The results of our retrospective study highlighted a significantly higher clinical pregnancy rate after blastocyst transfer (33.3%) than after cleaved embryo transfer (25.3%; p < 0.01) in the general IVF/ICSI population; the same was true for the live birth rate (32.1 vs. 22.8%, respectively; p < 0.01). The same difference was found in Glujovsky et al.’s [9] meta-analysis of 27 randomized studies (covering a total of 4031 women or couples); the OR [95%CI] was 1.30 [1.14–1.47] for clinical pregnancies and 1.48 [1.2–1.82] for births (in 13 studies) after D5/D6 blastocyst transfer. For D5/D6 blastocyst transfer, Wang et al. [8] found an OR of 1.43 [1.15–1.78] for clinical pregnancies (in seven randomized studies) and an OR of 2.15 [1.57–2.94] for births (in four studies). Wang et al. also found that blastocyst transfer was associated with a lower miscarriage rate (OR [95%CI]: 0.51 [0.3–0.87]), relative to cleaved embryo transfer; this was not the case in our study (with values of 11.6% vs. 13.8%, respectively, in the general population) or in the meta-analysis by Glujovsky et al. (OR [95%CI]: 1.15 [0.88–1.50]) [9]. In our study, we transferred blastocysts on D5 only; this yields significantly more pregnancies than on D6, as shown by Bourdon et al.’s [15] meta-analysis (OR [95%CI]: 2.38 [1.74–3.24] for clinical pregnancies and 1.74 [1.37–2.20] for live births). Other researchers have not found any difference in pregnancy rates per transfer between blastocysts and cleaved embryos: with a serum progesterone level > 1.5 ng/ml on the trigger day, the rates were respectively 39.6% and 40.1% in Levi-Setti et al.’s study [16] and 35% vs. 39% in Demirel et al.’s study [17]. In our selected “top cycle” population, we did not observe any difference in the clinical pregnancy and live birth rate when comparing the blastocyst transfer and cleaved embryo transfer groups. Likewise, the miscarriage rates were similar: 8.3 vs. 8.0% in the blastocyst and cleaved embryo transfer groups, respectively. Any difference in embryo culture failure rates in the general and selected IVF/ICSI populations was reported in our study, whereas Glujovsky et al. [9] found a greater risk of no embryo transfer for D5 blastocyst versus D3 embryo.
In our study, the main predictive factor for pregnancy (whether after blastocyst transfer or cleaved embryo transfer) was the serum progesterone level on the eve of the trigger day and on the day itself; the levels were significantly higher among women who did not become pregnant in both the general and selected populations (p < 0.01 to < 0.001). Follicular phase progesterone facilitates the estrogens’ action on the pituitary gland, thus enabling the ovulatory peak of LH and FSH. It also has an essential role in decidualization of the endometrium and the opening of the implantation window via the structures or molecules that it controls (the pinopodes, insulin growth factor binding protein 1 and glycodelin levels, etc.) [10]. Hence, in a study of GnRH antagonist - FSH protocols, Van Vaerenbergh [18] reported differences in endometrial gene transcription between serum progesterone classes (≤ 0.9, 1-1.5, and > 1.5 ng/ml). Two pregnancies were found in the first class (out of 3 women), with two in the second (out of 6) and none in the third (out of 5). The largest number of gene transcription differences were observed when comparing the third group with the first group (n = 1388) and the second group (n = 819). The changes involved the serine protease PAPP-A, the IL-17 receptor, thrombospondin, and dickkopf homolog 3 (Xenopus laevis). Labarta et al. [19] found that a serum progesterone level > 1.5 ng/ml was associated with differences in the expression of proteins involved in cell adhesion, immunity, and growth. During the proliferative phase, progesterone levels are usually < 0.5 ng/ml, with a luteinization induction threshold of between 1 and 10 ng/ml. Filicori et al. [20] showed that the elevation in progesterone depended on the granulosa cells and (as also reported by other researchers [21, 22, 23]) was associated with the number of oocytes retrieved, the dose of FSH, and the serum oestradiol level. This premature elevation led some researchers [24, 25] to recommend an earlier trigger. The elevated serum progesterone level on the trigger day affects the endometrium but not embryo quality, as shown in Huang et al.’s study [26] and by the results of frozen embryo or donor embryo transfers [13, 21, 27]. Furthermore, the very high serum oestradiol level accelerates endometrial maturation (with no pregnancies if the advance exceeds 3 days [28]) and leads to luteal-phase LH deficiency, luteolysis, myometrial hypercontractility, and differences in gene transcription. Saadat et al. [29] found that the mean ± SD acceleration in endometrial maturation was the same for antagonist protocols (5.8 ± 0.4 days) and agonist protocols (5.9 ± 0.7 days) and persisted up until 7 days after the trigger.
