**6. Strategies for Euploid**/**Mosaic Blastocysts Transfer**

Implantation is considered to be an essential step for the success of assisted reproduction techniques and mainly depends on endometrial receptivity, embryo quality, and synchrony between them. However, the process of ovarian stimulation with elevated estrogen level, together with a possible progesterone premature growth, might reduce the expression of genes involved in the implantation process and negatively modify embryo-endometrium communication [175].

These negative effects can be responsible of decreased clinical results and adverse obstetrics and perinatal outcomes [175,176]. It has been suggested, indeed, that, after a fresh embryo transfer in a stimulated IVF cycle with E<sup>2</sup> levels >2724 pg/mL at the time of hCG administration, the risk of abnormal placentation and low birth weight [177] as well as the risk of obstetric hemorrhage [178] is definitely higher.

Euploid embryos can be transferred in a fresh stimulated cycle, performing biopsy of expanded blastocysts on day 5 and waiting for PGT result for a fresh transfer on day 6. Unfortunately, slower growing embryos, which cannot be biopsied on day 5, will not be included in PGT and considered for the fresh transfer. On the other hand, the freeze-all strategy involves cryopreservation of all biopsied embryos, including blastocysts developed on day 5, 6, or also 7, and then waiting for the PGT results in preparation for a further FTET. Undoubtedly, the uncertainty of having euploid embryos available for fresh transfer might enhance stress perception, strongly present in all patients with infertility problems. At the same time, the awareness that a fresh transfer is not planned, and that the whole cohort of embryos will be carefully evaluated, might relieve patient concerns [179].

Embryo cryopreservation is often only considered as a strategy to enhance the overall pregnancy rate per oocyte retrieval, when surplus embryos are available. However, a randomized controlled trial by Coates et al. [179] compared the clinical outcomes from frozen and fresh embryo transfers, finding that where the implantation rate was similar (75% vs. 67%), the ongoing PR (80% vs. 61%) and LB rates (77% vs. 59%) were significantly higher in the frozen group with respect to the fresh one. Based on these observations, it is possible to suggest the freeze-all strategy as a routine IVF approach when PGT is performed.

Frozen-thawed embryos can be transferred in different protocols: natural cycle (NC), modified natural cycle (modified-NC), and hormone replacement cycle (HR), but the best strategy should be still identified [180]. The authors of a retrospective cohort study including 389 cycles with 24-chromosome day 5/6 PGT-A found that the ongoing pregnancy rate obtained from a single euploid FTET in NC was significantly higher when compared to that in HR regimen. No difference was documented in the miscarriage rates [181].

Several studies investigated whether the duration of estrogen therapy or progesterone values before euploid embryo transfer might influence clinical outcomes in HR programs. A retrospective cohort study suggested that the duration of estradiol treatment before progesterone initiation does not affect the immediate FTET results. However, every additional day of estrogen administration is associated with a reduction of gestational age at delivery, not exceeding the criteria of preterm birth [182].

Gaggiotti-Marre et al. [183] examined the role of progesterone levels on the day before euploid embryo transfer, still in HR protocol, and documented that patients with its values over 13.1 ng/mL have a significantly higher miscarriage rate and lower live birth rate. On the other hand, a minimum threshold of progesterone concentration must be reached on the day of ET. It was documented that the progesterone level on the day of ET was predictive of clinical success and resulted in being significantly higher (28.0 ng/mL) in pregnant women, when compared to those with negative pregnancy test (16.4 ng/mL). The optimal cut-off value suggested was at least 20.6 ng/mL [184].

At the same time, a prospective controlled randomized trial documented that HR and modified-NC protocol for FTET lead to comparable clinical results, as suggested by overall pregnancy (61.9% vs. 62.3%), clinical pregnancy (50.4% vs. 54.1%), and implantation rates (50.4% vs. 54.1%). The live-birth rate of 45.8% in modified-NC was comparable to that observed in HR (41.5%). These overlapping results suggest that the choice of endometrial preparation should be based on women menstrual characteristics or, otherwise, the need for FTET planning [180].

Nevertheless, a study conducted by Litwicka et al. observed that, in modified-NC, the timing of HCG administration for ovulation induction might be relevant. The HCG injection when LH reaches values higher than 13 mIU/mL is associated with significantly lower overall and clinical pregnancy rates (45.4% vs. 73.3%) with respect to those in cycles with LH < 13 mIU/mL on the day of ovulation induction (36.4% vs. 65.9%). The authors suggested that, if natural ovulation process is already started (LH level ≥ 13 mIU/mL), hCG administration should be avoided, postponing the embryo transfer after spontaneous follicular rupture [17].
