1 National Laboratory of Medical Genetics of China and 2 Reproductive Medical Center of Xiangya Hospital, Central South University, Changsha, 410078, People's Republic of China
4 To whom correspondence should be addressed at: National Laboratory of Medical Genetics of China, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, China. Email: nlmglcy{at}xysm.net
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: assisted hatching/blastocyst/inner cell mass/safety/zona pellucida
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
However, some other studies indicated no benefit to aged women by zona drilling AH (Bider et al., 1997) and laser AH (Horng et al., 2002
). An increasing number of controversial clinical outcomes of AH have been reported and there is now debate as to whether AH really has advantages in defined subgroups of patients.
It has been shown that small holes resulting from application of mechanical zona dissection inhibited completion of the hatching process rather than improving implantation rates (Cohen and Feldberg, 1991). However, we further noted in vitro that a zona opening with usual/moderate size, in some cases, can also trap hatching blastocysts, making some blastocysts unable to be integrally hatched out. If this hatching trap can also take place in vivo, that would lead to the delay or the failure of complete hatching of blastocysts, and cause instability or failure of embryo implantation. We hypothesize that inappropriate size of zona opening would be a potential defect of conventional AH methods.
We now investigate a new AH technique to create a zona slit of controlled size, and perform experiments to test whether or not the zona opening of a moderate size, corresponding with the size of current AH methods, would be large enough for the complete hatching of blastocysts in vitro, and whether the new technique could improve the in vitro effect of hatching, as well as decrease the technical manipulation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Manipulation using new technique
A new technique called controlled zona dissection (CZD) was established as described in Figure 1. In the CZD, micro-pipettes are modified. The classical vertical opening of the holding pipette is altered to bevelled opening with an angle of 65° (Figure 1) made by using a pipette puller (Narishige, Japan) and microforge (Japan). The tip of the hatching needle is blunted by a gentle heating on the microforge, and is thin at the front, just like a conventional ICSI needle that can also be used as the hatching needle in this new procedure. In the CZD, the procedure is modified as described in Figure 1. In brief, using the modified holding pipette with a bevelled opening can reduce the time required to force the needle through the embryo's side, enabling easy and stable manipulation while puncturing the ZP from the side of the embryo, even using a blunted hatching needle. The blunted hatching needle can then push the embryonic cells aside with no damage to cells and so can create a large perivitelline space, even in the case of a blastocyst which usually has no perivitelline space. The hatching needle can puncture the zona to a controlled size, followed by dissecting the punctured zona with the arc of the needle cutting against the bottom of the dish. The ZP dissection by CZD needs only one attempt to obtain a slit of the type. We shall refer to CZD creating a slit size of about two-fifths of the embryo's diameter as moderate zona dissection (MZD), and a slit size beyond two-thirds of the embryo's diameter as long zona dissection (LZD).
|
The CZD-AH in human blastocysts
Fifty-nine human blastocysts were manipulated by AH at middle-blastocyst stage with a selective size of zona opening by CZD: LZD group with slit size beyond 100 µm and MZD group with slit size between 55 and 65 µm as described in Table II. Embryos were allocated to different groups at random with respect to quality but not with respect to number. Considering the very limited number of donated human embryos, more embryos were allocated to the MZD group, as we were more concerned about the disadvantage of zona opening with usual/moderate size. Embryo hatching was observed and photographs were taken from day 5 post-fertilization.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During AH, the internal pressure generated by expanding blastocysts is released through the zona opening, and is too small to slit the zona opening to a larger size. So the size of the zona opening during hatching is basically determined by the original zona opening size created by AH. Thus, zona opening with originally usual/moderate size tends to cause blastocysts to fail complete hatching, especially in cases of good embryos with large ICM (Figure 2D, Table II). Despite years of investigation, the exact mechanism of zona escape in vivo has yet to be elucidated. If the embryos developed in vivo following AH, the ICM trapping would occur in the same way as in vitro. That would create a danger of the embryo being damaged or slit, which has been suspected as the cause of the monozygotic twinning following AH (Cohen et al., 1990; Alikani et al., 1994
). So, a good AH method evaluated in vitro must produce a large enough zona opening during hatching and achieve a high rate of complete hatching of blastocysts, whereas zona opening with originally moderate or small size by AH may be defective in blastocyst hatching. Mantoudis et al. (2001)
have reported that higher implantation rates can be obtained by zona thinning rather than zona opening, and further higher implantation rate can be obtained as the thinned zona area increases (Mantoudis et al., 2001
). This improved outcome might be due to the larger zona opening during hatching in vivo, because the accumulated internal pressure of hatching the blastocyst may break through the thinned zona area and achieve a larger zona opening than the zona opening from AH, after which blastocysts cannot accumulate internal pressure during hatching.
