Revisiting embryo assisted hatching approaches: a systematic review of the current protocols

Abstract

Zona pellucida (ZP) manipulation, termed “assisted hatching” (AH), has been introduced in order to favor embryo hatching and ultimately improve assisted reproductive technology success but with poor proofs of safety and biological plausibility. We herein provide a systematic review of clinical outcomes following the application of different methods of ZP manipulation on fresh or frozen/thawed embryos at different developmental stages in different groups of patients. Out of the 69 papers that compared the clinical outcomes deriving from hatched versus non-hatched embryos, only 11 considered blastocysts while the rest referred to cleavage stage embryos. The ZP thinning of fresh embryos either by chemical or laser approach was shown to provide very limited benefit in terms of clinical outcomes. Better results were observed with procedures implying a higher degree of zona manipulation, including zona removal. Studies comparing the mechanical or chemical procedures to those laser-mediated consistently reported a superiority of the latter ones over the former. Literature is consistent for a benefit of ZP breaching in thawed blastocysts. This review provides the current knowledge on the AH procedure in order to improve its efficacy in the appropriate context. Embryologists might benefit from the approaches presented herein in order to improve Assisted Reproduction Technologies (ART) outcomes.

Introduction

The assisted hatching (AH) procedure was first described by Cohen et al. who in 1988 reported the first pregnancy after AH on The Lancet [1]. In that study, the partial zona dissection was performed on mature oocytes using the mechanical force in male factor couples.

After more than 25 years of its application on human embryos, the performance of AH in terms of efficacy is still unclear. In 2014, the Practice Committees of the American Society for Reproductive Medicine and of the Society for Assisted Reproductive Technology, based on a Cochrane meta-analysis including 31 randomized controlled trials (RCTs) involving 2933 women whose embryos received an AH treatment and 2795 women in the control group [2], have concluded that there was insufficient evidence to suggest that AH may improve live birth rate [3]. Moreover, due to its association with an increased risk of multiple pregnancies, the routine use of AH for all patients undergoing in vitro fertilization (IVF) or for poor prognosis patients was not recommended. However, there was significant heterogeneity among studies, suggesting that combining trials might not be appropriate.

Most of the confusion surrounding this topic is probably related to the fact that, because AH procedures highly vary in terms of time and mode of application, they are likely to produce completely different consequences on the hatching dynamics of the embryos. Procedures include manipulations of the zona pellucida (ZP) such as (i) making holes, slots or thinning of different sizes; (ii) drilling, cutting, digesting, or melting the zona mechanically, chemically or with a laser beam; (iii) on fresh or frozen/thawed embryos (iv) at different developmental stages (v) in different groups of patients [4, 5]. Although the Cochrane meta-analysis has undertaken some subgroup analyses in order to disentangle issues related to the different methods of AH and the degree of zona manipulation [2], it was not able to address all the possible multifaceted aspects of this topic. Moreover, although the laser system has recently become the most common technology for AH, the potential use of the other methods has not been disproved [6]. The general conclusion drawn from the evaluation of the overall effectiveness may indeed withhold some differences.

Assisted hatching is a microsurgical procedure performed on the embryo and, hence, evaluating its efficacy without stratifications for indications and methods is equivalent to evaluate the efficacy of a surgical procedure independently from its indication and from the technique used; important information might be left out.

A review taking into account most, if not all, of the results stemming from the different AH protocols utilized to date is currently needed. Thus, the purpose of this review was to systematically evaluate the results of all available publications according to the different techniques and protocols used, the different stages of the embryo, the type of cycle, and the indications to the procedure. The ultimate aim is to provide the embryology community with the knowledge necessary for the refinement of AH and the potential improvement of Assisted Reproduction Technologies (ART) outcomes.

Materials and methods

Literature search methodology

This review was performed according to the PRISMA statement [7]. No institutional review board approval was needed because only published, de-identified data were analyzed. All authors participated in the design of the search strategy, inclusion and exclusion criteria.

PubMed and Medline were systematically searched, from inception until 2017 (last research 30 March 2017) using the following keywords and medical subject heading (MeSH) terms: “assisted hatching” alone or in combination with “zona pellucida,” “assisted reproduction technology,” “IVF,” “laser,” “mechanical,” “chemical,” “acidified Tyrode’s,” “partial zona dissection,” “thinning,” “breaching.” All pertinent articles were carefully assessed and their reference lists were evaluated to detect other studies that could be included in this review. All the authors reviewed the articles and discrepancies were resolved by consensus. The reviewers were not blinded to the names of investigators or sources of publication. The eligibility of the studies was firstly based on the titles and abstracts. Full manuscripts were obtained for all selected papers and the decision for the final inclusion was made after detailed evaluation of the articles.

Study selection

Peer-reviewed, English-language journal articles were included in this systematic review. The search included English and human as limits although for the first descriptive part some studies in animals were also cited. During the first screen, titles and abstracts were investigated and studies with lack of any relevance were excluded (Fig. 1). For the AH techniques part, analyses not reporting outcomes were excluded, as well as studies not reporting original data including reviews, meta-analyses, or comments. Full papers of all remaining items were collected and for each study, the following information was collected: first author’s last name, year of publication, research objective, design of the study, outcomes investigated, and conclusions. Specifically, the following outcomes were extracted: implantation, clinical pregnancy, abortion, multiple pregnancy, and live birth rates. Randomized controlled trials (RCTs), prospective cohort studies, case-control studies, and retrospective cohort studies were screened where available. Small case series (< 10 cases) were considered only if they provided highly valuable information. Letters to the editor, case reports, and abstracts accepted in conferences were excluded from the review.

Fig. 1.

Flow diagram of the search strategy, screening, eligibility, and inclusion criteria

The search detected 131 studies eligible for inclusion. Of these, 88 articles were finally included (Fig. 1). Given the characteristics of the included studies and the heterogeneity of the available articles in terms of methodology, no meta-analysis was attempted.

Assisted hatching techniques

Assisted zona hatching is a procedure of assisted reproductive technology in which a small hole is made in the zona pellucida, using a micromanipulator, thereby facilitating for zona hatching to occur. This definition is by far limited considering the variety of protocols and manipulations introduced over the years ranging from zona breaching, whereby a gap of varying sizes is introduced via different methods to the ZP, to zona thinning, whereby chemical solutions or non-contact photo-ablation laser systems are used.

Regarding the common thinking behind the different strategies, the complete perforation of the ZP is thought to cause adverse effects, such as an abnormal expansion process, a higher risk of infectious or immunologic aggression of the exposed embryo, and blastomere loss through the breached ZP [8, 9]. Trapping phenomena are not so infrequent following ZP breaching of the blastocyst (Fig. 2). A significant relation between the size of the zona opening and completion of the in vitro hatching process has been described in animal studies. In the mouse blastocyst, if the size of the opening is < 10 μm, complete hatching is impaired [10] while in cattles, holes smaller than 40 μm in length caused more trapping phenomena of the blastocyst than larger ones [11].

Fig. 2.

Trapping events. Blastocysts were laser-hatched to favor the biopsy. a The embryo is hatching from two openings. b, c The embryo splits in two parts with the ICM trapped and degenerated into the ZP

Advantages of a zona thinning over the creation of a hole would include (i) the prevention of blastocyst hatching without expansion, (ii) the prevention of any potential risk to the embryo due to the retaining of the resilient inner layer of the zona, and (iii) the prevention of embryo splitting to produce monozygotic twinning [12]. On the other hand, the ability of ZP thinning to enhance human embryo implantation has been questioned [13] based on the idea that the human ZP needs to be fully breached to enhance embryo implantation ability [14].

Data from a murine model indicate that the blastocyst repeatedly contracts and re-expands during the hatching process. The physiological role of blastocyst contraction is not well understood but strong contractions in the blastocyst stage seem to have a negative influence on hatching compared to weak contractions [15]. It is plausible to speculate that AH might help the blastocyst hatching process in order to avoid strong contractions and limit great energy expenditure.

The following chapters will illustrate in details these methods and their outcomes on fresh and frozen embryos at different developmental stages.

Chemical methods: protocols and results

Chemical thinning of the ZP utilizing acidified Tyrode’s solution (pH 2.5 ± 0.3) [16, 17] or pronase [18] were first reported to initiate the hatching process at the blastocyst stage in mice [19]. These techniques were carried out based on the acknowledged bilayer nature of the ZP consisting of an easily digested outer layer and a more compact inner layer [20]. The solution (i) is expelled with a micropipette until one-third of the ZP is dissolved to perform the thinning (Fig. 3), (ii) is expelled by a stream procedure until a trough ranging between 20 and 40 μm in width is created in order to perform the drilling [4, 21] (Fig. 4), (iii) is used in a microdroplet containing the embryo to perform a circumferential thinning [16, 22, 23]; and (iv) is used in a microdroplet to prolong the exposure of embryos to a controlled acidified environment to obtain the zona removal [24, 25].

Fig. 3.

Procedure commonly used for embryo chemical thinning: [1] the embryo is held by a holding pipette and approached by an AH needle filled by acidified/enzymatic solution; [2] the solution is released gradually in vertical fashion; [3] the solution release is stopped immediately once desired thinning is achieved

Fig. 4.

