3.1.2. Blastocyst Hatching

Embedding of the blastocyst into the maternal endometrium requires hatching from the zona pellucida, which otherwise would prevent adhesion of the embryo to the uterine wall. Blastocyst hatching exposes the trophectoderm and allows the blastocyst to implant in the maternal uterus. The crucial event for blastocyst hatching is the formation of a nick into the zona pellucida, and proteases, such as serine-, cysteine-, and metallo-proteases have been proposed to play a major role in this event depending on the species [218–223]. Cathepsins, belonging to the ubiquitous cysteine proteases family [224], have been demonstrated to be involved in blastocyst hatching and zona lysis in mice; the expression of cathepsin L and P (mRNA and protein) and their natural inhibitor, Cystatin C, has been demonstrated in mouse peri-hatching blastocysts [225]; treatment of golden hamster embryos with Cystatin C is able to block blastocyst hatching [221]. The process of murine blastocyst hatching from the zona pellucida is also regulated by two mouse-specific proteinases, Strypsin (ISP1) and Lysin (ISP2). ISP1 and ISP2 are two related S1-family serine proteinases, which are tandemly localized in a cluster of tryptase genes [226,227]. The ISPs are co-expressed in the mouse preimplantation embryos and in the mouse uterine endometrium during the WOI, indicating that they could play a role in the process of blastocyst implantation [226,228]. Expression of ISP genes is positively regulated by progesterone and TH [219,223,226] and ISPs are secreted by the blastocyst and the endometrial glands into uterine fluid just prior to implantation [229]. The use of antibodies against ISP1/ISP2 abrogate murine embryo hatching and outgrowth, ascribing a crucial role for ISPs in this process [228]. This is further supported by our recent observations using mouse blastocysts cultured in the presence of TH, with or without endometrial cells used as the feeder layer. In the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching (Figure 1) by upregulating the expression of blastocyst produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. On the contrary, in the absence of the endometrial feeder layer, TH does not affect blastocyst hatching, suggesting that TH is one of the players involved in the bidirectional crosstalk between the blastocyst and the endometrium during the WOI [223]. Human homologs of ISPs have not been so far identified, and it is possible that other proteases might be involved in blastocyst hatching in humans. hatching exposes the trophectoderm and allows the blastocyst to implant in the maternal uterus. The crucial event for blastocyst hatching is the formation of a nick into the zona pellucida, and proteases, such as serine-, cysteine-, and metallo-proteases have been proposed to play a major role in this event depending on the species [218–223]. Cathepsins, belonging to the ubiquitous cysteine proteases family [224], have been demonstrated to be involved in blastocyst hatching and zona lysis in mice; the expression of cathepsin L and P (mRNA and protein) and their natural inhibitor, Cystatin C, has been demonstrated in mouse peri-hatching blastocysts [225]; treatment of golden hamster embryos with Cystatin C is able to block blastocyst hatching [221]. The process of murine blastocyst hatching from the zona pellucida is also regulated by two mouse-specific proteinases, Strypsin (ISP1) and Lysin (ISP2). ISP1 and ISP2 are two related S1-family serine proteinases, which are tandemly localized in a cluster of tryptase genes [226,227]. The ISPs are co-expressed in the mouse preimplantation embryos and in the mouse uterine endometrium during the WOI, indicating that they could play a role in the process of blastocyst implantation [226,228]. Expression of ISP genes is positively regulated by progesterone and TH [219,223,226] and ISPs are secreted by the blastocyst and the endometrial glands into uterine fluid just prior to implantation [229]. The use of antibodies against ISP1/ISP2 abrogate murine embryo hatching and outgrowth, ascribing a crucial role for ISPs in this process [228]. This is further supported by our recent observations using mouse blastocysts cultured in the presence of TH, with or without endometrial cells used as the feeder layer. In the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching (Figure 1) by upregulating the expression of blastocyst produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. On the contrary, in the absence of the endometrial feeder layer, TH does not affect blastocyst hatching, suggesting that TH is one of the players involved in the bidirectional crosstalk between the blastocyst and the endometrium during the WOI [223]. Human homologs of ISPs have not been so far identified, and it is possible that other proteases might be involved in blastocyst hatching in humans.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 9 of 30

pellucida, which otherwise would prevent adhesion of the embryo to the uterine wall. Blastocyst

