Cumulative IVF Pregnancy Rates After Transfer of Day 5 Fresh and Frozen Embryos in Agonist Cycles

INTRODUCTION

Blastocyst transfer (BT) has been shown to significantly enhance implantation and clinical pregnancy rates, contributing to improved outcomes in fertility treatments. However, performing fresh BT cycles for all patients undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) is not always practical. This is often due to factors such as poor endometrial receptivity or the onset of ovarian hyperstimulation syndrome (OHSS). In such situations, the blastocysts are cryopreserved for use in future cycles. Additionally, if a fresh BT does not result in pregnancy, these frozen embryos can be used in subsequent attempts. Vitrification has become the preferred method of cryopreservation over conventional slow-freezing, primarily because it prevents ice crystal formation and is technically easier to perform.^[1] Traditionally, assisted reproductive technologies (ART) that use controlled ovarian stimulation (COS) followed by fresh embryo transfers have faced issues such as lower implantation rates (IRs) and a higher risk of OHSS. Recent population-based and large-scale studies have indicated that pregnancies from COS with fresh transfers may be linked to adverse perinatal, neonatal, and long-term health outcomes, largely due to the higher incidence of multiple pregnancies. Importantly, newer evidence suggests that even singleton ART pregnancies may carry increased risks.^[2] A robust embryo cryopreservation programme is essential to improving the overall success of IVF. One key benefit of frozen embryo transfers (FETs) is that the endometrium is not subjected to the elevated hormone levels seen in COS cycles. Furthermore, frozen transfers may offer a wider Window of Implantation (WOI). Randomised controlled trials consistently demonstrate that FETs result in higher pregnancy rates and better perinatal outcomes compared to fresh transfers.^[3–5] The primary aim of the study was to review the data of the outcome of the patients undergoing in vitro fertilisation-embryo transfer (IVF-ET) cycles with the conventional agonist downregulated protocol. Hence, a retrospective study evaluating the overall efficacy of an IVF programme that included conventional agonist stimulation cycles with a Human Chorionic Gonadotropin (HCG) trigger, followed by fresh and/or BTs in subsequent frozen-thawed cycles, was conducted.

MATERIAL AND METHODS

This retrospective study was conducted at Kamlesh Tandon Test Tube Baby Centre, Agra, from July 2022 to April 2024. All the patients who underwent IVF cycles during the above period were included in the study. Donor programmes with corresponding recipient cycles were excluded from the study. Patients underwent COS followed by oocyte retrieval and fresh embryo transfers. Patients with high serum oestradiol levels and over 15 oocytes were deferred for a fresh embryo transfer. A freeze-all policy was followed by a subsequent freeze-thawed embryo transfer cycle. The Clinical Pregnancy Rates (CRPs), Implantation Rates (IRs), Abortion Rates (ARs), Ectopic Pregnancy Rates (EPRs), Live Birth Rates (LBRs), Cumulative Clinical Pregnancy Rate (CCPR), and Cumulative Live Birth Rate (CLBR) served as the primary end measures. The Cumulative Pregnancy Rates and the LBRs were calculated. Stimulation was done with either Human Menopausal Gonadotrophin (HMG) or Recombinant Follicle Stimulating Hormone (rFSH) (Humog HP or Folligraf, Bharat Serums and Vaccines Limited, Thana, Maharashtra, Mumbai). Serum oestradiol levels and transvaginal ultrasonography were used to evaluate the ovarian response and modify the gonadotrophin dosage. When patients had two or more follicles more significant than 18 mm in diameter, a 250 μg injection of recombinant HCG (Ovitrelle, Merck Serono Amsterdam, The Netherlands) was given. A transvaginal ovum pick-up was scheduled to be done 34–38 hours following the HCG injection. After about an hour of oocyte retrieval, all mature MII oocytes were injected following oocyte denudation.