Procedure commonly used for embryo chemical breaching: [1] the embryo is held by a holding pipette and approached by an AH needle filled by acidified solution; [2] the solution is released gradually and from a distance to assure no harm occur to embryos; [3] the solution release is stopped immediately once a hole is created

Fresh embryo transfer: zona breaching

The use of chemical AH utilizing zona drilling on embryos has been analyzed in 21 studies (Table 1). None considered AH at blastocyst stage. Results from the various studies are very controversial.

Table 1.

Results of ZP breaching/thinning using chemical-assisted hatching in fresh embryo transfer

	Method	Opening size	Time of AH	Age (mean ± SD) AH/control	PR	CPR	IR	AbR	MPR	LBR	P/PR/R study
Cohen et al. 1992 [1]	Acidic Tyrode’s solution zona breaching	20 ± 6.7 μm	Day 3 embryos	–	Improved	Improved	Improved	–	Unchanged	–	PR
Liu et al. 1993	Acidic Tyrode’s solution zona breaching	20 ± 6.7 μm	Day 3 embryos	–	Improved	Improved	Improved	–	Unchanged	–	R
Tucker et al. 1993	Acidic Tyrode’s solution zona thinning	–	Day 3 embryos	34.1 ± 4.8 34.2 ± 4.1	–	–	Unchanged	–	–	–	PR
Schoolcraft et al. 1994	Acidic Tyrode’s solution zona breaching	30 μm	Day 3 embryos	38.5 ± 3.3 37.2 ± 3.1	Improved	Improved	Improved	–	–	–	R
Tucker et al. 1996	Acidic Tyrode’s solution zona breaching	10–12 μm	Day 3 embryos, 4 h before ET	33.5 ± 4.3 35.3 ± 4.2	–	Improved in ≥ 35 age	Unchanged	–	–	–	PR
Hu et al. 1996 [2]	Acidic Tyrode’s solution zona thinning + mechanical breaching	5–7 μm final opening	Days 2–3 embryos	–	–	n.a.	n.a.	–	–	n.a.	R
Bider et al. 1997	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos	41.9 ± 2.8 41.1 ± 2.2	Unchanged	–	Unchanged	Unchanged	–	Unchanged	R
Hurst et al. 1998	Acidic Tyrode’s solution zona breaching	–	Day 2 embryos	28.2 ± 1.0 25.3 ± 0.9	Unchanged	–	Reduced	–	–	Unchanged	R
Lanzendorf et al. 1998	Acidic Tyrode’s solution zona breaching	20 μm	Day 2 embryos, 55 h post insemination	38.3 ± 0.3 38.5 ± 0.2	–	Unchanged	Unchanged	–	Unchanged	–	PR
Meldrum et al. 1998	Acidic Tyrode’s solution zona breaching	20 μm	Day 3 embryos	–	–	Unchanged	Improved in 35–39 and 40–42 age	Unchanged	Unchanged	Unchanged	R
Magli et al. 1998	Acidic Tyrode’s solution zona breaching	25 μm	Day 3 embryos	40.5 ± 2.4 39.5 ± 1.7a	Improved	–	Improved	Unchanged	–	–	PR
				32.7 ± 3.2 32.2 ± 3.2b	Improved	–	Improved	Unchanged	–	–
				39.6 ± 1.5 40.5 ± 2.4c	Unchanged	–	Unchanged	Unchanged	–	–
Mahadevan et al. 1998	Acidic Tyrode’s solution zona breaching		Day 3 embryos	39.7 (39–43) 40.1 (39–43)	Unchanged					Unchanged	R
Graham et al. 2000	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos	36.4 ± 2.9 32.7 ± 3.5d	–	Unchanged	Unchanged	–	–	Unchanged	R
				35.2 ± 2.4 31.5 ± 3.3e	–	Unchanged	Unchanged	–	–	Unchanged
Hsieh et al. 2002 [3]	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos	39.9 ± 1.2 40.9 ± 1.9f	Reduced	Unchanged	Reduced	Unchanged	–	Reduced	PR
Balaban et al. 2002 [4]	Acidic Tyrode’s solution zona breaching	30 μm	Day 3 embryos	34.6 ± 3.2 29.8 ± 3.0	Unchanged	Unchanged	Unchanged	Unchanged	Unchanged	–	R
	Pronase zona thinning			34.5 ± 3.1 29.8 ± 3.0	Unchanged	Unchanged	Unchanged	Unchanged	Unchanged	–
Joris et al. 2003 [5]	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos for biopsy	32.3 ± 4.4 32.2 ± 4.9f	Unchanged	Unchanged	Unchanged	–	Unchanged	Unchanged	R
Dayal et al. 2006	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos	35.2 ± 3.6 34.4 ± 3.8	Improved	Improved	Improved	–	–	Unchanged	R
Ma et al. 2006	Acidic Tyrode’s solution zona breaching	10–12 μm	Day 3	34.5 ± 4.7 35.5 ± 3.8	Unchanged	Unchanged	Improved	Unchanged	Unchanged	Unchanged	PR
Yano et al. 2007 [6]	Acidic Tyrode’s solution zona thinning	–	Days 2–3 embryos	34.5 ± 3.3 36.4 ± 2.1	–	Improved	Improved	Unchanged	Unchanged	–	PR
Grace et al. 2007	Acidic Tyrode’s solution zona breaching	20–30 μm	Day 3 embryos	35.7 ± 3.9g 35.8 ± 4.6h	Improved	Improved	Improved	–	–	Improved	R
Lazendorf et al. 2007 [3]	Acidic Tyrode’s solution zona breaching	20 μm	Day 3 embryos	34.0 ± 4.5 35.2 ± 4.6f	Unchanged	Unchanged	Unchanged	–	Unchanged	Unchanged	PR
Feng et al. 2009 [7]	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos	35.0 ± 2.1 34.6 ± 2.7	–	Improved	Unchanged	–	–	–	R
Hagemann et al. 2010	Acidic Tyrode’s solution zona breaching	20 μm	Day 3 embryos	32.1 ± 3.0 31.2 ± 3.5	–	Unchanged	Unchanged	Unchanged	Unchanged	Unchanged	PR
Kim et al. 2012 [8]	Acidic Tyrode’s solution zona breaching	–	Day 3 embryos for biopsy	31.6 ± 2.5 31.3 ± 2.7i	–	Reduced	Reduced	–	–	–	R

[1] Overall results of 3 trials. [2] No control group provided. [3] Compared to laser-assisted hatching. [4] Comparison of 4 different techniques including laser and PZD. [5] Compared to laser-assisted hatching in PGD cycles. [6] Compared to circumferential thinning and controls. [7] Compared to PZD and laser-assisted hatching. [8] Compared to PZD in PGD cycles

ET embryo transfer, PR pregnancy rate, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective, n.a. not applicable, PZD partial zona dissection, PGD pre-implantation genetic diagnosis

^aAdvanced maternal age group

^bRepeated unexplained IVF failures group

^ca + b

^dDay 3 transfer

^eDay 5 transfer

^fMean age of the laser-hatched group

^gMean age of the IVF failure group with at least one optimal embryo replaced in a previous cycle without HA

^hMean age of the IVF failure group with only suboptimal quality embryos replaced in a previous cycle without HA

ⁱMean age of the PZD hatched group

Retrospective analyses generally did not support the benefit of the procedure [26–28] except for some subgroups of patients—poor prognosis [29], older age [30], or with previous failed IVF cycles [31, 32]—or again, results were not assessable due to the lack of a control group [33]. Even an impairment in embryo quality and implantation rate was observed in the AH group compared with the control group in a retrospective contribution including a small sample of young patients [34].

