Assisted Hatching for In Vitro Fertilization-Embryo Transfer: An Update

Assisted Hatching for In Vitro Fertilization-Embryo Transfer: An Update

Shahryar K Kavoussi*
Austin Fertility & Reproductive Medicine/Westlake IVF, 300 Beardsley Lane, Bldg B, Suite 200, Austin, Texas, USA
*Corresponding author: Shahryar K Kavoussi, M.D, M.P.H, Austin Fertility & Reproductive Medicine/Westlake IVF, 300 Beardsley Lane, Bldg B, Suite 200, Austin,
Texas, USA, Tel: 001-(512) 444-1414; Fax: 001-(512) 444-5621; E-mail: austinfertility@gmail.com
Received date: 06-02-2014; Accepted date: 13-03-2014; Published date: 17-03-2014
Copyright: © 2014 Kavoussi SK. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.



Abstract
Assisted Hatching (AH) is a technique performed after In Vitro Fertilization (IVF) and involves the artificial thinning or opening of the Zona Pellucida (ZP) prior to Embryo Transfer (ET) as an attempt to improve the probability of embryo implantation. AH can be performed by embryologists via mechanical, chemical, or laser-assisted means. A few studies suggest that a larger size of ZP opening/thinning as well as a site near the ICM may be associated with a greater probability for complete hatching. It is not recommended to apply AH to all IVF cycles universally. Subgroups of patients that may benefit from AH include those with prior implantation failure, those undergoing Frozen-Thawed Embryo Transfer (FET) cycles, and women who are 38 years of age or older. IVF programs should identify subgroups of women within their patient population who may benefit clinically from AH.



Keywords: Embryo; Blastocyst; Assisted hatching; Zona pellucida

Introduction
In utero, as an embryo at the blastocyst stage expands and thins its
surrounding glycoprotein covering, the Zona Pellucida (ZP), rupture
of the ZP eventually occurs with the aid of proteases known as lysins.
The embryo hatches and begins the implantation process via
interaction with endometrial cells. The inefficiency of Implantation
Rates (IR) after Embryo Transfer (ET) may be attributed to various
factors including the potential failure of a euploid blastocyst to hatch
from the ZP. Assisted Hatching is a technique performed after in vitro
fertilization and involves the artificial thinning or opening of the zona
pellucida by the embryologist prior to ET as an attempt to improve the
probability of embryo implantation [1].

Methods of AH
AH has been in use since the first report by Cohen in 1988 [2] and
has since evolved in terms of modality by which the ZP is artificially
thinned or opened. AH can be performed by mechanical, chemical, or
laser-assisted means. When AH is performed to create an opening in
the ZP, the procedure may be performed via the use of acidified
Tyrode’s solution, partial zona dissection with a glass microneedle,
laser photoablation or piezomicromaniupulation. When AH is
performed to thin and not breach the ZP, acidified Tyrode’s solution,
proteolytic enzymes, or laser-assistance may be used [1].

The effect of the size of ZP breaching or thinning on AH
outcomes
Several studies have suggested that the larger size of ZP breaching
or thinning may confer a clinical benefit. In a study by Hiraoka et al.,
101 consecutive frozen-thawed embryo transfer procedures were
grouped into no Laser-Assisted Hatching (LAH) (n=30), LAH with 40
μm ZP opening (n=40), and LAH with 50% circumferential ZP
opening (n=31). Cleavage stage embryos were thawed and grown to
blastocyst stage in patients who had failed both fresh and frozen
cleavage stage ET; LAH was performed at the expanded blastocyst
stage. The Pregnancy Rates (PR), Implantation Rates (IR), and Live
Birth Rates (LBR) were higher in the group with 50% ZP opening
(74%, 52%, 65%) when compared with the control group (17%, 10%,
13%; P<0.01) and the group with 40μm ZP opening (43%, 27%, 38%;
P<0.04) [3]. The same authors conducted a study in order to examine
the effect of the size of ZP thinning on clinical outcomes in vitrifiedwarmed
cleavage stage FET. Random assignment of 120 cases was
made to 50% ZP thinning and 25% ZP thinning groups. Clinical
Pregnancy Rates (CPR) (46.7 vs 25.0%; P=0.0218) and IR (32.0 vs
16.2%, P=0.0090) were significantly increased in the 50% ZP thinning
group [4]. Zhang et al. studied the effect of the size of ZP thinning by
LAH on the clinical outcome of cleavage stage FET [5]. Among 122
consecutive procedures, 31 were in the control group (no AH), 34
were in the AH group with 40μm ZP thinning, and 57 were in the AH
group with 80μm ZP thinning. PR and IR were significantly higher in
the 80 μm AH group as compared with control (40.3 vs 16.1%, P=0.03;
21.5 vs 7.5%, P=0.007, respectively). IR was significantly higher in the
80μm AH than the 40μm AH group (21.5 vs 9.4%, P=0.024). The
authors concluded that the greater size of ZP thinning may matter for
PR and IR in cleavage stage FET [5].