However, zona thinning by chemicals (Cohen et al., 1992) or laser (Mantoudis et al., 2001
) may pose potential harm to embryos. Furthermore, laser AH is too expensive for many IVF laboratories. PZD (Cohen et al., 1990
) is a simple and economical method without the risk of chemical or optical harm to embryos inherent in the other two methods, but it is difficult to obtain a satisfactory size of zona opening (Cieslak et al., 1999
; Balaban et al., 2002
). Even though the 3D PZD (Cieslak et al., 1999
) has improved the zona opening size by using two crossing PZD, the opening is still not large enough to achieve a high rate of complete hatching of blastocysts (Table I). Furthermore, in PZD or 3D PZD, mechanical harm, such as squeezing or injury to embryos, may occur during the manipulation, especially during ZP rubbing, while the hatching needle may slip from holding the pipette and strike the embryo on the holding pipette, causing a rapid mechanical pressure to the embryo. The mechanical change of hydrostatic pressure has potential harmfulness to spindle microtubules in living cells (Macas et al., 1996
). Benefited by the use of the modified holding pipette with bevelled opening and the use of modified zona dissection procedure, the manipulation of LZD is stable and easy with no squeezing or buffeting the embryo, even using a hatching needle with a blunted tip. LZD therefore causes little or no distortion and injury to embryos, and is independent of the size of perivitelline space (PVS), as it can produce PVS by pushing embryonic cells aside under the ZP using a hatching needle with blunt tip. LZD can also apply to blastocysts for AH, even to the expanding blastocysts with no PVS under ZP, with no harm to blastocysts. However, it is difficult for other methods to achieve AH at blastocyst stage, since they all depend on the size of PVS to select for AH site, so as to reduce mechanical, chemical or optical damage to embryos during manipulation. So, compared with traditional PZD or 3D PZD, the LZD technique not only allows an easier manipulation to create a markedly longer zona slit, which enhances the rate of complete hatching of blastocysts in vitro, but also decreases the potential risk of mechanical harm to embryos. However, the improved hatching rates and improved manipulation using LZD in vitro do not predict better implantation rates in vivo, and further investigation by clinical trials is needed to evaluate this new technique.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balaban B, Urman B, Alatas C, Mercan R, Mumcu A and Isiklar A (2002) A comparison of four different techniques of assisted hatching. Hum Reprod 17, 12391243.
Bider D, Liversushits A, Yonish M, Yemini Z, Mashiach S and Dor J (1997) Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod 12, 317320.[Abstract]
Blake DA, Forsberg AS, Johansson BR and Wikland M (2001) Laser zona pellucida thinningan alternative approach to assisted hatching. Hum Reprod 16, 19591964.
Carroll J, Depypere H and Mathews CD (1990) Freeze-thaw-induced changes of the zona pellucida explain decreased rates of fertilization in frozen-thawed mouse oocytes. J Reprod Fertil 90, 547553.[ISI][Medline]
Cieslak J, Ivakhnenko V, Wolf G, Sheleg S and Verlinsky Y (1999) Three-dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching. Fertil Steril 71, 308313.[CrossRef][ISI][Medline]
Cohen J (1991) Assisted hatching of human embryos. J In Vitro Fertil Embryo Transfer 8, 179190.[CrossRef][ISI][Medline]
Cohen J and Feldberg D (1991) Effects of the size and number of ZP openings on hatching and trophoblast outgrowth in the mouse embryo. Mol Reprod Dev 30, 7080.[CrossRef][ISI][Medline]
Cohen J, Elsner C, Kort H, Malter H and Massey J (1990) Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 5, 713.[ISI][Medline]
Cohen J, Alikani M, Trowbridge J and Rosenwaks Z (1992) Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 7, 685691.[Abstract]
De Felici M and Siracusa G (1982) Spontaneous hardening of the zona pellucida on mouse oocytes during in vitro culture. Gamete Res 6, 107113.[CrossRef][ISI]
Horng SG, Chang CL, Wu HM, Wang CW, Cheng CK, Huang HY, Wang HS and Soong YK (2002) Laser-assisted hatching of embryos in women of advanced age after in vitro fertilization: a preliminary report. Chang Gung Med J 25, 531537.[Medline]
Loret De Mola JR, Garside WT, Bucci J, Tureck RW and Heyner S (1997) Analysis of the human zona pellucida during culture: correlation with diagnosis and the preovulatory hormonal environment. J Assist Reprod Genet 14, 332336.[ISI][Medline]
Macas E, Imthurn B, Rosselli M and Keller PJ (1996) The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection. Hum Reprod 11, 24962501.[Abstract]
Mantoudis E, Podsiadly BT, Gorgy A, Venkat G and Craft IL (2001) A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 16, 21822186.
Meldrum DR, Wisot A, Yee B, Garzo G, Yeo L and Hamilton F (1998) Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J Assist Reprod Genet 15, 418421.[ISI][Medline]
Selva J (2000) Assisted Hatching. Hum Reprod 15 (Suppl 4), 6567.
Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J and Fisch B (1995) Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril 63, 838841.[ISI][Medline]
Submitted on November 27, 2004; resubmitted on January 21, 2005; accepted on January 25, 2005.
|