Similarly, first prospective studies have initially shown an increase in implantation and clinical pregnancy rates only in certain conditions. Cohen et al., combining 3 randomized trials, concluded that AH by means of chemical zona drilling was effective in improving clinical pregnancy and implantation rates in case of embryos with a thick zona (≥ 15 μm), especially in women over 38 and with elevated basal FSH. On the contrary, embryos with a thin zona (< 13 μm) appeared to be damaged from the procedure [35]. The next year, the same group analyzed retrospectively the pregnancies of the above-mentioned three trials and reported that implantation occurred significantly earlier in the AH group than in non-hatched controls, perhaps by allowing an earlier embryo-endometrium contact [36]. In a prospective randomized trial involving 100 couples, AH was performed on day 3 utilizing acidified Tyrode’s solution and while the treatment did not have an overall significant impact on intracytoplasmic sperm injection (ICSI) outcomes, in the small group of patients aged ≥ 35 years, AH exhibited a significant benefit in terms of clinical pregnancy rate (45.2% in treated versus 16.9% in non-treated controls, p < 0.05) [37]. These results were not supported by a subsequent randomized double-blinded study conducted on patients aged ≥ 36 with a similar sample size [38]. In a larger prospective randomized study, Magli et al. [39] examined AH efficacy in three groups of poor prognosis patients: (i) advanced maternal age (≥ 38), (ii) ≥ 3 previous IVF failures, (iii) a combination of the two aforementioned conditions. Clinical pregnancy rate per cycle was significantly higher in the AH study groups i (31%) and ii (36%), compared with the respective non-hatched control groups (10 and 17% respectively, p < 0.05). The same trend was observed for group iii, but the difference was not statistically significant possibly because of the low number of patients in this group. Similarly, the implantation rate was statistically higher in the first 2 groups (11.5 and 15% compared to the respective controls 4 and 6.3%, p < 0.02) but not in the last group. More recently, the efficacy of chemical AH was re-evaluated in two prospective randomized trials, both including broad-spectrum patients. Ma and coworkers [40] included all patients with an indication for ICSI. Regardless of age, the implantation rate was higher in the AH group compared to the control non-hatched group (16 vs 8%; p < 0.01). However, a sub-analysis based on the woman’s age (≤ 34 years and ≥ 35 years old) showed that only women aged ≥ 35 years maintained a higher implantation rate compared to the same aged control group. Focusing only on young patients (≤ 38 years old), Hagemann and coworkers [41] could not find any statistical difference in ART outcomes between the hatched and the control group. Nevertheless, this study had some important limitations, in particular the inadequate power. Moreover, embryos with a ZP thickness ≤ 13 μm were not hatched, so that some transfers in the hatched group may have included non-hatched embryos, and embryos from patients with good prognosis in day 3 were left untouched and transferred on day 5, skewing the randomization.

Therefore, in general, the evidence showcased a tendency to exclude the benefit of the procedure in all patients but rather suggested that better outcomes are limited to subgroups of poorer prognosis patients.

Some studies have compared the chemical AH to other hatching methods. None of these demonstrated a superiority of chemical hatching compared to the laser AH and, notably, there were some controversies in the comparison of the latter with the mechanical method [42–45]. Lanzendorf and coworkers [46] did not find a difference in clinical outcomes when comparing laser or acidified Tyrode’s solution hatching. Once again, embryos from patients with a good prognosis in day 3 (≥ 4 embryos at 7–8 cells stage) were left untouched and transferred in day 5. Interestingly, the implantation rate for day 5 non-hatched embryos was significantly greater than both the laser and the acid treatment groups. On the contrary, Feng and colleagues [47] found that the AH procedures, performed by laser, acidified Tyrode’s solution or mechanically, improved the clinical pregnancy rate compared with the control non-hatched group. Their findings, additionally, showed that both the chemical and the laser AH were more effective in enhancing clinical pregnancy rate than Partial Zona Dissection (PZD). None of the studies have reported differences in multiple pregnancies in patients who received chemical hatching of the embryos.

Fresh embryo transfer: zona thinning

Chemical AH with the aim to thin the ZP in fresh embryos has been object of limited interest (Table 1) after the prospective randomized study of Tucker et al. [20] who failed to show any improvement in implantation rates in a non-selected group of 218 patients. Also, various sub-analyses including an age limit of 34 years old, high FSH levels or variability in the zona thickness, did not show any improvement in the clinical outcome. Interestingly, according to these authors, embryos with a thicker ZP implanted as well as those with a thinner zona regardless the AH procedure. As previously mentioned, a large retrospective comparative study comparing four different methods including laser, PZD, chemical breaching by acidified Tyrode’s solution, and ZP thinning using pronase, showed no significant improvement in either implantation or clinical pregnancy rate when compared to non-hatched control groups [42]. Finally, Yano et al. [23] randomized 163 patients that experienced ≥ 2 cycles of IVF or ICSI in three groups: (i) partial zona thinning using acidified Tyrode’s solution, (ii) circumferential zona thinning, and (iii) intact zona demonstrating, in disagreement with the previous study, the superiority of partial zona thinning compared to controls without AH in terms of implantation and clinical pregnancy rates. Circumferential zona thinning did provide some benefits but without statistical significance.

The results of these studies are inconclusive with regards to the efficacy of the thinning technique in favoring fresh embryo implantation but no other studies have been published using the chemical approach since 2007. The introduction of laser has probably substituted this approach in the following years.

Frozen embryo transfer: zona breaching

In 1995, a case was published confirming successful implantation of cryopreserved human embryos after AH with acidified Tyrode’s solution. Later on, a retrospective analysis by Check et al. [48] was conducted on surplus cryopreserved embryos from 158 patients (Table 2). The implantation rate as well as the clinical pregnancy rate were significantly higher in the AH group compared to the non-AH group. However, since culture conditions and times were different between the two groups, this improvement could not be clearly attributed to AH. The protein supplementation during culture was also different. A pioneer study examined the effect of AH on day 2 frozen/thawed surplus embryos in a small population of patients meeting at least one of the following criteria: (i) > 38 years old, (ii) thick zona (> 17 μm), (iii) poor embryo quality, and (iv) multiple embryo transfer failures. The small sample size and the very poor clinical outcomes in the control group hamper seriously the value of this study [49]. A larger prospective blinded randomized study comprising 253 frozen/thawed cycles at day 2 was subsequently conducted by Gabrielsen and coworkers [50]. Embryos underwent drilling treatment using acidified Tyrode’s solution 24 h after thawing. Implantation rate in the AH group was significantly improved (11.4%) when compared with controls (5.8%; p < 0.005) but no improvement in pregnancy rate was observed.

Table 2.

Results of ZP breaching/thinning using chemical-assisted hatching in frozen embryo transfer

	Method	Opening size	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Check et al. 1996	Acidic Tyrode’s solution zona breaching	–	≥ 8 cells, post thawing	34.5 34.9	Improved	Improved	–	Unchanged	–	R
Tao et al. 1997	Acidic Tyrode’s solution zona breaching	10–13 μm and 15–18 μma	Day 2 embryos, 1–1.5 h before ET	36 40b	Unchanged	Unchanged	Unchanged	–	–	P
Gabrielsen et al. 2004	Acidic Tyrode’s solution zona breaching	~ 30 μm	Day 3 embryos, post thawing	33.1 ± 4.2 23.8 ± 4.1	Unchanged	Improved	–	–	–	PR
Sifer et al. 2006	Pronase thinning	14.5 ± 2.75 μm	Day 3 embryos, 15 min before ET	32.3 32.0	Unchanged	Unchanged	–	–	–	PR

ET embryo transfer, PR pregnancy rate, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective

^aInner and outer diameters, respectively

^bMedian age

Frozen embryo transfer: zona thinning

Chemical zona thinning was evaluated in a prospective randomized study [9], including 125 frozen transfer cycles in patients with various infertility indications (Table 2). Surplus embryos were cryopreserved in day 2 or day 3 and partial enzymatic digestion by pronase was performed post thawing on day 3 stage embryos. Despite the statistically significant decrease of ZP thickness after pronase treatment [(mean ± SD) 18.5 ± 2.25 versus 14.5 ± 2.75 mm; p < 0.0001], neither implantation (9.6 versus 9.2%) nor clinical pregnancy rates (18.0 versus 17.2%) were modified by the treatment.

In summary, based on presented findings, studies on embryo AH using chemical methods, which were largely published over a decade ago, did not consider the procedure on blastocysts and were mostly focused on fresh embryos. Randomized controlled studies found some benefit of zona breaching for subgroups of poorer prognosis patients. None of the studies found a superiority of the chemical approach to the use of laser.

Mechanical methods: protocols and results

The partial zona dissection (PZD) has been the precursor of these procedures. The embryo is held firmly in position by the holding pipette and an opening is made by introducing an injection/dissecting pipette through the ZP, followed by rubbing the embryo gently against the holding pipette until the embryo is released [45, 51, 52] (Fig. 5). A modified procedure of PZD called three-dimensional PZD (3D-PZD) was introduced because an inadequate size of the opening could be responsible for embryo entrapping and incomplete hatching (Fig. 6). Detailed description of this procedure can be found elsewhere but, essentially, a cross-shaped opening is made with a microneedle through maneuvers of embryo rotating and cuts. PZD or 3D-PZD openings are usually of 30–40 μm in human embryos [53].

Fig. 5.

Procedure commonly used for embryo PZD: [1] the embryo is rotated with the microneedle until the area with the largest perivitelline space is visible at the 12 o’clock position; [2] ZP is pierced by a needle at the 1 to 2 o’clock position; [3] the embryo is released from the holding pipette and rubbed against it until the embryo is released

Fig. 6.

Procedure commonly used for embryo 3D-PZD: [1] the embryo is rotated with the microneedle until the area with the largest perivitelline space is visible at the 12 o’clock position; [2] the ZP is pierced by a needle at the 1 to 2 o’clock position; [3] the embryo is released from the holding pipette and rubbed against it until the embryo is released; [4] the embryo is held again and rotated vertically until the first slit is visible at the 12 o’clock position; [5] steps from 1 to 3 are repeated until the embryo is released from the piercing needle

While 3D-PZD has improved the zona opening size, reasons to limit the mechanical zona dissection use include (i) the opening may be not large enough to achieve a high rate of complete hatching of the blastocyst [53]; (ii) the potential mechanical injury during the manipulation, such as squeezing or injury to embryos; and (iii) the change in hydrostatic pressure, which is potentially harmful to spindle microtubules [54].

