Authors: Paula Brígido, Shuyana Deba, Javier Del Río and Isabel Sánchez
Implantation is one of the key steps in human reproduction, and hatching of the blastocyst is a critical point in the sequence of physiological events that lead to implantation (2). It has been estimated that only 15-20% of embryo transfers culminate in implantation, and as a consequence clinical pregnancy and live birth rates are quite low (3, 4). The potential of an embryo to implant is related to its own nature, the quality of gametes and the endometrial receptivity. But failure in blastocyst hatching is also an important factor that prevents implantation (2, 3, 4). With the aim of solving this problem and in order to improve both implantation and pregnancy rates after Assisted Reproductive Technology (ART) procedures, scientists developed assisted hatching (AH) (2, 3, 4). AH consists of an artificial alteration of the zona pellucida (ZP) either by slimming or breakage. This technique was first described on 1988 by Cohen et al., who reported the first pregnancy after AH (4), and successive works have since shown its efficiency (2, 3).
Because AH does not seem to present clear advantages to all patients, it should not be applied, in principle, to all of them as a rule (3). Some studies have shown that AH has effectively improved ART outcomes in patients with bad prognosis, like those with a history of 2+ implantation failures, bad embryo quality, aged patients (38+), patient with frozen embryos or those who have oocytes with a thick ZP (2, 3, 4, 6).
ART have some actual consequences on gametes and embryos, one of them being hardening of the ZP by the use of certain culture media or by cryopreservation. These and other negative effects may hamper blastocyst hatching, which might be solved through AH. Overall, this approach might be useful considering the early implantation window in women treated with exogenous gonadotrophin stimulation, compared to a natural cycle. Additionally, benefits from AH include the possibility of ZP breakage improving the embryo-endometrium communication (5).
In order to help the embryo hatch through the ZP before implantation, a variety of techniques over the years have been developed (7). As a commonality for all of them, it is important to minimize the time of the embryo being outside the incubator and to optimize the methodologies to reduce both pH and temperature variations (3).
MECHANICAL ASSISTED HATCHING (PARTIAL ZONA DISSECTION)
This method does not require any new equipment, and it can be easily performed by an embryologist handling a micromanipulator (8). During this procedure, the embryo is held by the suction exerted by the holding pipette, while the microneedle is passed through the zona pellucida at the largest visible portion of the perivitelline space and introduced in a tangential trajectory to the embryo. Then, in order to tear the ZP apart, the embryo is released from the holding pipette and rubbed against it (7).
However, one of the main disadvantages of this technique is the difficulty of creating a hole of significant size without abruptly damaging the embryo. Moreover, it is not a fast procedure, which means embryos need to remain outside the incubator for a long time, and so this may affect development of the transferred embryos (7, 9).
ACID TYRODE'S ASSISTED HATCHING
For this procedure, first the embryo is stabilized with the holding pipette. Second, a pipette containing acid Tyrode is approached to the embryo, oriented at the 3 o`clock position, next to an area of empty perivitelline space. Finally, the acidic solution is gently expelled over a small area (~30 µm Ø) and immediately washed away (7). This technique allows the formation of a hole of significant size; nevertheless, the exposure to acid is problematic, since this compound may be potentially embryotoxic. Furthermore, this technique requires practice and expertise from the operator (7, 10).
Laser-assisted hatching (LAH) is designed for easy positioning of the embryos, focus and shooting. It can be executed with a single click of the mouse controller. The diameter of the drilled holes vary between 5 and 10 µm according to how many shots are performed and the irradiation time (7). From the technical point of view, this method is easier, better controlled and more precise. Without physical contact with other embryos, the procedure can be completed faster than other methods. So, time of the embryos out of the incubator is shorter than for other techniques (9).
Even though the equipment may result expensive, the laser-assisted technique seems to represent the lowest potential risk for the embryo, and it is relatively simple to perform with consistency between operators (3).
EFFICACY OF AH
Although it has been performed for more than 20 years, up to date results are still inconclusive. Ma and co-authors performed one of the first trials to determine the overall effect of this technique. They concluded that implantation rates could be enhanced by performing ICSI along with AH, but differences were not statistically significant (11).