**Figure 1.** Thyroid hormone (TH) supplementation stimulates mouse blastocyst hatching *in vitro*. (**A**) Schematic representation of the *in vitro* model developed to assess TH role in implantation. (**a**) Co-**Figure 1.** Thyroid hormone (TH) supplementation stimulates mouse blastocyst hatching in vitro. (**A**) Schematic representation of the in vitro model developed to assess TH role in implantation. (**a**) Co-culture of murine blastocysts and endometrial primary cells as the feeder layer; (**b**) blastocysts

from Piccirilli et al. [223].

cultured on plastic; and (**c**) endometrial cells cultured without blastocysts. (**B**) Representative images of the cultures. Scale bar 50 µm. (**C**,**D**) Graphs summarizing the results shown in B: percent of hatched blastocysts after co-culture on endometrial cells (**C**) or on plastic (**D**). Reproduced with permission from Piccirilli et al. [223]. culture of murine blastocysts and endometrial primary cells as the feeder layer; (**b**) blastocysts cultured on plastic; and (**c**) endometrial cells cultured without blastocysts. (**B**) Representative images of the cultures. Scale bar 50 µm. (**C**, **D**) Graphs summarizing the results shown in B: percent of hatched blastocysts after co-culture on endometrial cells (**C**) or on plastic (**D**). Reproduced with permission

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 10 of 30

#### *3.2. Apposition 3.2. Apposition*

Histological analysis of uteri of pregnant women allows to recognize three different levels of blastocyst adhesion to the uterine wall, which correspond to the three stages of blastocyst implantation (Figure 2) [230,231]. Histological analysis of uteri of pregnant women allows to recognize three different levels of blastocyst adhesion to the uterine wall, which correspond to the three stages of blastocyst implantation (Figure 2) [230,231].

. **Figure 2.** Blastocyst apposition, adhesion and invasion. The diagram shows a preimplantation-stage (**A**, **B**) and invading (**C**) blastocyst (about 9 to 10 days after conception) and the processes and factors required for uterine receptivity and blastocyst apposition (**A)**, adhesion (**B**) and invasion (**C**). hCG denotes human chorionic gonadotropin, LIF leukemia inhibiting factor, IL-1β interleukin-1 beta, EGFlike growth factors epidermal growth factor-like growth factors, AREG amphiregulin, EREG epiregulin, PG progesterone, COX-2 cyclooxygenase-2, PGE2 prostaglandin E2, CSF-1 colony stimulating factor-1, NOTCH1 Notch receptor 1, OPN osteopontin, MUC-1 mucin-1, MMPs metalloproteinases, EGFL7 epidermal growth factor-like domain 7, MAPK mitogen activated protein kinase, AKT protein kinase B, PA plasminogen activator, TGFβ transforming growth factor beta, **Figure 2.** Blastocyst apposition, adhesion and invasion. The diagram shows a preimplantation-stage (**A**, **B**) and invading (**C**) blastocyst (about 9 to 10 days after conception) and the processes and factors required for uterine receptivity and blastocyst apposition (**A)**, adhesion (**B**) and invasion (**C**). hCG denotes human chorionic gonadotropin, LIF leukemia inhibiting factor, IL-1β interleukin-1 beta, EGF-like growth factors epidermal growth factor-like growth factors, AREG amphiregulin, EREG epiregulin, PG progesterone, COX-2 cyclooxygenase-2, PGE2 prostaglandin E2, CSF-1 colony stimulating factor-1, NOTCH1 Notch receptor 1, OPN osteopontin, MUC-1 mucin-1, MMPs metalloproteinases, EGFL7 epidermal growth factor-like domain 7, MAPK mitogen activated protein kinase, AKT protein kinase B, PA plasminogen activator, TGFβ transforming growth factor beta, TIMPs tissue inhibitor of metalloproteinases, PAI-1 plasminogen activator inhibitor-1.

TIMPs tissue inhibitor of metalloproteinases, PAI-1 plasminogen activator inhibitor-1.