The controlled zona dissection (CZD) represents a modified form of PZD that is carried out using modified micro-pipettes. It includes a moderate zona dissection (MZD), with a hole measuring about two-fifths of the embryo diameter, or a long zona dissection (LZD) for larger holes. The opening of the holding pipette beveled with an angle of ∼ 65°. The hatching needle is similar to the ICSI injection needle, thinner at the front and with a blunted tip. Detailed description of the LZD procedure can be found elsewhere [55]. Some maneuvers performed using both the holding and the hatching needle are required (Fig. 7). For blastocyst AH, the zona opening site can be selected at the site opposite to the inner cell mass (ICM), near the ICM or other designated sites.

Fig. 7.

Procedure commonly used for embryo LZD: [1] the embryo is held at about the 8 o’clock position and then is pierced with the tip of the hatching needle at about the 5 o’clock position. Pushing the cell membrane aside by using the needle tip allows to obtain space in the perivitelline area; [2] the needle advances until it touches the opposite side of the ZP; [3] once there, the needle is withdrawn a little from the ZP, then the embryo is released from the holding pipette and rotated; [4] the embryo is then held from a new site and the needle is moved forward to cut the ZP. Repeating the step of rotating the embryo and piercing a new site helps obtaining a cut ZP with a controlled size; [5] the curve of the needle is pushed against the bottom of the dish to cut the pierced ZP

The piezo-micromanipulation represents a modification of the PZD technique, in which a vibratory motion of a needle produced by a piezoelectric pulse is used so that both zona thinning and breaching can be achieved simultaneously in a specific area. A 20-μm hole can be produced in the thinned zona [42]. Finally, an increase in the hydrostatic pressure inside the embryo represents a natural mechanism that may favor the hatching process through a mechanical expansion of the ZP. Such hydrostatic pressure can be artificially induced by injecting some medium into the perivitelline space resulting in the stretching of the ZP. The technique has been rarely used.

Fresh embryo transfer: zona breaching

Data on outcomes derived from the mechanical zona breaching at cleavage stage during fresh embryo transfer are very inconsistent. The studies with the greater sample size tend to exclude the usefulness of the technique and its superiority respect to others (Table 3). Hellebaut and coworkers [56] were the first to publish a randomized study using PZD in all patients undergoing an ART cycle failing to find differences in outcomes related to the procedure. Conversely, four subsequent studies focused on patients with repeated IVF failures demonstrating that zona breaching using PZD or piezo-micromanipulator could provide some benefits in terms of implantation and clinical pregnancy rates [52, 57–59]. Interestingly, the same group that has firstly found a benefit of PZD in terms of clinical outcomes in patients older than 38 years [52] some years later reported that the same technique could reduce the clinical pregnancy rate in recurrent implantation failure patients younger than 35 and no difference was found in older participants [60]. In a small retrospective study considering patients who underwent PZD for (i) advanced age, (ii) two or more IVF failures, or (iii) presence of embryos with an increased zona thickness and control patients who did not undergo AH, clinical outcomes were similar among the various groups evaluated [61].

Table 3.

Results of ZP breaching using mechanical assisted hatching in fresh embryo transfer

	Method	Opening size	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Hellebaut et al. 1996	PZD	–	Day 2 embryos, before ET	30.9 ± 4.3 30.8 ± 3.9	Unchanged	Unchanged	–	Unchanged	–	PR
Stein et al. 1995	PZD	–	4–6 cell stage	> 38	Improved	–	–	–	–	P
Parikl et al. 1995	PZD	20 μm	6–8 cell stage	> 38	–	Improved	–	–	–	R
Chao et al. 1997	PZD	–	Day 2 embryos, 4–6 h before ET	36.5 ± 5.2 34.0 ± 3.9	–	Improved	–	–	–	PR
Edirisinghe et al. 1999	PZD	–	Day 3 embryos	–	Unchanged	Unchanged	Unchanged	Increased	–	R
Cieslak et al. 1999 [1]	3D–PZD	–	Day 3 embryos, before ET	34.0 33.8	Unchanged	Unchanged	Unchanged	Unchanged	–	PR
Nakayama et al. 1999 [2]	Piezo-micro manipulator	20 μm	Days 2–3 embryos, before ET	35.4 ± 4.5 35.9 ± 4.0	Improved	Improved	–	–	–	PR
Balaban et al. 2002 [3]	PZD	–	Day 3 embryos, before ET	34.4 ± 3.3 29.8 ± 3.0	Unchanged	Unchanged	Unchanged	Unchanged	–	R
Rufas-Sapir et al. 2004	PZD	–	Days 2–3 embryos, before ET	< 35	Reduced	–	–	–	–	PR
Makrakis et al. 2006 [4]	PZD	5–10 μm	Day 3 embryos, before ET	41.0 ± 1.5 40.9 ± 1.5	Unchanged	Unchanged	–	–	Unchanged	PR
Kim et al. 2012 [5]	PZD	–	Day 3 embryos	31.3 ± 2.7 31.6 ± 2.5	Improved	Improved	–	–	–	R

[1] Compared to PZD. [2] In good quality embryos. [3] Compared to acidified Tyrode’s solution, laser diode, and pronase zona thinning. [4] Compared to laser AH. [5] Compared to acidified Tyrode’s solution

ET embryo transfer, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective, PZD partial zona dissection, 3D-PZD three-dimensional partial zona dissection

Subsequent studies have mostly compared different AH techniques.

Cieslak and coworkers (1999) have compared PZD versus 3D-PZD zona breaching in an unselected population finding a non-significant improvement of clinical outcomes for the three-dimensional procedure. The mechanical procedure did not demonstrate any improvement over the laser-mediated AH at least in selected patients [42, 51] whereas in a single recent small retrospective contribution, PZD has shown superiority over acidified Tyrode’s solution [45] in terms of implantation, clinical and ongoing pregnancy rate.

Frozen embryo transfer: zona breaching

Zona breaching by means of PZD in frozen/thawed embryos was used for the first time by Tucker and coworkers in 1991 in a prospective randomized study [62] (Table 4). No significant improvement in clinical pregnancy rate was observed in the hatched group compared to non-hatched controls, probably due to the small sample considered. It needs to be underlined, however, that embryos were frozen/thawed and transferred at different stages (zygote and day 3) and that in the AH group, more one-cell embryos (zygotes) were frozen/thawed and transferred compared with the control group, potentially affecting the results. Another study, attempting to evaluate the usefulness of PZD performed after thawing of frozen day 3 embryos, failed to demonstrate a beneficial effect in patients with (i) advanced age, (ii) two or more IVF failures, or (iii) presence of embryos with an increased zona thickness [61]. Results from frozen/thawed blastocysts were more encouraging (Table 4). In a retrospective analysis, artificial opening of the ZP on blastocysts originating from embryos of different qualities demonstrated a significant improvement of the transfer rate and of the implantation and clinical pregnancy rates [63].

Table 4.

Results of ZP breaching using mechanical assisted hatching in frozen embryo transfer

	Method	Opening size	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Tucker et al. 1991	PZD	–	Zygotes and day 3 embryos, after thawing	33.9 ± 3.9 36.0 ± 3.7	Unchanged	Unchanged	–	–	Unchanged	PR
Edirisinghe et al. 1999	PZD	–	Day 3 embryos, after thawing	–	Unchanged	Unchanged	–	–	–	PR
Vanderzwalmen et al. 2003	PZD	10–15 μm	Day 5 blastocysts, after warming	–	Improved	Improved	–	–	–	R
Fang et al. 2010	Mechanical expansion	–	Day 3 embryos, after thawing	–	Improved	Improved	–	–	–	PR
Sun et al. 2012 [1]	LZD	2/3 ZP diameter	Day 5 blastocysts, after warming	≤ 38	Improved	Improved	–	–	–	PR

[1] Compared with PZD

ET embryo transfer, PR pregnancy rate, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective, PZD partial zona dissection, LZD long zona dissection

In a relatively small prospective randomized study comparing LZD with PZD, significantly higher implantation and pregnancy rates [64] were found to be associated with the LZD procedure together with increased complete hatching rates. Mechanically-induced zona expansion has been examined in day 3 frozen/thawed embryos in patients with fresh IVF failures or in those who did not undergo fresh embryo transfer, randomly allocated to AH or a control group. Mechanical expansion of the ZP increasing hydrostatic pressure after thawing could increase the implantation and clinical pregnancy rate compared to the group of women in whom AH was not performed [22].

In summary, based on the findings provided, a strong inconsistency characterizes studies AH using the mechanical method on fresh cleavage stage embryos. Rather interestingly, these studies have been published more than 10 years ago. Although none of the studies have provided a definitive and strong message in this regard, the lack of recent evidence suggests that the mechanical procedure is no longer commonly used on fresh embryos. The benefit of the zona opening on frozen embryos needs to be further investigated.