All fertilised oocytes were cultured for 5 days in culture media (global total LP, Life Global Group LLC, Scundview Road, Guilford, CT 06437 US), with a change in fresh media droplets on day 3. The grading of cleavage-stage embryos and blastocysts was done as per the Istanbul consensus.^[6] In patients where Fresh Embryo Transfer (Group 1) was planned, the luteal phase support was given in the form of a natural micronised vaginal progesterone pessary 200 mg thrice daily (Susten vaginal pessary, Sun Pharma Laboratories Ltd, Assam, India) along with injectable progesterone 100 mg SC every third day (Inj. Susten 100 mg IM, Sun Pharma India) and Tab. Dydrogesterone 10 mg thrice a day (Duphaston 10 mg, Abbott India Ltd, Puducherry, India) from the day of oocyte retrieval and was continued till the 11th day of testing of serum β-HCG levels. Serum β-HCG values of more than 100 mIU/ml post 11 days of embryo transfer were considered positive pregnancies. In these cases, the luteal phase support was continued until the clinical pregnancy evaluation was carried out by transvaginal ultrasonography. The luteal phase support was gradually tapered from 12 weeks onwards over 2 weeks. All the embryos were vitrified in the women who were assigned FET (Group 2). In the planned cycle of the FET cycle, after the baseline hormonal tests were conducted, the patients were put on Hormone Replacement Therapy (HRT) for endometrial growth in the form of oestrogen tablets (Tab. Progynova 2 mg, Bayer Zydus Pharma, Thane, Mumbai, India). The dose was increased incrementally, and an ultrasound was conducted on day 9 to check the endometrial thickness. Once the endometrial thickness crossed 7 mm, good triple-line luteal phase support was started, in a similar protocol used for the fresh embryo transfer cases. Information regarding the pregnancy outcome was obtained by reviewing obstetrical and neonatal medical records.

RESULTS

The CPRs, IRs, ARs, EPRs, LBRs, CCPR, and CLBR served as the primary end measures. The ratio of the number of transferred embryos to the number of detected gestational sacs on Ultrasonography (USG) was used to compute the IR. Clinical Pregnancy was defined by measuring HCG levels 11 days after embryo transplantation. Identifying an intrauterine gestational sac visualisation served as the benchmark for clinical pregnancy. The analysis considered the following factors: age, number of attempted cycles, type of infertility, number of retrieved and mature (MII) oocytes, number of 2PN, cleaved embryos, blastocyst generated, blastocyst transferred, and blastocyst frozen. Table 1 shows patient details related to age, days of stimulation, total gonadotropin consumption, ovarian response, and endometrial thickness in both groups: Fresh embryo transfer cases in Group 1 and FET cases in Group 2.

During the retrospective study period, 142 oocyte retrievals with 122 fresh embryo transfers and 32 FET cases were done. As shown in Table 2, in Group 1, a total of 1477 follicles were aspirated in the 122 total oocyte retrieval instances, resulting in an average of 12.10 follicles per retrieval oocyte recovery rate (ORR). Of the 1477 Oocyte Cummulus Complex (OCC) obtained, 959 mature oocytes (metaphase II/M2) were obtained, yielding an oocyte maturity rate (OMR) of 65.00%. These 959 mature oocytes underwent ICSI, and 767 2PN were generated, yielding a fertilisation rate (FR) of 80.00%. After these, 767 2PN were further cultured, and 613 cleaved embryos were obtained, giving a cleavage rate (CR) of 80.00%. All embryos were kept for extended cultures, and 463 blastocysts were obtained, yielding a blastocyst rate (BR) of 75.50%. In Group 2, a total of 488 follicles were aspirated in the 32 total oocyte retrieval instances, resulting in an average of 15.25 follicles per retrieval ORR. Of the 488 OCC obtained, 338 mature oocytes (metaphase II/M2) were obtained, yielding an OMR of 68.00%. These 338 mature oocytes underwent ICSI, and 268 2PN were generated, yielding a FR of 80.00%. After these, 268 2PN were further cultured, and 214 cleaved embryos were obtained, giving a CR of 80.00%. All embryos were kept for extended cultures, and 185 blastocysts were obtained, yielding a BR of 86.00%. As shown in Table 2, the OMR, FR, CR, and BR were comparable, with not much difference between the groups.

