In this retrospective cohort study, we demonstrated that the embryo pooling could achieve significantly lower cumulative live birth rate when compared with the conventional non-pooling strategy. Moreover, the time to live birth was significantly longer in the embryo pooling group than the non-pooling group.
The concept of accumulation of oocytes through repeated IVF cycles was first published by Cobo et al[14]. In this report, oocytes were retrieved through multiple conventional antagonist cycles and vitrified until at least 5 embryos were anticipated to be obtained for subsequent ET. They found significantly higher cumulative LBRs/patients compared to those who had single cycle with fresh ET (36.4% vs 23.7%). Our embryos pooling strategy was similar to the Cobo et al[14]. which stipulate all couples were offered several consecutive stimulating cycles until accumulate at least five embryos to be transferred. Different from our strategy, Datta et al[17]. described in their study to determine the cumulative clinical outcomes of transferring vitrified-thawed embryos from a pool of embryos accumulated over 3 consecutive modified natural cycles (regardless of the number of embryos obtained) in women with low ovarian reserve where the chance of obtaining an embryo is uncertain. The LBRs following 1 ET of this ‘triple freeze programme’ were compared with those of a single modified natural-IVF/ICSI cycle and 1 fresh ET. The LBR was significantly higher in the 3 ORs followed by 1 ET in the study group compared to 1 OR with 1 ET in the control(30.6% vs 13.3%; p = 0.002). Additionally, the per patient cumulative CPRs and LBRs including all ETs was also significantly higher with this study protocol (23.2%) than that of patients in the control group who had single NM cycle followed by ET (8.6%, p = 0.0003). However, their analysis was based on an ET or ETs undertaken from the pooled embryos through 3 ORs when compared with a fresh ET following a single cycle, which was actually not a fairly comparison between the two group. In our study there was no restriction on the number and type of ovarian stimulation in both arm, and among the participants recruited in either group, all cycles finished within 2 year after first ovarian stimulation were analysis—whether cancelled, pregnant, or non-pregnant.
Poor outcomes in the treatment of women with POR with a single IVF cycle is usually due to high cycle cancellation rates or unavailability of good quality embryos for transfer, and there is still no consensus on the optimal ovarian stimulation protocols for POR patients[22]. Similar to our study, Greco et al.[15] selected a mild IVF protocol in preference to conventional high-dose protocols for women with POR, in order to make repeated IVF cycles more‘patient friendly’ and thus to prevent women from dropping-out of treatment. In our study, more women in the pooling group were adopted PPOS and mild stimulation protocol for the embryo accumulation. PPOS and mild stimulation with clomiphene would lead to the impossibility of fresh embryo transfer because of a severe impairment in endometrial receptivity[23, 24]. Thus, PPOS or most of the mild stimulation protocols have to adopt ‘freeze-all’strategies, that is the freezing of the entire cohort of embryos. The modern technology of vitrification allows safe cryopreservation of oocytes and embryos with a high post-warming survival rate. Hence the transfer of fresh embryos to a uterus that has been subjected to hormonal stimulation is no longer required. The transfer of cryopreserved–thawed/ warmed embryos in the freeze-all embryo protocol has been reported in some studies resulting in improved pregnancy outcomes compared to fresh transfer[25, 26]. However, in this study we found the comparable biochemical pregnancy, clinical pregnancy, ongoing pregnancy and live birth rate per transfer between two groups, even the total implantation rate was inferior in the embryo pooing group in which freeze-all embryo protocol was dominant. One randomized trial[27] compared use of medroxyprogesterone versus a GnRH antagonist in oocyte donation cycles found the clinical pregnancy rate was 31% versus 46% (P = 0.006) and the ongoing pregnancy rate 27% versus 40% (P = 0.015) for medroxyprogesterone and GnRH antagonists, respectively. This suggests a possible impairment of oocyte quality when medroxyprogesterone was used in ovarian stimulation. On the contrary, different from PPOS, GnRH antagonist can compromise endometrial receptivity during ovarian stimulation, and made transfer of fresh embryos possible. Majority women in the non-pooling group used antagonist protocol, and embryos can be transferred either in fresh or in frozen cycles, many women underwent fresh ET and achieved pregnancy finally leading to live birth, and this was the major reason for time to live birth in the control group was significantly shorter than the embryo pooling group.