Laser methods: protocols and results

Laser-mediated assisted zona drilling was first used by Tadir et al. [65] and Palanker et al. [66], who employed the laser beam technology to create a precise breach in the ZP. Laser represents an ideal tool for microsurgical procedures, as the energy is easily focused on the targeted area producing a controlled and precise hole, consistent between operators. Two methods can be used. In the first method, the contact mode, the laser is guided through optical fibers touching the embryo and employs ultraviolet (UV) wavelength delivered by a glass pipette, or alternatively infrared (IR) wavelength delivered with a quartz fiber. Although the procedure initially led to encouraging results in terms of clinical outcomes [67], it requires a certain level of technical expertise and the re-sterilization of pipettes and fibers. In the second method, the non-contact mode, the laser beam is directed using an optical lens tangential to the embryo through the ZP. The non-contact mode using various laser wavelengths was preferred for gamete micromanipulation. UV radiation is potentially mutagenic and some technical advantages associated with the non-contact mode 1.48 μm diode IR laser have led to its general preference [68, 69]. In fact, the 1.48-μm laser radiation can be focused through the conventional optics of an inverted microscope within a polystyrene culture dish filled with culture medium, providing an easy non-touch and objective-driven access of the laser light to specific cellular subcomponents such as the embryo ZP. The procedure can be done without particular technical skill with the aid of computer programs to precisely define the exact location, duration and increment of the energy to be delivered. Finally, a laser instrument designed for the clinical practice must consider the possible thermal effects and should minimize pulse duration and laser intensity [70].

Laser use allows to thin or to make actual holes in the ZP (Fig. 8a–f). Both light electron microscopy and scanning electron microscopy have revealed no ultrastructural degenerative alterations of oocyte and embryo ZP following laser-mediated assisted zona drilling [71].

Fig. 8.

Procedure commonly used for embryo laser HA. The embryo is thinned with a single hole without reaching the inner membrane [a] or is thinned irradiating at one point and continuing until one quarter [b] or half [c] of ZP; the embryo is breached with a single hole completely through the ZP [d], or is breached for one quarter [e] or for half of ZP [f]; the blastocyst is breached with a single hole completely through the ZP [g], or is breaching for one quarter [h] or for half of ZP [i]

Fresh embryo transfer: zona breaching

There are four randomized clinical trials, a prospective study and two retrospective analyses reporting clinical outcomes after fresh embryo transfer of laser breaching embryos (Table 5) with very conflicting results. Also these studies have evaluated the outcomes at the embryo stage. Older studies are in general more supportive regarding the efficacy of the procedures in terms of improvements of IVF outcomes than the more recent ones. The older randomized study using non-contact UV laser instead of IR laser showed statistically increased clinical pregnancy and implantation rates in treated patients with ≥ 2 previous failures but the method for allocation of randomization was not defined and the sample size was limited [72]. Further, a prospective non-randomized study revealed an age-related benefit after zona breaching. Higher implantation and pregnancy rates were reported among women < 36 years, but not among older women (≥ 36 years), compared to a control group [73]. An important bias for this study was that only two out of three transferred embryos were subjected to laser zona drilling in order to minimize the hypothesized increase of monozygotic twinning. Moreover, AH seemed to be beneficial for embryos with thin zonae (≤ 16 μm) but not with thick zonae (≥ 17 μm) [73]. The more recent randomized study by Valojerdi and coworkers was based on a large sample size of patients with a poor prognosis (205 patients ≥ 37 years and 398 patients with recurrent implantation failures) and concluded that neither patients with advanced age nor with recurrent implantation failures may benefit from transfer of laser-breached embryos [74]. No benefit was also demonstrated in a good prognosis population of 118 women whose embryos were laser-assisted hatched compared to 82 control women [75] and in 182 unselected randomized patients undergoing their first ICSI cycle [76]. None of these randomized studies could detect significant differences in multiple pregnancies in patients who received fresh laser-breached embryos [74–76]. Conversely, in a retrospective contribution, Ghannadi and coworkers have observed an increased clinical pregnancy rate in both patients ≤ 35 years old (50.0 and 30.7% in treated and untreated women, respectively) and in those > 35 years (27.7 and 16.4% in treated and untreated women, respectively). Multiple pregnancy rate was increased as well in younger patients (22.3 and 5.9% in treated and untreated women, respectively) [77].

Table 5.

Results of ZP breaching using laser-assisted hatching in fresh embryo transfer

	Laser indication (irradiation time)	Opening size	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Antinori et al. 1996	15–20 pulse/s with 2–5 mJ	15-20 μm	Day 2 embryos, immediately before ET	38.2 ± 1.3 37.8 ± 1.5	Improved	Improved	Unchanged	Unchanged	Unchanged	PR
Ali et al. 2003	25–750 ms	8-10 μm at the inner part of ZP; 15–17 μm at outer part of ZP	Day 2 embryos, before ET	≤ 36	Improved	Improved	–	–	–	P
				> 36	Unchanged	Unchanged	–	–	–
Kanyo et al. 2003	2–3 shots of 10–15 ms	5-10 μm	Day 2 embryos, 24 h before ET	35.3 33.2	Unchanged	Unchanged	Unchanged	–	Unchanged	R
Sagoskin al. 2007	6–10 bursts of 500 μs	20 μm	Day 3 embryos, immediately before ET	34.0 ± 3.3 34.0 ± 3.2	Unchanged	Unchanged	Unchanged	Unchanged	Unchanged	PR
Valojerdi et al. 2008	several pulses of 0.5 ms	40 μm	Day 2 embryos, immediately before ET	39.7 ± 2.2 39.9 ± 2.1a	Unchanged	Unchanged	–	Unchanged	–	PR
				32.8 ± 5.3 32.2 ± 5.5b	Unchanged	Unchanged	–	Unchanged	–
Ghannadi et al. 2011	2–3 pulse of 0.8 ms with 400 V	5-10 μm	Day 2 embryos, before ET	≤ 35	Improved	–	–	Increased	–	R
				>35	Improved	–	–	Unchanged	–
Razi et al. 2013	605 μs	10-12 μm	Day 2 embryos in the morning and left until ET	32.9 ± 0.5 31.6 ± 0.4	Unchanged	–	–	Unchanged	Unchanged	PR

ET embryo transfer, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective

^aIn patients with advanced female age

^bIn patients with recurrent implantation failure

Discrepancies among studies might be related to differences in the study power, study design, or variations in AH techniques. Time of AH may vary from 24 h before transfer to immediately before transfer and similarly, irradiation time and size of the hole (10–12 μm, 20 μm, until 40 μm) were very different (Table 5). It should be also emphasized that the live birth rate was either not evaluated or was not affected.

A single offspring follow-up study has analyzed the effects of laser AH and found no increase in major congenital malformations or in chromosomal aberrations. However, the number of children evaluated was limited (n = 134) [78].

Fresh embryo transfer: zona thinning

Using six ablations of 15 ms made successively around the ZP to achieve a depth of 50–80% of the zona thickness for a total length of 80 μm, the hatching and complete hatching rate in vitro were shown to be significantly increased when laser-mediated thinning of the blastocysts was performed compared to non-treated control embryos [79] (Fig. 8).

Eight prospective randomized studies and a retrospective analysis have reported clinical outcomes after fresh embryo transfer of laser-thinned embryos with quite consistent results (Table 6). All the studies except one [80] have evaluated the outcomes of the procedure performed at the embryo stage. Four published papers focused on the application of laser zona thinning in a population aged ≤ 37 years [81–84] without revealing any significant beneficial effect of laser zona thinning on clinical pregnancy, implantation, miscarriage, and live birth rates. Two papers analyzed the effects of zona thinning in a population aged > 37 years, again showing that laser AH had no impact on the IVF outcomes [13, 85]. The only result deserving attention refers to a prospective randomized clinical trial that considered patients with previous implantation failures [86]. For patients with ≥ 2 previous implantation failures, but not in patients with one previous implantation failure, the implantation rate was significant higher for laser-thinned embryos than for the control group (10.9 and 2.6%, p = 0.02) [86]. None of the studies have reported differences in multiple pregnancies in patients who received fresh laser-thinned embryos [81]. Moreover, in a prospective randomized study specifically addressing the usefulness of AH in patients with endometriosis, the technique did not result in a significant improvement suggesting that endometriosis would not be an indication [14]. The only contribution considering both fresh laser-thinned embryos and blastocysts has evaluated a retrospective cohort and showed that laser AH did not increase the clinical pregnancy or live birth rates but the sample size was very small [80].

Table 6.

