Gonadotropin Releasing Hormone (GnRH) agonists, used for down-regulation in long agonist In Vitro Fertilization (IVF) cycles, work by preventing premature surges of endogenous Luteinizing Hormone (LH) through pituitary suppression, allowing time for a larger number of oocytes to reach maturity prior to harvesting. Even though the agonist treatment ends abruptly on day of Human Chorionic Gonadotropin (hCG) trigger, endogenous LH secretion can remain suppressed for as long as 10 days after treatment with a long acting GnRH agonist ends. Due to prolonged suppression of endogenous LH secretion, luteal function is frequently inadequate in amount and duration and subsequent progesterone and estradiol secretion is abnormal. Without proper progesterone or oestrogen support, endometrial receptivity is compromised, leading to decreased implantation and pregnancy rates. If luteal phase support is not provided in assisted reproduction technique cycles, the serum estradiol and progesterone levels drop, thus leading to a decrease in implantation rates and pregnancy rates.
Theoretically, hCG might offer advantages over other forms of luteal support because it stimulates the corpus luteum, both estradiol and progesterone levels are increased, along with any other factors which may facilitate implantation. However in comparative trials, its efficacy appears no greater than progesterone and several studies have demonstrated that hCG supplementation increases the risk of ovarian hyperstimulation syndrome.
The only consensus is that luteal phase supplementation improves outcomes in IVF cycles. Numerous clinical trials have compared clinical, ongoing, or delivered pregnancy rates and spontaneous miscarriage rates between groups receiving treatment with different luteal phase support regimens, with varying results. Progesterone supplementation during luteal phase is a routine treatment throughout the world with different routes and doses of administration [1].
The corpus luteum produces not only progesterone but also estradiol during the luteal phase. This has led to emergence of concept of addition of estradiol during luteal phase in IVF cycles.
The ideas about using estradiol for luteal phase support are conflicting; some reports have favoured the addition of estradiol supplementation [2-5], while others have failed to observe any beneficial effect [6-10]. Hence the role of addition of estradiol to progesterone as luteal phase support in women undergoing IVF is not yet clear. Also, no such study has been done in Indian population till now. To the best of our knowledge, our study is the first study being done in Indian population, to determine efficacy of estradiol along with progesterone as luteal phase support in women undergoing IVF. The present study was attempted to determine the effect of additional luteal phase support with estradiol on the hormonal profiles and pregnancy rates in women undergoing IVF.
Materials and Methods
Patient Population: The study was a randomized controlled trial conducted in the IVF and Reproductive Biology Centre of a tertiary care institute and medical college of Delhi from October 2011 to March 2013. Sample size was calculated as 150, it was the sample size of convenience.
Out of 187 women screened, 150 women fulfilling the inclusion and exclusion criteria of the study were recruited and randomly divided into two groups of 75 women each (using computerized randomization).
The inclusion criteria were: 1) Age 20 to 39 years, 2), Women undergoing IVF by Long agonist protocol.
The exclusion criteria was: 1) Serum Ethinyl Estradiol (E2) level >3000 pg/mL on day of oocyte retrieval {because of risk of Ovarian Hyperstimulation Syndrome (OHSS)}; 2) Diminished ovarian reserve (Day 2 serum Follicle Stimulating Hormone(FSH)> 10 mIU/mL); 3) Endometriosis greater than stage 2; 4) Severe male factor (<5 million/mL motile spermatozoa); 5) Endocrine disorders; 6) Medical or surgical illness.
Ethical clearance was taken from the institutional ethical committee vide letter number F.No.11/IEC/MAMC/2011/246. Trial was registered with Clinical Trial Registry of India with the number CTRI/2017/10/010083. Written informed consent was obtained from all the women enrolled in the study.