As shown in Tables 3 and 4, in Group 1, 122 cases of fresh BT were done, wherein 288 blastocysts were utilised, 147 surplus blastocysts were frozen, and 28 were unsuitable for freezing were discarded. 122 cases received 288 blastocysts, averaging 2.36 blastocysts per embryo transfer. Out of the 122 fresh transfers, 63 cases had a clinical pregnancy, giving a CPR of 51.63% (CPR-Fresh), 7 cases were aborted, and there was one case of ectopic pregnancy. A total of 55 cases were delivered, with 38 cases of singletons and 17 cases of twins, yielding an LBR of 45.08%. In Group 2, the total number of blastocysts thawed in the FET cycle was 105; 41 blastocysts were discarded due to post-thaw non-survival, and the balance of 64 was transferred in 32 cases, yielding an average of 2.0 blastocysts per embryo transfer. Out of the 32 cases in this group, 21 cases had clinical pregnancy, 3 cases aborted, and there were no cases of ectopic pregnancy, resulting in a CPR of 65.62%. There were 18 delivered cases with 11 singleton and 7 twin pregnancies, yielding an LBR of 56.25%. 32 cases received 64 blastocysts, averaging 2.00 blastocysts per embryo transfer. The IRs were 25.69% and 43.75% in Group 1 and Group 2, respectively [Figure 1]. As seen in Table 4, the p-value for IRs and LBRs is significant in our study.

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Figure 1:

Comparative analysis of the outcomes in fresh and FET.

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Summarising the results, as shown in Table 5, gives us a CCPR of 68.85% and a cumulative birth rate of 47.40%, respectively. Thus, it points out that frozen ET has a better outcome than fresh ET.

We analysed the data further into the laboratory outcomes and obstetrics outcomes for the frozen transfer group into two subgroups: one of the cases with freeze-all and the other with a repeat frozen cycle, where the first embryo transfer failed to give a pregnancy. As seen in Table 6, only the oocyte maturity and CRs had significant p-values.

As analysed in Table 7, the CPR, IR, AR, LBR, and Twin rates had no statistically significant p-values.

As analysed in Table 8, the groups, the RR, the 95% CI values, and the corresponding p-values shown indicate that Group 1 is less likely to have an outcome compared to Group 2. So, there is a trend towards Group 2, but the p-value is not statistically significant.

A short summary is shown above in Figure 2.

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Figure 2:

Short summary of blastocysts. A total of 154 embryo transfer (ET) cases were studied, 122 fresh ET and 32 frozen-thawed ET cases. In the fresh ET group, 1,477 oocytes were retrieved, resulting in 463 blastocysts. Of these, 288 were transferred, 28 discarded, and 240 frozen. In the frozen-thawed ET group, 488 oocytes yielded 185 blastocysts, of which 64 were transferred and 41 discarded.

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As summarised in Table 9, out of the total 11 cases of OHSS, the maximum number of cases belonged to the age group between 26 and 30 years, and the maximum number of cases had an oocyte number between 21 and 25. The total number of cases needing paracentesis was 3, of which 2 cases needed it twice. Two cases required ICU admission for 5 days and the rest of the 9 cases were managed on a daily out patient department (OPD) basis.