Results of ZP thinning using laser-assisted hatching in fresh embryo transfer

	Laser indication (irradiation time)	Length/depth of ZP	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Baruffi et al. 2000	12 ms	16–18 μm/not stated	Day 2 embryos, immediately before ET	31.8 ± 3.6 31.4 ± 3.6	Unchanged	Unchanged	Unchanged	–	–	PR
Petersen et al. 2002	1–2 irradiation of 10 ms applied to 4 different sites on the ZP	15–20 μm/60–90%	Days 2–3 embryos, immediately before ET	39.8 ± 1.3 40.0 ± 1.9	Unchanged	Unchanged	Unchanged	–	Unchanged	PR
Petersen et al. 2005	Maximum of 8 ablations of 9 ms	25%/50–80%	Days 2–3 embryos	34.6 ± 4.6a 34.1 ± 5.3	Unchanged	Unchanged	Unchanged	–	Unchanged	PR
				35.7 ± 3.8b 35.3 ± 5.1	Unchanged	Improved	Unchanged	–	Unchanged
Nadir Çiray al. 2005	Not stated	25%/not stated	Day 3 embryos, 2–3 h before ET	33.1 ± 4.2 34.0 ± 3.7	Unchanged	Unchanged	–	–	–	PR
Frydman et al. 2006	10 ms	25%/not stated	Day 2 embryos, immediately before ET	39.0 (37.0–42.3)c 38.5 (37.0–41.6)c	Unchanged	Unchanged	–	–	Unchanged	PR
Ge et al. 2008	Few milliseconds	25%/50%	Days 2–3 embryos, immediately before ET	31.1 ± 4.7 30.4 ± 4.2	Unchanged	Unchanged	–	–	Unchanged	PR
Balakier et al. 2009	2 ms	20–40 μm/60–80%	Day 3 embryos, 1–3 h before ET	32.5 ± 3.8 33.8 ± 3.2	Unchanged	Unchanged	Unchanged	Unchanged	Unchanged	PR
Kutlu et al. 2010	4 ms	Not stated/ZP thickness was not less than 5 μm	Day 3 embryos	29.9 ± 2.9 28.9 ± 3.4	Unchanged	Unchanged	–	–	–	PR
				38.0 ± 2.3 37.4 ± 2.4	Unchanged	Unchanged	–	–	–
Lu et al. 2015	2.8 ms	25%/not stated	Days 2–5 embryos/blastocysts	37.5 ± 3.9 37.1 ± 5.5	Unchanged	–	–	–	Unchanged	R

ET embryo transfer, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective

^aPatients with one previous implantation failure

^bPatients with repeated implantation failures

^cMedian (range)

It should indeed be considered that most of these studies did not have the adequate power to enable detection of subtle changes in clinical pregnancy and live birth rates and were characterized by a great variability in study design, inclusion and exclusion criteria, and in the laser technique used (Table 6).

Laser pulse times to thin the zona varied among the studies while the length of zona thinning could differ from 16–18 μm [82] to 20–40 μm [81]. In some studies, the irradiation was initiated at one point and continued along the ZP starting at the 9 o’clock position and reaching the 12 o’clock position (Fig. 8b) [13, 83, 86] while in others, the laser was applied to four different sites of the ZP in which length thinning was ≤ 20 μm [84]. Depth of zona thinning could vary from 50 to 90% of the initial ZP thickness without complete breaching [81, 83, 85, 86].

Finally, some comparative studies of different laser-based techniques have been published. Mantoudis and coworkers [87] have compared three types of laser procedures on day 2 embryos: (i) the partial laser AH whereby a single hole has been created without reaching the inner membrane (Fig. 8a), (ii) the thinning of a quarter of the ZP (Fig. 8b), and (iii) the total AH whereby one single hole has been created through the ZP (Fig. 8d), in a very heterogeneous group of patients (advanced age or with recurrent implantation failures or undergoing frozen embryo replacement cycles or poor responders). Results obtained were in favor of the partial procedure and the quarter thinning with a significant clinical pregnancy rate of 18.3 and 22.1% respectively, compared to total breaching of the ZP (5.2%) [87]. However, the choice of the various techniques among the patients was not defined and some selection biases cannot be excluded. Nevertheless, their finding is consistent with a retrospective study by Ghobara et al. [88], where higher clinical pregnancy, implantation and live birth rates predominantly in women < 38 years were associated with partial AH, in particular one-quarter thinning of the cleavage stage embryo (Fig. 8b), in contrast to total breaching of the ZP. Finally, in another prospective randomized study, the strategy to perform the ZP thinning of just the inner layer of the ZP from inward to outward was compared to the ZP opening and conventional ZP thinning. Implantation rates, clinical pregnancy rates and also multiple clinical pregnancy rates were statistically improved when the laser was used to remove only the ZP inner layer [40].

Frozen embryo transfer: zona breaching

Three prospective randomized studies have evaluated the use of laser AH in frozen embryos through the performance of a breaching (Table 7). Two have evaluated AH performed at the cleavage embryo stage and one at the blastocyst stage. A European multicenter prospective study has shown significantly lower pregnancy and implantation rates with laser AH compared to controls in patients who initiated their first transfer cycle of frozen/thawed embryos [8]. This is still the only study on breached frozen embryos showing a dramatic decrease in implantation rate to 1.6% in the laser AH group [8]. Conversely, and in contrast to their own data on the effectiveness of laser-mediated embryo thinning [89], Valojerdi and coworkers have demonstrated that laser AH by ZP breaching could improve the clinical pregnancy and implantation rates in patients with frozen/thawed embryos [74].

Table 7.

Results of ZP breaching using laser-assisted hatching in frozen embryo transfer

	Laser indication (irradiation time)	Opening size	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Primi et al. 2004	1–4 laser shorts by 12–30 ms	20 μm	Days 1–2–3 frozen/thawed embryo, few hours before ET	32.8 ± 4.2 32.6 ± 3.8	Reduced	Reduced	Unchanged	Unchanged	Unchanged	PR
Valojerdi et al. 2008	several pulses of 0.5 ms	40 μm	Days 2–3 frozen/thawed embryo	31.2 ± 4.9 31.8 ± 5.4	Improved	Improved	–	Unchanged	–	PR
Hiraoka et al. 2008	500 μs	40 μm	Days 2–3 frozen/thawed embryo, 3 h before ET at the blastocyst stage	32.4 ± 3.8 31.2 ± 2.9	Improved	Improved	–	Unchanged	Improved	R
		50% of ZP		33.1 ± 3.7 31.2 ± 2.9	Improved	Improved	–	Unchanged	Improved
Wan et al. 2014	400 μs	One quarter of ZP	Days 5–6 vitrified/warmed embryo	33.1 ± 3.7 32.6 ± 3.4	Improved	Improved	Unchanged	Unchanged	Unchanged	PR

ET embryo transfer, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective

Recently, a prospective randomized study has focused on vitrified/warmed blastocysts developed from low-grade cleavage stage embryos suggesting that implantation of these blastocysts and clinical pregnancy rates but not live birth rate were significantly increased after laser AH [90]. This study deserves some attention because this is the first and only report assessing the effects of laser AH on clinical and implantation rate using vitrified/warmed blastocysts. Vitrified blastocysts were warmed on the day of embryo transfer and an opening of one quarter of the ZP was formed (Fig. 8h). When the clinical outcomes were evaluated taking into account the developmental day in which blastocysts were vitrified, the implantation, clinical pregnancy, and live birth rates associated with the laser AH group were significantly increased for day 6 but not day 5 vitrified blastocysts. However, it is necessary to consider that the sample size of the various groups was very small [90].

In a retrospective study, Hiraoka et al. [91] have, firstly, evaluated the results associated with two different sizes of ZP breaching (a hole of 40 μm versus 50% of ZP opening) by laser AH in frozen cleavage stage embryos cultured to blastocyst after thawing in patients with multiple implantation failures. In both cases, higher clinical pregnancy, implantation and delivery rates were observed compared to those from the control group in whom the laser AH was not performed [91]. Furthermore, this study suggested that 50% of the ZP opening (Fig. 8f) improved the clinical outcomes of frozen embryos compared with the 40-μm opening, confirming that higher pregnancy and implantation rates may be observed when the area of ZP thinning (Fig. 8c) or breaching increases up to half (Fig. 8f) [91, 92].

In line, in a double-blind randomized study, Ng et al. [93] compared the clinical outcomes of frozen/thawed cleavage stage embryo transfer derived from laser ZP thinning or laser ZP breaching. Implantation and ongoing pregnancy rates were significantly higher in the thinning group in which more than a quarter of the ZP outer half-diameter was removed to reach a thickness of about 5 μm, while in the breaching group, a hole of about 30 μm was created.

It should be mentioned that both the site of AH and the size of ZP breaching seem to be important. Indeed, in a prospective randomized study, AH by laser breaching in vitrified/warmed blastocysts (Fig. 8g), performed at the site near the ICM or opposite to the ICM, had no influence in terms of clinical pregnancy, implantation, and live birth rates [94]. However, these results were not in line with those from a preliminary report showing that laser-mediated AH of vitrified blastocysts at a site close to the ICM resulted in higher rate of complete hatching, while the procedure performed at the opposite site could cause ICM trapping phenomena. Clinical outcomes were however not considered [95].