Ovulation Induction: All women underwent controlled ovarian hyperstimulation by the “long” agonist protocol. Down regulation of the cycle was achieved by injection Leuprolide acetate (Sun Pharmaceuticals, India) 0.5 mg per day, subcutaneously, starting from day 21 of menstrual cycle. On the second day of next cycle, controlled ovarian stimulation was started using recombinant or urinary gonadotropins or human menopausal gonadotropins. The starting dose was 225 IU/ day of recombinant FSH (Bharat Serums and Vaccines Limited, India). The response to stimulation was monitored with serial measurements of serum estradiol and trans vaginal ultrasound imaging of ovarian follicles. Most women required a total of 9-12 days of stimulation. It was aimed to have at least two follicles measuring 17-18 mm in mean diameter, ideally accompanied by a few others in the 14-16 mm range, and a serum estradiol concentration that is consistent with the overall size and maturity of the cohort (approximately 200 pg/mL per follicle measuring 14 mm or greater). Once the targeted thresholds of response were met, hCG (10000 IU) was administered subcutaneously to induce follicular maturation. Oocyte retrieval was performed after 34-36 hours of hCG trigger, transvaginal ultrasound guided aspiration under general anesthesia being the technique. Fertilization was achieved by conventional microinsemination or by ICSI in case of male factor. Embryo transfer (ET) was performed three days after oocyte retrieval; transcervical ultrasound guided, using Sydney IVF Embryo Transfer Catheter Set (K-JETS-7019-SIVF, manufactured by Cook’s Medical, USA). Urine Pregnancy Test (UPT) and serum level of beta hCG was done 14 days after ET. Positive UPT and /or serum beta hCG more than 50 mIU per mL indicated successful implantation. Women with positive UPT were followed up for detection of cardiac activity after two weeks.
Luteal Phase Support: Luteal phase support was started on the day of ET in both groups of women as follows:
Study group: Estradiol valerate, 2mg tablet orally, twice daily (BayerZydus Pharmaceuticals Private Limited, India) and Micronized progesterone, 400mg, softgel capsule, vaginally, twice daily (Sun Pharmaceuticals, India).
Control group: Micronized progesterone, 400mg, softgel capsule, vaginally, twice daily (Sun Pharmaceuticals, India).
Specimen Collection and Hormone Analysis: Blood samples (4 mL) for estimation of serum E2 was collected in plain vials on the day of oocyte retrieval (here after referred to as Day 0), on the day of ET (Day 3), three days after ET (Day 6), on the day of confirmation of biochemical pregnancy (Day 17). Serum levels of E2 were estimated by Radio Immuno Assay.
Statistical Analysis
Statistical analysis of difference between study and control group was done using Statistical Package for Social Sciences (SPSS) Version 16.0. The two groups were analyzed using the Pearson chi-square test, Mann-Whitney U test and student t-test, as indicated. The p-value of less than 0.05 was considered significant.
Results
There was no difference in the two groups with respect to demographic features, duration, type and etiological factors for infertility [Table/Fig-1].
Characteristics of the two study groups.
| Variable | Study Group(E+P)N=75 | Control Group(Only P)N=75 | p-value |
|---|
| Age (years) | 31.62±4.15 | 30.68±4.01 | 0.16 |
| Duration of infertility (years) | 7.98±4.40 | 8.38±4.10 | 0.58 |
| Type of Infertility (%)(Primary/Secondary) | 70.7/29.3 | 78.7/21.3 | 0.26 |
| Aetiological factors for infertility (%) |
| Tubal factor | 17.3 | 12 | 0.17 |
| Male factor | 45.3 | 62.7 |
| Unexplained | 30.7 | 22.7 |
| Endometriosis | 6.7 | 2.7 |
The overall pregnancy rate in the study was 38.7% (58/150). The pregnancy rate was observationally higher in the study group (41.3%) as compared to the control group (36%) but the difference was not statistically significant (p-value = 0.50).
The serum level of E2 in the study and control group on days 0, 3, 6, 17 were compared, as depicted in [Table/Fig-2] and also in women who conceived and those who didn’t conceive, in [Table/Fig-3].
Comparison of Serum E2 levels in the two groups.
| Study Group(E+P)N=75 | Control Group(Only P)N=75 | p-value |
|---|
| E2 level (pg/mL) |
| Day 0 | 2028.16±480.26 | 2059.95±511.23 | 0.47 |
| Day 3 | 1697.78±434.91 | 1633.78±434.82 | 0.40 |
| Day 6 | 1425.08±389.02 | 1204.44±312.50 | <0.001 |
| Day 17 | 460.93±418.19 | 312.98±268.43 | 0.03 |
| Percentage E2 decline from Day 0 to Day 6 (%) | 29.98±8.61 | 40.92±8.82 | <0.001 |
| Day 0 E2: Day 6 E2 ratio | 1.45±0.22 | 1.73±0.28 | <0.001 |
Comparison of Serum E2 levels in pregnant and non pregnant women.