DISCUSSION

Fresh or frozen transfer has always been a topic for debate, with a lot of controversy among fertility specialists. In our current study, pregnancy outcomes were observed in the case of IVF cycles using fresh and/or FET. In a study conducted by Wang et al. ^[7] the frozen embryo group (52%) showed good pregnancy outcomes as compared to the fresh embryo group (45.3%). They concluded that the IR and pregnancy rates are reasonable in FETs compared to fresh embryo transfers. The results of the study conducted by Roque et al.^[8] showed the pregnancy outcome in FETs was better than that of fresh embryo transfers, with the conception rate for the FETs group being 46.4% and the fresh embryo transfers group being 35.9%. According to our study, which aligns with the previously mentioned results, the CPR was considerably higher in the frozen embryo group (65.62%) than in the fresh embryo group (51.63%). Contrary to our study, in the Canadian study conducted by Gunby et al. ^[9] the pregnancy success rate for fresh embryos was approximately 33%; however, it was reported to be 24% for frozen embryos. According to a comprehensive meta-analysis review study by Roque et al. ^[10] IVF using frozen embryos produced superior reproductive outcomes than fresh embryos. They postulated that the improved access to synchronisation between the endometrium and embryo in the FET technique may cause these outcomes. Compared to women who underwent IVF using fresh embryo transfer, those who underwent IVF using FET demonstrated greater favourable reproductive and pregnancy results. As previously indicated, the gonadotrophin releasing hormone-antaginist (GnRH-ant)protocol was the COS protocol in all earlier investigations comparing fresh ET with FET. No report is available regarding the comparison of the effectiveness of fresh ET and FET using the GnRH-agonist protocol. We selected the gonadotrophin releasing hormone-agonist (GnRHa) protocol because, despite the antagonist’s availability, it remains the industry standard in numerous countries. Currently, no clinical evidence supports the indiscriminate application of the freeze-all method to every patient.^[11] We selected the first FET cycle because, as with fresh ET, the best-quality embryos were often transferred during the first FET. A second FET cycle was added to evaluate the cumulative rates for every variable. Since implantation is one of the most crucial stages in achieving a live delivery, it is a crucial sign of the IVF treatment’s effectiveness. The quality of the embryo and the endometrial receptivity are the two primary factors in the implantation process.^[12] A randomised trial conducted by Hershko et al. revealed no differences in embryo quality between the gonadotrophin releasing hormone-agonist (GnRHa) and gonadotrophin releasing hormone-antaginist (GnRH-ant) groups.^[13] Regarding endometrial receptivity, Hernandez et al. have observed that GnRH-antagonists may interfere with an auto/paracrine loop necessary for the endometrial epithelial cells’ mitotic programme, which could result in a decrease in the rate of pregnancy and an increase in the rate of abortions.^[14] In comparison to cycles treated with gonadotrophin releasing hormone-agonist (GnRHa) or the natural cycle, Rackow et al. observed that in cycles treated with GnRH-ant, endometrial stromal cell expression of HOXA10, an important regulator of endometrial receptivity, was much lower.^[15] According to research by Ruan et al., GnRH agonists may help mice’s uterine receptivity and partially restore endometrial physiological secretion.^[16] gonadotrophin releasing hormone-antaginist (GnRH-ant), compared with gonadotrophin releasing hormone-agonist (GnRHa) treatments, significantly reduced endometrial receptivity, according to a comparative proteomic analysis. The endometrial receptivity of the gonadotrophin releasing hormone-agonist (GnRHa) protocol may be superior to the gonadotrophin releasing hormone-antaginist (GnRH-ant) protocol in new ET cycles, according to the results of the studies above.^[14–16] In our study, there were 15 cases of freeze-all cases, out of which 11 cases were moderate OHSS (7.74%). One case insisted on an FET cycle by demand; in 2 cases, the cervix was not negotiable on the day of ET, and in one case, they did not take post-retrieval treatment, respectively. Of these 11 cases, only 2 needed hospital admission (1.40%). Both cases were of pregnancy with late OHSS. The rest of the cases were managed well on an OPD basis. Despite benefits, cryopreservation may harm embryos due to ice crystal formation and cryoprotectant toxicity, which can affect gene expression.^[17–20] Thus, when these risks outweigh uterine benefits, fresh transfers may result in better LBRs. Furthermore, freeze-all strategies may prolong treatment and increase costs. Ectopic pregnancy and pregnancy loss rates also differ between groups. While some studies suggest higher ectopic risk with fresh transfers, others found no difference.^[21–23] Our study showed a higher EPR in the fresh group (1.58%), while pregnancy loss was greater in the FET group (14.28%). The LBR was 56.25% for FET and 45.08% for fresh ET using the GnRH-a long protocol. Our CCPR and CLBR were consistent with existing literature.^[24,25] With the recent trends of using antagonist cycles, we could find little data on the use of agonist cycles. In a meta-analysis from Cochrane Database Syst Rev 2025 by Charalampos S Siristatidis et al., there is evidence that the long protocol may improve CPRs overall, but there is uncertainty about any difference in OHSS and miscarriage rates.^[26] The study holds its limitation first by small sample size, and secondly, it would be an ideal scenario if a similar cohort with different formulations of agonist combined with gonadotrophin stimulation, as done in a conventional IVF cycle (as in our study) and antagonist stimulation protocol were compared with the rest of the parameters being kept the same.^[27,28]

CONCLUSION

Patients who had IVF by FET showed a higher pregnancy rate and more successful fertility and pregnancy outcomes compared to patients who underwent IVF by fresh embryo transfer. The study holds its limitation first by a small sample size, and secondly, it would be an ideal scenario if a similar cohort with an antagonist stimulation protocol were compared with the rest of the parameters being kept the same.