Frozen embryo transfer: zona thinning

There were seven randomized studies and two retrospective analyses using zona thinning as a technique for aiding hatching in frozen embryos, again with conflicting results (Table 8). Unexpectedly, all the studies but two [96, 97] performed the procedure at the embryo stage and not at the blastocyst stage. Live birth rate was never modified by the procedure in randomized studies. Balaban et al. [98] have observed that laser AH prior to the transfer of frozen/thawed embryos could increase implantation and pregnancy rates. The strength of this study was represented by the sample size (n = 183) and AH was performed only on embryos showing evidence of cleavage 24 h post thawing [97]. These findings were consistent with data from Ge et al. [83] showing an improvement of pregnancy and implantation rates using this procedure before frozen/thawed embryo transfer. Zhang et al. [97] observed a clinical improvement only when a wide portion of ZP circumference was thinned. Multiple pregnancy and live birth rates were conversely not affected. Very recently, in a prospective randomized study, a significantly increase of clinical pregnancy rate was observed in patients > 37 years subjected to laser AH but not in the overall population [99].

Table 8.

Results of ZP thinning using laser-assisted hatching in frozen embryo transfer

	Laser indication (irradiation time)	Length/depth of ZP	Time of AH	Age (mean ± SD) AH/control	CPR	IR	AbR	MPR	LBR	P/PR/R study
Ng et al. 2005	90 mV for 6 ms	> 25%/outer half	Day 2 frozen/thawed embryo	34.0/34.0	Unchanged	Unchanged	Unchanged	Unchanged	–	PR
Petersen et al. 2006	9 ms	25%/50–80%	Days 2–3 frozen/thawed embryo	31.7 ± 4.8a 32.5 ± 4.4	Unchanged	Unchanged	Unchanged	–	Unchanged	PR
				32.1 ± 3.9 b 30.1 ± 4.3	Unchanged	Unchanged	Unchanged	–	Unchanged
Balaban et al. 2006	Not stated	25%/not stated	Day 3 frozen/thawed embryos, the day after thawing prior to ET	32.4 ± 3.3 32.7 ± 3.1	Improved	Improved	Unchanged	Increased	Unchanged	PR
Ge et al. 2008	Few milliseconds	25%/50%	Days 2–3 frozen/thawed embryos, immediately prior to ET	31.8 ± 3.9 30.7 ± 4.4	Improved	Improved	–	Unchanged	Unchanged	PR
Zhang et al. 2009	Several pulses of 550 μs	40 μm/more than half of thickness	Day 3 frozen/thawed embryos, prior to ET at the blastocyst stage	30.9 ± 0.7 31.1 ± 0.7	Unchanged	Unchanged	–	Unchanged	–	R
		80 μm/more than half of thickness		30.3 ± 0.5 31.1 ± 0.7	Improved	Improved	–	Unchanged	–
Valojerdi et al. 2010	8–10 pulses of 0.5 ms	40 μm/50–80%	Days 2–3 vitrified/warmed embryo	30.9 ± 5.8 29.9 ± 5.1	Reduced	Reduced	–	Unchanged	–	PR
Debrock et al. 2011	7–13 irradiations of 3.6–9.0 ms	120°/1/3 of ZP thickness	Days 1–2–3 frozen/thawed and vitrified/warmed embryos and blastocysts, the day after thawing prior to ET	32.3 ± 4.4 32.7 ± 4.3	Unchanged	Unchanged	Unchanged	Unchanged	Unchanged	PR
Zhou et al. 2014	500 μs	Not stated/2/3 of ZP thickness	Day 3 frozen/thawed and vitrified/warmed embryos	29.7 ± 4.0 29.9 ± 4.0	Improved	Improved	Reduced	Increased	Improved	R
Kanyo et al. 2016	10–15 ms	5–10 μm	Day 3 frozen/thawed embryos, immediately before ET	33.3 ± 3.3 33.0 ± 3.1	Unchanged	–	Unchanged	Unchanged	–	PR

ET embryo transfer, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective

^aPatients with all supernumerary embryos cryopreserved

^bPatients with all embryos cryopreserved due to risk of ovarian hyperstimulation syndrome

On the other hand, others failed to demonstrate any benefit of the procedure [96, 100, 101]. In particular, no benefit derived from laser AH was found by Debrock et al. [96] in a large randomized study considering a laser AH group (n = 302) and a control group (317). All frozen embryo transfer cycles in day 2, day 3, and day 5 embryos were included. In this case, the ZP was thinned to about one-third of its initial thickness and laser thinning was initiated at the 1 o’clock position with consecutive irradiations generated in a clockwise direction to the 5 o’clock position [96].

It should be noted that while slow freezing represented the most common procedure to cryopreserve embryos in the studies considered, the first randomized study assessing the effect of laser AH performed on vitrified/warmed embryos at the cleavage stage even reported a detrimental effect compared to the control group [89]. A total of 400 vitrified/warmed embryo transfer cycles were randomized and clinical pregnancy and implantation rates were significantly affected from 43.0 to 28.5% (p < 0.004) and from 16.7 to 11.2% (p < 0.002), respectively.

The variability among the various studies represents a burden in the interpretation. The timings of thawing, AH methods, and day of transfer could vary. Transfer was performed either on the same morning as AH [83, 89, 100, 101] or with approximately 20 h from thawing [96]. The quality of frozen embryos might have also affected the results: some centers have frozen all supernumerary embryos [101] while others froze only good quality embryos [100]. Furthermore, differences among these studies could also be related to endometrial preparation protocols.

Recently, a retrospective cohort study has analyzed the obstetrical and neonatal outcomes of babies born from cryopreserved day 3 embryos with and without laser AH [102]. Embryos had been both slow frozen or vitrified. Higher implantation, clinical pregnancy rate, and live delivery rates were observed in the laser-thinned embryo group. No difference was found between 292 babies born in the AH group and 100 children in the control group in mean gestational age, mean birth weight, malformation rate, and even mean Apgar score.

Studies comparing sizes of the zona thinning are too few to draw conclusions. As previously mentioned, Zhang et al. [97] found significantly higher implantation and clinical pregnancy rates following frozen/thawed cleavage stage embryo transfer in association with a length of zona thinning of 80 μm respect to 40 μm and compared to a non-AH group, suggesting that the size of thinning could influence the clinical outcome.

In line, a prospective randomized study has demonstrated a clinical benefit of a 50% (Fig. 8c) as opposed to a 25% thinning of ZP (Fig. 8b) performed on vitrified/warmed embryos at the cleavage stage. No differences in multiple pregnancy and miscarriage rates were found between the two groups [92].

In summary, even using the laser-mediated approach, it is unclear whether fresh cleavage stage embryos benefit from zona breaching or thinning. Studies so far referred largely to embryos at cleavage stage and, results, especially on the breaching procedure, were very inconsistent. There is some consistency for a non-evident advantage of the thinning approach. The benefit on frozen embryos or blastocysts is still controversial.

Total removal of the ZP

The rationale for completely removing of the zona is based on the observation that, independently from the technique used, there is a possibility that low-viability blastocysts are not able to hatch after ZP thinning or ZP breaching. Vajta and colleagues argued that escaping through the AH holes could require a considerable energy-consuming effort from blastocysts and suggested that complete ZP removal would increase the implantation rate compared to partial ZP removal [103]. The first studies, which focused on the effects of total removal of ZP on clinical outcomes, compared zona-free and zona-intact fresh blastocyst transfer. Five prospective randomized studies analyzed the effect of enzymatic treatment by pronase or with acidic Tyrode’s solution to remove totally the zona before blastocyst transfer (Table 9) [18, 24, 104–106]. However, results from one of these studies were not assessable due to lack of controls [18]. The others [24, 104, 106] except one [105] reported some degree of benefit in ART outcomes with a complete AH especially in patients having poor-quality blastocysts [106], with high risk for severe hyperstimulation syndrome [104] or performed in embryos reaching the morula or early blastocyst stage [24]. Chemical removal of the ZP on day 3 embryos showed no difference in clinical pregnancy, miscarriage, and live birth rates in patients < 40 years undergoing their first ICSI attempt, while could improve clinical pregnancy rates in > 40 years old women with a poor prognosis and/or at least two previous failed ICSI attempts [25].

Table 9.

