters when pregnant cycles were compared to non-pregnant cycles (data not shown). A similar number of embryos were replaced in pregnant and non-pregnant cycles. The hormonal profiles were also not different between pregnant and non-pregnant cycles on day 0 and day 10 (Table IVGo).


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Table IV. Comparison of hormonal profile of pregnant and non-pregnant cycles in patients receiving progesterone supplementation
 
The 25th, 50th and 75th centiles of oestradiol ratio (day 0/day 10) in these cycles were 3.5, 6.3 and 11.4 respectively. The outcomes were categorized into four groups according to the quartiles of the above oestradiol ratio: Group A <3.5; Group B 3.5–6.29; Group C 6.3–11.4 and Group D >11.4. Pregnancy rate, implantation rate, multiple pregnancy rate, and outcome of pregnancy were not significantly different among these four groups (Table VGo). Difference in pregnancy and implantation rates did not reach statistical significance when the oestradiol ratio (day 0/day 10) was categorized into <=5.0 and >5.0 (Figure 1Go). The oestradiol ratio (day 0/day 10) was not correlated with the total number of eggs obtained and day-of-HCG oestradiol concentrations, after log-transformation of the data.


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Table V. Comparison of IVF outcomes with different oestradiol ratio (day 0/day 10) in patients receiving progesterone supplementation
 


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Figure 1. Comparison of pregnancy (PR) and implantation (IR) rates of cycles with oestradiol ratio <=6.3 and >6.3 in patients receiving progesterone supplementation.

 
HCG versus progesterone as luteal support
Compared with cycles receiving HCG supplementation: oestradiol on day 0, progesterone on day 10, oestradiol/progesterone on day 10 and oestradiol ratio (day 0/day 10) were significantly higher in cycles receiving progesterone supplementation (Table VIGo).


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Table VI. Comparison of hormonal profile of cycles with human chorionic gonadotrophin (HCG) and progesterone supplementation
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of oestradiol in the luteal phase seems to differ from species to species. In rodents, oestradiol appears essential for implantation. Blastocysts of rats and mice could implant only after the rise of luteal oestradiol around day 4 of gestation and they would remain in a state of metabolic quiescence as long as enough progesterone was available (McLaren, 1973Go; Finn, 1977Go). In monkeys, the data on this issue are more controversial (Meyer et al., 1969Go; Bosu and Johansson, 1975Go; Ghosh et al., 1994Go).

When spontaneous cycles of normal fertile women were studied, significantly higher oestradiol concentrations were shown in serum (Stewart et al., 1993Go), in saliva (Lipson and Ellison, 1996Go) and in urine (Baird et al., 1997Go) 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., 1982Go). 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 IVF–embryo transfer. Luteal phase serum oestradiol and progesterone concentrations were not related to the outcome of IVF treatment (Muasher et al., 1984Go; Mettler and Michelmann, 1987Go; Nylund et al., 1990Go). 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 IVF–embryo 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; 2.5th–97.5th centiles: 2–3) was significantly higher than the non-pregnant group (median 3; 2.5th–97.5th centiles: 1–3). 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 (50–100 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, 1990Go; Stassart et al., 1995Go; Zegers-Hochschild and Altieri, 1995Go). 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., 1992Go; Younis et al., 1994Go). 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., 1992Go). 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, 1995Go).

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 IVF–embryo transfer cycles (Ng et al., 2000Go). Excess embryos from cycles with different oestradiol concentrations, however, had similar outcomes in frozen–thawed 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., 1988Go).

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., 1993Go).

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
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. E-mail: nghye{at}hkucc.hku.hk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 7, 2000; accepted on June 2, 2000.