The effect of the site of AH on outcomes
Since two observational studies had shown that the natural hatching
site of the human blastocyst is near the inner cell mass (ICM) whereas
that of the mouse is at the side opposite to the ICM [6,7], a prospective
randomized study was conducted in order to study whether or not the
choice of AH site is important to complete hatching. The authors of
this preliminary report demonstrated a significantly higher rate of
complete hatching when LAH was performed on thawed blastocysts
near the ICM as compared to the side opposite to the ICM [8]. Ren et
al. evaluated the effect of LAH site in vitrified-warmed blasts on
clinical outcome of FET. A total of 16 women were randomized to a
group with AH near site of ICM or to a group with AH at the site
opposite to the ICM. LAH was performed within 20-30 min after
blastocyst thaw once the ICM was detected. There was no difference in
IR, PR, LBR, or monozyogotic twin rate between the two groups [9].

The effects of AH on clinical outcomes
Unselected patients
The American Society for Reproductive Medicine (ASRM)
committee opinion states that the existing literature does not support
the universal application of AH to all IVF cycles [1]. The majority of
studies that have evaluated the effect of AH on the IR and PR of
unselected IVF patients have shown no increase in these outcome
measures [10-16] and a recent systematic review and meta-analysis by
Martins et al concluded that AH was unlikely to improve CPR in
unselected fresh ET cycles whereas AH in unselected FET cycles was
efficacious [17].

Advanced maternal age
It has been theorized that ZP hardening may occur due to
endocrine changes or the absence of lysins as a function of oocyte
aging in women of advanced maternal age. Although some studies
suggest benefit of AH in women of advanced maternal age, some show
no difference in IR and CPR [17-22]. Subgroup analysis of Advanced
Maternal Age (AMA) patients in the review by Martins showed no
benefit of AH in women of AMA who underwent fresh ET [16].

Thick ZP
A prospective, randomized, double-blinded, crossover study sought
to determine whether AH impacts clinical outcomes in women
younger than 38 years whose embryos have a thickened ZP, defined as
> 13 μm (n=121). Patients were randomized to a control group (no
AH) or group that had AH performed by acidic Tyrode’s solution.
There was no difference in IR, CPR, or LBR between groups; therefore,
this study suggests that AH does not appear to provide benefit to
women under age 38 who undergo IVF [23,24].
Prior implantation failure
In women with prior implantation failure, AH has been shown to
significantly increase the CPR [16,25]. LBR was not proven to be
increased; however, because most trials did not report LBR, there may
have been insufficient data to draw conclusions regarding the effects of
AH on LBR. The ASRM Committee Opinion found that AH may be of
benefit to women with > 2 failed IVF cycles [1].
Frozen-thawed embryo transfer (FET)
It has been suggested that the processes of embryo cryopreservation
and thawing may lead to changes in the microarchitecture of the ZP
with associated ZP hardening. If such changes theoretically impair the
chance of the embryo rupturing from the ZP, it has been thought that
AH may be indicated. The recent systematic review and meta-analysis
of randomized clinical trials by Martins et al showed that AH was
associated with a significant increase in IR and PR in unselected or
poor-prognosis women with frozen-thawed ET [16,26-33].
Endometriosis
There has been controversy about whether oocyte/embryo quality
or endometrial receptivity is predominantly impaired in women with
endometriosis, some studies have suggested that the former is related
to endometriosis-associated subfertility [34]. In order to determine if
AH improves IR in women with endometriosis, Nadir Ciray et al.,
conducted a prospective randomized study of 60 women with

endometriosis who had LAH performed for their embryos and 30
women with endometriosis who did not have LAH. There was no
difference in PR (28.3% LAH group, 40% control group) or IR (17.8%
LAH group, 19.4% control group) between groups. The authors
concluded that AH does not improve outcome in women with
endometriosis [35].
Multiple gestation
Monozygotic twinning has been found to be more common in IVF
cycles and the incidence is increased in pregnancies following AH
[36,37]. The recent review by Martins showed that AH increased
multiple gestation rates [16]. The 2012 Cochrane Review found that
there was low quality evidence showing a significant increase in
multiple gestation rates per woman [25].
Conclusions
AH is a technique that has been used in IVF laboratories for over 25
years. Several studies suggest that a larger size of artificial ZP opening/
thinning as well as performing AH at a site close to the ICM may be
associated with a greater propensity for complete hatching. Universal
application of AH to all fresh IVF cycles is not recommended in
accordance since the existing literature shows no difference in
outcomes. Although LBR data may be insufficient at this time, AH
seems to increase IR and CPR in cases of prior implantation failure as
well as in FET cycles. Although there is some conflicting data about
improved outcomes among women of advanced maternal age, data
exists that supports AH in women 38 years of age or older. As
recommended by ASRM, each IVF program should assess their own
patient characteristics and determine whether or not AH may provide
benefit to certain subgroups of their patients.
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