Results of ZP total removal using laser-assisted hatching compared to zona-intact

	Method	Time of AH	Age (mean ± SD) zona-free/zona-intact	CPR	IR	AbR	MPR	LBR	P/PR/R study
Fong et al. 1998	Pronase	Day 5 blastocysts, few hours before ET	32.6 ± 5.2	n.a.	n.a.	n.a.	n.a.	n.a.	PR
Isik et al. 2000	Pronase	Day 5 blastocysts, 30′–60′ before ET	30.5 ± 5.2 29.1 ± 3.6	Unchanged	Unchanged	–	Unchanged	Unchanged	PR
Mansour et al. 2000	Acidic Tyrode’s solution	Day 3 embryos, 2 h before ET	32.1 ± 2.5a 33.2 ± 1.4	Unchanged	–	Unchanged	Unchanged	Unchanged	PR
			37.3 ± 5.6b 36.3 ± 5.2	Improved	–	Unchanged	Unchanged	Unchanged
Urman et al. 2002	Pronase	Day 5 blastocysts, 30′–60′ before ET	31.8 31.5	Unchanged	Improved	Unchanged	Unchanged	–	PR
Kinget et al. 2002	Pronase	Day 5 blastocysts, few hours before ET	31.0 ± 3.9 32.0 ± 4.0	Improved	Improved	–	Unchanged	Unchanged	PR
Jelinkova et al. 2003	Acidic Tyrode’s solution	Day 5 blastocysts, 20′ before ET	32.3 ± 4.2 32.1 ± 3.1	Improved	Improved	–	Unchanged	–	PR

ET embryo transfer, CPR clinical pregnancy rate, IR implantation rate, AbR abortion rate, MPR multiple pregnancy rate, LBR live birth rate, P prospective, PR prospective randomized, R retrospective, n.a. not applicable

^aPatients with < 40 years old undergoing their first ICSI attempt

^bPatients with ≥ 40 years old and/or at least two previous failed ICSI attempts

Two retrospective studies compared clinical outcomes between total removal and ZP thinning/breaching. In fresh cycles of unselected IVF patients, Lan et al. [107] showed that blastocysts subjected to the chemical removal of the zona on day 5 implanted at a similar rate as those with laser AH breaching at the cleavage stage and culture up to day 5. In frozen/thawed cycles, Hiraoka and colleagues reported that AH with total removal of the ZP using laser/mechanical pipetting significantly improved clinical pregnancy, implantation, and delivery rates of vitrified blastocysts compared to partial opening of the ZP using acidified Tyrode’s solution (67 versus 42%, p < 0.02; 55 versus 30%, p < 0.01; 56 versus 36%, p < 0.04, respectively), while no difference in multiple pregnancies was observed [108]. Assisted hatching was performed immediately after warming of collapsed blastocysts.

Discussion

A very recent review of the literature by a panel of famous embryologists has emphasized the need for carefully monitoring and assessing the methodological changes introduced in an IVF laboratory. As in the case of other methodologies, AH has been introduced in IVF laboratories without the rigorous approach that should be committed when dealing with human embryos [109, 110].

Although significant amount of information is available on the composition, assembly, and structure of the ZP, its role in human oogenesis, egg-sperm interaction, and pre-implantation development is somehow not notoriously clarified. The paucity of the native human material [111] and the limitations associated with the common approaches of using recombinant proteins mostly from E. coli or insect cells [112–115] or studying transgenic animals expressing human ZP subunits [116, 117] have hampered a rapid progress in this context. For this reason and considering the limited literature, the functional significance of a possible ZP hardening is even less clear. Evidence for the induction of ZP hardening, supportive of an AH intervention, following prolonged human embryo culture or freezing/thawing procedures are based on few controversial data, often performed on the ZP of the oocyte [118–120]. Thus, there are no convincing elements in humans sustaining the biological plausibility for performing an embryo AH during ART. It should however be considered that, at least in some mammalian species, the process of in vivo embryo escaping from the ZP has been recognized to be strongly different from the in vitro phenomenon [103] and it is in this context that any potential benefit of a manipulation should be viewed.

As a matter of fact, recommendations for the use of the procedure from both the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine have never been concrete from the beginning and became even fainter over the years in favor of the notion that strong consolidation to support the efficacy of the technique has yet to be provided. The last published meta-analysis including 36 randomized studies with 6459 participants reported a borderline significant improvement in clinical pregnancy (OR = 1.16, 95%CI 1.00–1.36) and a significant increase in multiple pregnancy rate (OR = 1.50, 95%CI 1.11–2.01) but not in live birth rate when AH were compared to the controls [5]. Interestingly, when results were stratified by hatching method, significant results for clinical pregnancy were observed for chemical (OR = 1.26, 95%CI 1.01–1.57) and mechanical methods (OR = 1.68, 95%CI 1.17–2.42) but not for the laser technique (OR = 1.03, 95%CI 0.81–1.30). This was an unexpected result considering that none of the studies reported herein, which compared the mechanical or chemical procedures to those that were laser-mediated, reported a superiority of the former ones over the latter. Collectively, of the five studies, two of which were prospective randomized studies [43, 46], addressing the comparison between laser AH and the chemical approach [42–44, 46, 47] in terms of clinical outcomes, two reported better results for the laser procedure and three similar results. The single study that has compared outcomes deriving from laser AH to those based on the mechanical approach [51] did not find differences. However, the meta-analysis did not include studies comparing different techniques.

In line with the last published meta-analysis, the last Cochrane systematic review and meta-analysis including 31 trials and 5728 participants failed to find significant difference in live birth rate in the AH group compared with the control group [2]. Clinical pregnancy rate in women who underwent AH was slightly improved with a borderline statistical significance (OR = 1.13, 95%CI 1.01–1.27) while multiple pregnancy rates were significantly increased (OR = 1.38, 95%CI 1.11–1.70). The two meta-analyses were however discordant on results of clinical pregnancy rates deriving from subgroup comparisons. The benefit deriving from the application of AH in fresh versus frozen embryos transfers and in women with a history of recurrent failure was not consistently detected in the two analyses. Li et al. [5] observed significant results for clinical pregnancy among women who underwent transfer of frozen/thawed embryos without a failure history while no significant increase in frozen embryo transfer was observed in the Cochrane systematic review [2]. Discordance in the results of meta-analyses may derive from differences in the studies considered, in the model applied [5], and/or in the risk estimates. Moreover, they have the general limit not to consider the time factor in the assessment of the studies since this is an area where about 50% of the studies considered does not reflect the current practice [121]. Indeed, the laser-mediated technology has received a widespread diffusion in the last 10 years over the mechanical and chemical procedures. A very recent meta-analysis considering only 11 randomized controlled studies on the effect of laser-mediated AH in frozen cycles has demonstrated a significant difference in clinical pregnancy favoring the AH group but not the live birth rate [122].

Although, undoubtedly, according to the dogma of evidence-based medicine, it is essential to consider the collective evidence from the past and the present, and embryologists should interpret this information with attention with regard to the applicability and also the relevance of the older studies, which used AH protocols spanning from mechanical and chemical procedures to laser.

A concern in this regard is also represented by a limited number of studies that have evaluated the effect of AH on blastocysts. With the widespread introduction of the comprehensive chromosome screening (CSC) on blastocysts, the laser-mediated manipulation of the blastocyst ZP represents presently an almost routine procedure in the lab. But the opening of the zona with the aim to collect some blastomeres has a different significance than the AH procedure as intended in the present review and in the Cochrane systematic review.

For the obvious reason that blastocyst ZP is naturally thin, the idea to open or thin it in fresh cycles has even less biological significance. As a matter of fact, all the papers but one [79] considering fresh cycles as identified in this systematic review (Tables 1, 3, 5, and 6) referred to AH in cleavage stage embryos. The situation might be different in frozen cycles, especially if the blastocysts are collapsed before freezing in order to avoid ice crystal formation by reducing the fluid content of the blastocoel [123] or when they are subjected to biopsy for CSC. In these cases, the AH procedure may reduce trapping phenomena linked to the single hole. We have been able to identify only five studies addressing the clinical outcomes deriving from partial hatched blastocysts in frozen cycles compared to non-hatched controls [63, 90, 91, 96, 97]. Some benefits associated with AH emerged quite consistently from these studies. In the only study that failed to demonstrate a beneficial effect, the ZP was only thinned to about one-third and not breached [96]. However, considering the large and common use of laser in these years, the paucity of the studies addressing this issue is surprising.

In general, results from this review and the available meta-analyses [2, 5] tend to support greater benefits with procedures supporting a higher degree of zona manipulation compared to the simple ZP thinning. Interestingly, none of the studies that evaluated the effect of the zona removal could demonstrate reduced clinical outcomes. Conversely, a general support for the removal of ZP can be acknowledged from this analysis both in studies comparing the procedure to non-treated controls and in those comparing different hatching techniques. As a matter of fact, significant results of clinical pregnancy were observed among women who received AH in which the zona was completely removed in the meta-analysis by Li and coworkers [5]. Blastocyst outgrowth assay was recently used to understand the blastocyst implantation potential after complete ZP removal. Time for adhesion and outgrowth area improved in vitrified/warmed blastocysts with completely removed ZP compared with those with a single hole in the ZP [124]. Again, the poor evidence available in this regard cannot be explained completely especially in the CSC era. A potential explanation for the poor development in this area might be represented by the concern that embryos without ZP might be more fragile during routine procedures. Potential damage resulting from mechanical traumas, attachment to glass and plastic surfaces or loss of protection from infections may be viewed as possible drawbacks. On the other hand, according to some authors [103], these aspects should not represent an actual problem.

One of the most interesting outcomes of this systematic review is the identification of a major gap in the literature; a randomized trial should consider to perform a laser-mediated AH in frozen/thawed blastocysts comparing (i) non-AH to (ii) complete removal or (iii) partial removal of the zona.

Conclusions

This review summarizes the current knowledge on the biology of the ZP and the technical aspects to manipulate it in order to improve ART efficacy. ZP manipulation was introduced into the clinical practice without proofs of safety and with poor biological plausibility. The refinement of the technique based on results from evidence-based medicine represents a priority for the future. Although AH procedures will soon mark their 30th anniversary, they still lack consolidation from sufficient well-designed studies.

Funding

This work has not been supported by a grant.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.