| Pregnant(N=58) | Non Pregnant(N=92) | p-value |
|---|
| E2 level (pg/mL) |
| Day 0 | 2120.57±538.48 | 1995.82±461.31 | 0.23 |
| Day 3 | 1760.41±465.46 | 1606.17±405.27 | 0.06 |
| Day 6 | 1403.23±376.67 | 1258.98±354.31 | 0.02 |
| Day 17 | 541.80±498.01 | 289.34±171.94 | <0.001 |
| Percentage E2 decline from Day 0 to Day 6 (%) | 33.29±9.31 | 36.81±10.66 | 0.02 |
| Day 0 E2: Day 6 E2 ratio | 1.53±0.25 | 1.63±0.30 | 0.03 |
The percentage fall in E2 level from Day 0 to Day 6 was significantly lower in study group, as compared to control group. Also, the women who conceived had a significantly lower percentage fall in E2 levels, as compared to the women who did not conceive. The ratio of Day 0 E2 level to Day 6 E2 level was significantly lower in the study group compared to the control group. Also, the women who conceived had a significantly lower Day 0 E2: Day 6 E2 ratio compared to those who did not conceive [Table/Fig-2].
Discussion
Oestrogen during luteal phase has a modulatory effect on the endometrial progesterone receptor concentration and serves to replenish and maintain a sufficient level of progesterone receptors to mediate and complete progesterone response. This has been proved by studies on the effect of luteal phase oestrogen antagonism on endometrial development and luteal function in women. As mentioned earlier, the results of addition of estradiol as luteal phase support are conflicting. Even the results of meta-analyses are also not clear. After thorough search of literature, we did not find any study done in Indian population in this regard. Kolibianakis WM et al., Gelbaya TA et al., and Jee BC et al., suggested that the addition of E2 to P for luteal phase support does not improve IVF outcomes [11-13]. On the other hand, meta-analysis done by Huang N et al., and Pinhero LMA et al., reported conflicting results and suggested further trials to determine the beneficial effect of estradiol administration in luteal phase [14,15]. Another meta-analysis by Zhang XM et al., suggested improved pregnancy rates with addition of oestrogen [16]. In our study, the pregnancy rate was observationally higher in the study group (41.3%) as compared to the control group (36%) but the difference was not statistically significant.
The limiting factor in the success of IVF is implantation, which involves two major participants, embryos and endometrium. Endometrial development is driven by oestrogen secretion from the ovaries. Serum levels of estradiol and progesterone decline in the mid luteal phase in IVF cycles in which pituitary suppression is used during controlled ovarian hyper stimulation. This decline has been found to adversely affect implantation [17].
Significantly higher E2 levels were found on Day 6 and 17 in study group and in women who conceived. Ghanem ME et al., and Var T et al., also reported significantly higher E2 levels in E+P group compared to P only group on day 7 after hCG trigger [2,4].
The levels of serum E2 decrease in the luteal phase. It has been shown that this decline adversely affects implantation. It is found that lower luteal phase E2 level 10 days after oocyte retrieval lowers the chances of conception and the magnitude of decline in estradiol concentrations after oocyte retrieval may be important in predicting IVF success. It is postulated that endometrial integrity may become compromised when a dramatic drop in estradiol occurs by the mid-luteal period. The percentage decline in estradiol levels from the day of oocyte retrieval to the day of implantation was significantly lower in study group, as compared to control group and also in women who conceived as compared to those who did not conceive.
Ghanem ME et al., in their study have tried to predict the pregnancy rate according to the E2 peak to midluteal E2 ratio [2]. In our study, the Day 0 E2: Day 6 E2 ratio was significantly lower in the study group, compared to control group. Also, the women who conceived had a significantly lower Day 0 E2: Day 6 E2 ratio compared to those who did not conceive.
Limitation
It was found that although estradiol supplementation in the luteal phase did not improve pregnancy rates significantly, but it certainly improved the hormonal profile in the study group compared with the control group, hence a study with larger number of women is required to show a significant difference in pregnancy rate.
Conclusion
It was concluded that estradiol supplementation in luteal phase of IVF cycles in beneficial. It is also postulated that a further increase in dose of estradiol valerate to 6 mg/day may increase the pregnancy rate significantly, although a properly designed randomized controlled study with dose stratification (2 mg/day, 4 mg/day and 6 mg/day) needs to be conducted to prove this hypothesis. Also, it is postulated that beginning of estradiol supplementation from the day of oocyte retrieval may result in even better hormonal profiles and eventually translate into higher pregnancy rates, but this needs to be proven by a randomized controlled study with a larger sample size.