Two recent meta-analyses evaluating potential benefits of this technique have reported significant heterogeneity among results (12, 13), suggesting that effects of AH may differ depending on specific patient features (14).
Most researchers support the hypothesis that this technique improves clinical pregnancy rates in patients with previous failed IVF cycles or poor prognosis. However, there is insufficient evidence to affirm that AH improves live-birth rates in these populations, and so it remains uncertain whether AH is beneficial to other patients (6, 14, 16, 17).
IS THERE AN INCREASE IN MULTIPLE GESTATION RATE?
Even though certain reports associate artificial manipulation of the zona pellucida with multiple pregnancies, there is actually insufficient evidence to support an increased risk of monozygotic twinning after AH. In fact, the overall rate of monozygotic twin pregnancy in IVF with AH is less than 1% (6, 16).
MISCARRIAGE AND CONGENITAL MALFORMATIONS RATES
It has been reported that this technique may enhance implantation of abnormal embryos. Thus, the lower live birth rate observed, which is related to the high number of pregnancies ending in early miscarriage, may be due to chromosomal abnormalities of the embryos (17). However, Ma et al. performed cytogenetic tests on miscarriaged embryos and umbilical cord blood from newborn infants, where they found a similar incidence of major congenital malformations in ICSI-born patients compared to the general population (11), thus ruling out any direct relationship of AH and miscarriage.
EFFECTS OF ASSISTED HATCHING ON FROZEN CYCLES
Data show that cryopreservation may induce zona hardening as well as advanced female age and in vitro culture conditions (18). The answer to this problem could be AH, but results obtained on implantation and pregnancy rates after AH for frozen embryo transfer cycles are controversial, as well as for fresh IVF cycles (19). These discrepancies may be attributed to the type of AH, the extent of ZP microdissection, the number of patients and criteria for their selection, or even the quality and stage of embryos selected for AH performance.
In an earlier study, Primi et al. (2004) were unable to show any specific advantage of LAH in cryopreserved embryos. In this study, no embryo selection was reported, so this could explain why the implantation rates observed were lower than those achieved when embryos were selected (20).
Ng et al. (2005) also found a negative effect of LAH on frozen-thawed embryos in their randomised study (implantation rates: 9.0% vs. 12.5%; pregnancy rates: 6.8% vs. 15%). Although a subgroup analysis showed a higher implantation rate, differences were not statistically significant when LAH was performed on embryos with zona thickness of 1.6 mm (21).
In contrast, other studies using similar techniques of LAH were able to show improvement in implantation and pregnancy rates in the LAH group. Such was the case in a study by Balaban and co-authors (2006), in which their data revealed that implantation rates (20.1% vs. 9.9%) and pregnancy rates (40.9% vs. 27.3%) were significantly higher in the group in which embryos had been subjected to LAH before transfer, as compared to those from the control group (22). Valojerdi et al. (2008) in turn showed that LAH increased significantly both implantation and pregnancy rates in embryo cryopreservation cycles (23). These conclusions resemble those by Kanyo and co-authors (2016), who compared clinical pregnancy rates after using LAH technique on day-3 frozen-thawed embryos, and found a higher pregnancy rate after applying the LAH procedure (24).
Reports on patient populations are found that show benefits from AH, which include patients whose embryos present a thick zona, with elevated FSH, over 38 years old and cryopreserved cycles. It seems that the laser-based method is the most used among professionals and the most effective, although differences between are found between studies. However, no significant data show any increase in clinical pregnancy rates that could be translated into an encouragement of this technique to be routinely performed.
It should also be noted that some studies do not include specific important confounding factors, such as patient socioeconomic status, parity, duration of infertility, number of repeated failed cycles, embryo quality and/or smoking and alcohol intake, which might cause residual variance in the obtained results. Additional limitations of the study include the lack of information on the type of assisted hatching (mechanical, chemical, or laser), which may have changed over time or between clinics.
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