Elevated progesterone levels before the ovulation trigger are responsible for a decrease in pregnancy rates. The research question addressed in the literature concerns the serum progesterone threshold above which this effect appears. According to Bosch et al. [30], the threshold is 1.5 ng/ml, whereas Xu et al. [21] quoted values of 1.5 to 2.75 ng/ml, depending on the follicular response. In fact, Xu et al.’s study of 10000 cycles [21] found a fall in the rates of pregnancy above 2.75 ng/ml in strong responders (> 20 oocytes), above 1.75 ng/ml in normal responders, and above 1.5 ng/ml in nonresponders. In contrast, the pregnancy rates for frozen embryo transfers increased with the serum progesterone level. In our study, 77 couples in the selected population had frozen embryos after freeze-all (mean number of embryos frozen: 3.43 ± 2.14), which prompts us to expect even higher cumulative pregnancy and birth rates. Corti et al. [31] reported a difference in the clinical pregnancy rate above vs. below a serum progesterone threshold of 1.5 ng.ml. (50% vs 33.3%, respectively; OR [95%CI]: 2 [1.07–3.75]). Other researchers have found a lower serum progesterone threshold. Venetis et al.’s study of 60000 cycles [13] found an OR for birth of 0.39 when the serum progesterone level on the trigger day was between 0.4–0.6 ng/ml, with 0.79 between 0.8 and 1.1 ng/ml, 0.67 between 1.2 and 1.4 ng/ml, 0.64 between 1.5 and 1.75 ng/ml, and 0.68 between 1.9 and 3 ng/ml. Venetis et al. did not observe an impact of a serum progesterone level ≥ 1.5 ng/ml in poor responders (< 6 oocytes, n = 796 cycles) or strong responders (> 18 oocytes, n = 730). In 2015, the same research group found a threshold of 0.9 ng/ml for 3296 cycles [32]. Griesinger et al. [33] found that the prevalence of an elevated serum progesterone level varied with the follicular response: 4.5% for poor responders (≤ 5 oocytes) vs. 19% for strong responders (> 18 oocytes). The pregnancy rate was also low (OR: 0.55 [0.37–0.81]), especially in poor responders. Fanchin et al. [34] came to the same conclusion in poor responders for a serum progesterone threshold at 0.9 ng/ml but this level did not have an impact among normal responders. Requena et al. [35] found the same pregnancy rates in strong responders (> 20 oocytes and 3000 pg/ml) above and below a serum progesterone level of 1.5 ng/ml. In our study, the serum oestradiol level on the trigger day and the number of oocytes retrieved testified to a normal response to ovarian stimulation. Santos-Ribeiro et al. [36] found the best pregnancy rate (29.7%) when the serum progesterone level was between 0.76 and 1 ng/ml, after adjustment for female age, the number of oocytes retrieved, the serum oestradiol level, the FSH dose, the number of embryos transferred, and the embryo stage. Wang et al. [37] studied the pregnancy rates after transfers of fresh embryos (n = 1455) or frozen embryos (n = 1455). They observed than when the serum progesterone level ≤ 1 ng/ml, the pregnancy rates for fresh and frozen embryo transfers were respectively of 56.4% and 54.6% in women aged ≤ 35 and 45.1% and 48.9% in woman over 35. When the serum progesterone level exceeded 1 ng/ml, the rate after fresh embryo transfer was lower (46.1% for age ≤ 35; OR: 1.38 [1.11–1.71], and 35.2% for age > 35; OR: 1.73 [1.34–2.24]); these values are similar to those found in the present study for the general population vs. the “top cycle” population. Papanikolaou et al. [38] studied the clinical pregnancy rate in GnRH antagonist-rFSH protocols as a function of the serum progesterone level on the trigger day. In case of transfer of embryos on D3, the clinical pregnancy rate was 37.5% when the progesterone level was < 0.73 ng/ml, 34% for the range 0.74–0.90, 30.6% for the range 0.91–1.2, 25.9% for the range 1.21–1.53, and 15.7% when