The present study included 457 PGT-A cycles with 915 biopsied blastocysts from three reproductive centers and compared the results of blastocyst euploidy. Our results indicated that the GnRH-ant protocol was the most effective for PGT-A cycles. To our knowledge, this is the first study to analyze and compare blastocyst euploidy among the three commonly used COS protocols.

The results of a previous study showed that the euploidy rates of embryos formed from donor young donor eggs varied among reproductive centers [3]. Recently, another study revealed varying euploidy rates among physicians within a single center due to physician-specific COS protocols for donors [7]. Therefore, it is essential to choose an appropriate COS protocol for PGT-A to ensure that at least one euploid embryo is obtained. Numerous studies have compared COS protocols in terms of clinical pregnancy or live birth outcomes and have arrived at different conclusions. Despite these studies, the COS protocol that can achieve better embryo euploidy remains unclear.

Our study found that the PPOS protocol cycle had significantly lower numbers of euploid blastocysts and lower euploidy rates than those using the GnRH-a and GnRH-ant protocols. We noted a negative association between the GnRH-ant protocol and the mean number of euploid blastocysts with regression analysis while controlling for female age, BMI, male age, basal FSH, AMH, AFC, total Gn dosage, Gn days, E2, and LH levels on trigger days.

The PPOS protocol with orally administered progestins is beneficial economically compared to GnRH analogs. The higher patient comfort and lower cost of progesterone compared with GnRH make the PPOS protocol attractive for COS. However, we found a lower euploid rate regardless of whether the blastocyst was biopsied or per oocyte retrieved, even though the PPOS protocol had a higher MII rate than the GnRH-ant protocol and had fertilization rates similar to those of the GnRH-a and GnRH-ant protocols. Similar to our study, an RCT of oocyte donation cycles showed that lower biochemical pregnancy rate, clinical pregnancy rate, and live birth rate in the PPOS protocol group than in the the GnRH-ant protocol, although both groups obtained similar numbers of mature oocytes in oocyte donation cycles [8].

In contrast to our study, Marca et al. (2019) found that blastocysts and euploid blastocysts count per patient and the number of euploid embryos per injected oocyte were similar between the PPOS and GnRH-ant protocol [9]. Another study found that the euploidy rate per embryo biopsy was similar in patients undergoing the PPOS or GnRH-ant protocols [6]. While most studies have indicated that elevated progesterone levels on trigger day do not negatively affect the euploid rate and the outcome of FET [10,11,12], some reports have suggested that elevated progesterone levels negatively affect embryo development [13, 14]. However, blastulation time has been associated with euploidy [15, 16].

The GnRH-a and GnRH-ant protocols are well-established methods for treating COS. Current reports comparing the clinical outcomes of these two protocols based on prior meta-analyses and systematic reviews are inconsistent [17, 18]. Clinical outcomes were mainly limited to clinical pregnancy, ongoing pregnancy, live births, and birth defects. However, only one study has compared the effects of GnRH-a and GnRH-ant on embryonic aneuploidy. The results showed that the GnRH-ant protocol is associated with a higher aneuploidy rate in early aborted tissues and blastocysts compared to the GnRH-a long protocol [5].

Contrary to this study, we found a similar but increasing trend in the euploidy rate per blastocyst biopsied, per oocyte retrieved, and per MII oocyte in the GnRH-ant protocol. This discrepancy may be due to the inclusion of aborted tissues after IVF/ICSI-ET and PGT, including PGT-SR and PGT-A in the Wang et al. study, whereas our study included only patients who underwent PGT-A. Compared to the GnRH-a protocol, the GnRH-ant protocol had a shorter COS duration and lower Gn dosage. Therefore, the GnRH-ant protocol provides greater time and economic benefits.

Although COS aims to recruit several follicles for an increased likelihood of obtaining an euploid embryo that results in a healthy conceptus, several studies have suggested that high Gn stimulation might be embryotoxic and/or increase aneuploidy rates by enhancing the abnormal segregation of chromosomes during meiosis [19, 20]. Another study showed that the duration of the ovarian stimulation treatment was correlated with the aneuploidy rate [4, 21]. Our study found no significant association between Gn dosage, Gn duration, and blastocyst euploidy. Similarly, a retrospective cohort study with 2230 IVF/PGT-A cycles indicated that the Gn dosage, duration of ovarian stimulation, E2 level, follicle size at ovulation trigger, and the number of oocytes retrieved within certain ranges did not appear to significantly influence euploidy rates [22].

To our knowledge, this is the first study to investigate the association between three commonly used COS protocols and blastocyst euploidy. Originality, multiple centers, and relatively large sample sizes could be considered points of strength. The main limitation of our study was its retrospective design. Although additional testing was used to account for confounders and reduce potential bias in our population, there are weaknesses in the use of multiple testing. Moreover, the trigger reagents were not analyzed, and triggers with GnRH-a were used in partial cycles with the PPOS and GnRH-ant protocols. However, GnRH-a triggers are associated with higher euploidy rates in hyper responders [23].

Our study demonstrated that the GnRH-ant is the preferred COS protocol for PGT-A treatment. Further well-designed prospective studies with large sample sizes are required to validate our findings and provide more conclusive evidence for selecting a COS protocol for PGT-A treatment.