In this study we found a similar number of oocytes obtained, number of oocytes fertilized, number of cleaving embryos and number of transferable embryos per retrieval between the two groups although we used different ovarian stimulation between the two groups. More cycles canceled before oocyte retrieval in the study group than those in the control group was probably because of different protocols used in the two groups, but cancellation rate resulting in no available embryos to transfer was similar between the two groups, and almost all the patients had at least one available embryo for transfer after multiple oocytes retrievals in either group. So, the evidence in our study showed no extra advantages after embryo pooling over conventional non-pooling strategy in terms of reducing cancellation rate as proposed by Datta et al[17]., who claimed the beneficial effect of embryo pooling after comparing .‘triple freeze programme’ with those of a single IVF cycle.
Strategies with accumulation of embryos up to five by vitrification instead of immediate transfer could allow more IVF cycles in the same period as conventional treatment. Our study showed within 2 year of treatment, most patients undergoing standard treatment could complete up to 2.61 ± 1.47 cycles, whereas those undergoing the embryo pooling strategy could complete up to 5.31 ± 2.83 cycles. The accumulation of frozen embryos over multiple cycles would provide a greater number of embryos for transfer, however, it did not help to the improve the CLBR in POR patient. Cox regression model demonstrated that the number of oocytes obtained, and number of transferable embryos per woman and number of transfer cycles were not associated with the CLBR, but the number of stimulation cycles per woman was negatively associated with the CLBR after adjusting for other confounding factors. It can be argued that multiple oocytes retrievals and cryo-preservation of embryos in the embryo pooling strategy might have incurred in a higher cost compared to that with a single cycle followed by immediate transfers. And there is an obvious advantage of avoiding unnecessary further treatment cycles, in case live birth is achieved in the first or second cycle. Furthermore, embryo pooling strategy subjected women to repeated invasive oocytes retrievals procedures. Nevertheless, if the higher success with this strategy, couples could justify or benefit from it, outweighing the perceived risks.
The patient selection criteria in our study were different from those used in previous works. Cobo et al[14]. used the poor responder criteria described previously by Surrey and Schoolcraft[28], whereas Chatziparasidou et al.[13] included poor responders according to low AFC levels (AFC < 7) and candidates for PGD. In contrast, we used the Bologna Criteria to identify poor responders[18]; this may have caused a lower follicle pool in our study group, and therefore, a worse oocyte quality than in other studies. It is difficult to directly compare our results with previous studies as none of the available study evaluated the effect of embryo pooling on cumulative live birth rates nor assessed time to pregnancy in a period of time. In this study we reported cumulative live birth rates within 2 years in which multiple cycle was allowed. The duration of the study (24 months) allowed the majority women to accumulating enough embryos and undergo subsequent FETs. .CLBL is the outcome of interest for infertile couples, for not only just single fresh or FET cycle live birth, but also results from several IVF cycles including all subsequent frozen embryo cycles performed within 2 years period were evaluated, thereby giving the actual efficacy of these two strategies in the daily practice can be compared. Other strengths include none of the patients lost to follow-up in the study, leading to an increased reliability of our outcomes. Furthermore, we performed a Kaplan-Meier analysis to compare cumulative success rate in each group, as it assumed that women who did not return for subsequent FET cycles had the same chance of a pregnancy resulting in a live birth as those who returned for treatment[29]. Some patients in both groups with remaining frozen embryos and no live birth at the end of the study, and those patents were regarded as being censored. Time to pregnancy was much shorter in the non-pooling group which is also an important factor to evaluate the efficacy of IVF treatment[30], and further strengthen the overall result as embryo pooling is not beneficial with respect to the cumulative outcomes in two groups.
Our study is limited by its retrospective design and a small sample size and was performed at a single center so results may not be generalizable to other populations, clinics and embryology laboratories. Some imbalanced characteristics were found in this study and Cox regression analysis was carried out for controlling the basis. Another limitation might be the permitted between-cycle regimen adjustments in both strategy since patient or physician are apt to try another protocol for ovarian stimulation if failed in previous one. The the number of embryos transferred were different with more women underwent single embryo transfer in the non-pooling group as some of the patients only had one embryo to transfer after one IVF cycle, but this did not affect the final results that non-pooling strategy was in favour of pooling strategy. Further large randomized trials with adequate sample size would be needed to confirm these findings.