the level was > 1.53 ng/ml (p = 0.008). Following blastocyst transfer on D5, there was no significant difference in the pregnancy rates as a function of the serum progesterone level (< 0.67: 42.9%; 0.68–0.99: 39.5%; 1-1.15: 29.3%; 1.16–1.48: 40%, and > 1.48: 41.5%). The researchers concluded that the transfer of a good-quality blastocyst compensates for the high progesterone level. In 2012, the same research group [39] did not find a difference for a serum progesterone level above or below a threshold of 1.5 ng/ml in either GnRH antagonist or GnRH agonist protocols. In a study by Vanni et al. [40], the serum progesterone threshold for obtaining good-quality blastocysts was 1.49 ng/ml, and the yield was better below 1 ng/ml. Shapiro et al. [41] reported a lower pregnancy rate after fresh blastocyst transfer on D6 (relative to D5); this was probably due to transfer outside the implantation window, which is advanced by the ovarian stimulation (the pregnancy rate was the same for frozen embryo transfers). Healy et al. [42] compared D5 and D6 transfers and observed an influence of the serum progesterone level above 0.8 ng/ml. On D6, the relative decrease in the pregnancy rate was 8% when the serum progesterone level was normal and 17% when it was > 1.5 ng/ml. On D5, the best pregnancy rate was obtained when the serum progesterone level was < 1 ng/ml, with an OR [95%CI] of 0.75 [0.67–0.88] (p < 0.001) when considering the serum progesterone level as a continuous variable. According to Huang et al. [26], the best pregnancy rates were observed after embryo transfers on D3 or D5 when the serum progesterone level was between 0.5 and 0.74 ng/ml. Overall, 50.4% of the women became pregnant when the serum progesterone level was < 1 ng/ml, with 45.5% when it was between 1-1.49 ng/ml and 36.2% when it was ≥ 1.5 ng/ml (p < 0.01). In our study (with a threshold of 0.9 ng/ml), there was a significant difference in the clinical pregnancy rate between the blastocyst transfer and cleaved embryo transfer groups in the general population but not in the selected “top cycle” population (Fig. 2). As in the study by Papanikolaou et al. [38], it appears that the selection of “top cycles” reduced the impact of an elevated serum progesterone level on the trigger day. Hill et al. [43] studied the impact of elevated serum progesterone level on the hCG day as a function of various prognostic criteria, including female age below 35. This influence can be seen in our Fig. 3, where the clinical pregnancy rates were high in the selected population up to a serum progesterone level of 1.1 ng/ml for both blastocyst transfer and cleaved embryo transfer.
Lastly, the serum progesterone level was independent of the type of gonadotrophin and the duration of administration [35, 44, 45], as shown by the absence of a difference for these parameters in our study. In a report published in 2014, Lee et al. [46] showed that elevation of the progesterone level for 2 or more days was associated with a lower pregnancy rate (20.7%, vs. 39.4 for women with a normal level; p < 0.001). This might explain our results in the general population and the selected population, where the serum progesterone levels during the last two days of the stimulation were significantly higher in non-pregnant women (regardless of the embryo transfer stage). Nevertheless, Santos-Ribeiro et al. [47] did not find a lower birth rate when the serum progesterone level was above 1 ng/ml (even after more than a day with elevated levels before the trigger), whereas the rate fell when the serum progesterone level was > 1.5 ng/ml (30.3% in the absence of an elevated serum progesterone level, 20.4% for one day of elevation, and 20.5% for more than one day). However, a progesterone level > 1.5 ng/ml was very infrequent (1.9%) in Santos-Ribeiro et al.’s study.
We studied various ratios involving the serum progesterone level on the trigger day (Table 3): P/Ooc, P/E2, and PFI. The PFI was considerably higher in the general population than in the selected population, and considerably higher (p < 0.001) in the women receiving a cleaved embryo transfer (regardless of whether or not they became pregnant). Shufaro et al. [14] estimated that the PFI was more representative of follicular progesterone secretion and the cycle outcome than the mere number of oocytes collected. For 8649 cycles followed by cleaved embryo transfer, the researchers found that the mean PFI was 0.32 ± 0.25 nM, and that the chance of pregnancy was four times lower if the PFI was high (> 93rd percentile, i.e. a serum progesterone level > 4.2 nM and a PFI > 0.6). The pre-ovulatory serum progesterone level was 2.22 ± 1.33 nM in Shufaro et al.’s study. The researchers recommended using the PFI rather than the progesterone level on the trigger day to decide whether to continue a cycle (i.e. with a low PFI) or to abandon it (i.e. with a high PFI and, in some cases, a high serum progesterone level). In our study, the PFI was always above 0.6 (probably because of a low serum progesterone level) and was significantly higher in the selected population and in non-pregnant women after cleaved embryo transfer; these findings are in line with Shufaro et al.’s findings [14], which did not apply to cases of blastocyst transfer. The P/Ooc ratio even differed significantly when comparing pregnant and non-pregnant women after a cleaved embryo transfer in the general population (p < 0.01). Furthermore, and in contrast to the PFI, the P/Ooc ratio after cleaved embryo transfer was significantly lower in the selected populations than in the general populations – regardless of whether pregnancy was achieved. Grin et al. [48] showed that the P/Ooc ratio was inversely correlated with the clinical pregnancy rate (adjusted OR 95%CI: 0.063 [0.016–0.249]; p < 0.001) and the birth rate (0.036 [0.007–0.199]; p < 0.001), and that the 90th percentile of this ratio was 0.36 ng/ml/oocyte (clinical pregnancy rate: 8%; birth rate: 5.9%). It is noteworthy that the P/Ooc ratio was always well below 0.36 ng/ml/oocyte in our study. For the P/E2 ratio (indicating premature luteinization when > 1 [49] or ≥ 1.2 [50]), we only found a significant difference for blastocyst transfers in the general population. In Lai et al.’s study [50] of GnRH agonist protocols, a P/E2 ratio < 1.2 (mean: 0.6 ± 0.3 ng/ml) had no impact on the ongoing pregnancy rate (29.3%, vs. 34.5% for P/E2 > 1.2). The sensitivity was 75%, the specificity was 32%, the positive predictive value was 37%, the negative predictive value was 71%, and the area under the ROC curve [95%CI] was 0.534 [0.456–0.613], corresponding to poor predictive value for the P/E2 ratio in GnRH agonist protocols. In a study of women who responded strongly to a GnRH agonist protocol, Lee et al. [51] found that the highest pregnancy rates were achieved with a serum progesterone level ≤ 0.9 ng/ml or between 0.9 and 1.4 ng/ml, with mean ± SD P/E2 ratios of 0.18 ± 0.01 and 0.27 ± 0.01, respectively. It is probable that the premature luteinization threshold in Lee et al.’s study (P/E2 > 1) was low because the hyperstimulation had already contributed to an acceleration in endometrial maturation [52]. In a study of women with a serum progesterone level ≥ 1.5 ng/ml during GnRH antagonist protocols, Golbasi et al. [53] showed that the P/E2 ratio did not have predictive power (mean value: 0.73 ± 0.54 for births vs. 1.05 ± 1.38 in the absence of births; p = 0.158). In GnRH agonist protocols, effective blockade of LH elevation (in 95%-98% of the women) meant that the elevated serum progesterone level before the trigger must have been caused by another factor. Hence, in our study (where the P/E2 ratio was always below 0.6), the LH level was higher (in 75% of cases) among women with a low serum progesterone level on the trigger day. This finding was also described by Huang et al. [26]: the mean ± SD serum LH level on the trigger day after cleaved embryo transfer on D3 was 2.0 ± 1.3 IU/l when the serum progesterone level was < 1 ng/ml and 1.7 ± 1.0 IU/l when it was ≥ 1.5 ng/ml (p < 0.01). The use of hMG (with hCG activity) for ovarian stimulation might explain these results [54]; however, we did not find a significant difference between the pregnant and non-pregnant groups in this respect.
Our study was limited by its retrospective character and its low statistical power, relative to the literature [13]. Nevertheless, our random choice of the embryo transfer day enabled us to obtain two demographically similar populations. We excluded cases in which the serum progesterone level was > 1.5 ng/ml, which probably impacted the pregnancy rates. Likewise, the high incidence of “freeze-all” cycles in each group decreased the pregnancy rate per retrieval, and prompted us to only consider the pregnancy rate per transfer. The subsequent transfer of frozen embryos should result in even higher cumulative pregnancy rates. Lastly, our use of the same quality-controlled progesterone assay throughout our study minimized the variability in the concentration data. However, inter-assay differences are frequent, and so our results may differ from those of other studies.