THE JOURNAL OF EXPERIMENTAL ZOOLOGY 256232-236 (1990)

RAPID COMMUNICATION

Developmental Potential of Biopsied Mouse Blastocysts

MARTINE NIJS AND ANDRE VAN STEIRTEGHEM Centre for Reproductiue Medicine, VrtJe Uniuersitr?it Brussel, B-1090 Brussels, Belgium

ABSTRACT The health of a preimplantation embryo can be diagnosed in one or more cells biopsied from the conceptus. Here, we tried to evaluate the impact of biopsy of some trophectoderm cells from hatching mouse blastocysts on their further in vitro implantation and early egg cylinder formation. Of 374 blastocysts evaluated 112 hours after hCG, 34% initiated hatching with a small number of mural, polar, or intermediate trophectoderm cells. Half of 59 embryos that underwent induction of hatching by zona puncturing herniated some cells through this opening. After removal of cells with a glass microneedle from spontaneously hatching blastocysts, viability assessed by vital FDA staining was impaired, as well as the in vitro zona pellucida shedding and implantation. When polar trophectoderm cells were biopsied, a significantly lower number of embryos reached the egg cylinder stage.

The knowledge of human embryology vastly ex- tended since the techniques of in vitro fertiliza- tion, freezing and thawing of embryos, and trans- fer of gametes or embryos became routine practice. However, the transfer of a non-affected embryo cannot be guaranteed in couples at risk for a child with an inherited disease. The develop- ment of a pre-implantation diagnosis technique would make it possible to assess the health condi- tion of the embryo before transfer to the mother (Adinolfi and Polani, '89). One or more cells, biop- sied from the embryo, can be used for diagnosis of an inherited disease. Holding and Monk ('89) re- cently described the diagnosis of B-thalassaemia by DNA amplification in a single biopsied mouse blastomere. Using the same technique, Handy- side et al. ('90) were able to sex human embryos after biopsy of a single blastomere from 6- to 8- cell stage human embryos. In combination with the development of different microassays, the biopsy techniques for blastomeres from mamma- lian pre-implantation embryos require thorough evaluation, Hence, the technique for the removal of one or more cells from an embryo must warrant the normal in vitro development, in vivo implan- tation, or growth. In this study we tried to evalu- ate a technique for biopsy of trophectoderm cells from mouse blastocysts. Pre-implantation diag- nosis on the blastocyst has some advantages be- cause differentiation has already occurred and

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non-foetal tissues, such as the trophectoderm, can be biopsied. Normal fertile couples at risk for a child with an inherited disease no longer need to undergo an in vi tro fertilization procedure since Buster et al. ('85) described flushing of human blastocysts from Ibe uterus after in vivo fertiliza- tion. The aim of the study was to evaluate the impact of the biopsy of trophectoderm cells hatch- ing from the mouse blastocyst by vital staining of the manipulated embryos and by their in vitro culture to the early egg cylinder stage.

MATERIALS AND METHODS Mouse embryos and their culture

Female F1 hybrids (C57BL6j females x CBAca males) were superovulated with an intra- peritoneal injection of 5 units of PMSG (Intervet, Belgium) and 48 hr later with 5 units of human chorionic gonadlltrophin (hCG, Intervet, Bel- gium). The female mice were mated with a single F1 male (C57BUjj females x CBAca males) after hCG (day 0). Zygotes were collected 19 hr post hCG. The embryos were washed and placed into 10 pl drops of culture medium [Earle's, 0.4% sodiumlactate, 0.5% cristalline BSA (ICN, Bel- gium)] under li3ht paraffin oil (British Drug House, UK) in a 3002 petri dish (Falcon, Bel-

Received January 2, L990; revision accepted June 13, 1990.

PREIMPLANTATION BIOPSY OF MOUSE BLASTOCYSTS 233 - mural trophectoderm - intermediate trophectoderm

polar trophectdoderm

- mural trophectoderm - intermediate trophectoderm

polar trophectdoderm

Fig. 1. Classification of hatching trophectoderm cells (mural, polar, and intermediate trophectoderm).

gium), after the removal of the cumulus cells with hyaluronidase. The zygotes were cultured at 37°C in humidified air (5% Coal until day 5 in order to identify the exact moment of hatching from the zona pellucida at the blastocyst stage.

Evaluation of specific hatching site Blastocysts were evaluated under a n inverted

microscope (Nikon, Diaphot TMD) at 112 hr post hCG for the hatching of a few number of trophec- toderm cells from the zona pellucida (ZP) at a specific site. The blastocysts were classified ac- cording to the origin of the hatching trophec- toderm cells: polar when overlaying the inner cell mass (ICM), mural when covering the blastocoelic cavity opposite the ICM, and intermediate when situated between polar and mural region (Ya- mazaki and Kato, '89; Fig. 1).

Biopsy of cells from hatching blastocysts Hatching blastocysts were transferred to a 10

~1 manipulation droplet of PBS under oil and placed on the Nikon microscope table (37°C). Each blastocyst was fixed with a holding pipet of 50 pm diameter opposite the hatching site. A glass nee- dle was used to remove the herniating cells from the blastocyst (Defonbrune microforge, Leitz mi- cromanipulators, Drummond pipets). As a control group, blastocysts were placed in PBS under oil at 37°C for 15 min (time necessary for biopsy proce- dure).

Induction of site-spectfic hatching In mouse blastocysts that had not initiated in

vitro hatching at 112 h r post-hCG, hatching was induced at a selected site. The ZP was punctured with a microneedle at the mural, polar, or inter- mediate site. Four hours later, we evaluated whether herniation had occurred at the different puncture sites. Non-punctured control blastocysts were cultured in vitro to the early egg cylinder stage.

Viability assessment Viability of isolated cells and manipulated blas-

tocysts was evaluated immediately after biopsy, by vital staining with fluorescein diacetate (FDA). Embryos were exposed for 1 min to a solu- tion of 1.5 FM FDA PBS (37°C; Mohr and Troun- son, 'SO). Fluorescence was scored with B emis- sion under the Nikon Diaphot TMD; bright fluorescence ( + ) corresponded to normal viabil- ity; reduced or absent fluorescence ( - ) indicated an impaired cellular activity and/or membrane integrity. Control embryos were exposed to PBS solution under oil (37°C) for 1 min.

In vitro development The experimental and control embryos were

carefully washed and cultured up to day 10 in a Falcon 3002 petri dish (Hsu, '79) in order to study the early differentiation of the ICM and the in vitro implantation after biopsy. After biopsy (day 5), embryos were cultured 1 day in CMRL 1066 medium (Gibco, Belgium) supplemented with 10% FCS (Gibco, Belgium), and 1 day in CMRL 1066 supplemented with 20% FCS followed by daily changes of the medium by CMRL 1066 sup- plemented with 20% fresh decomplemented hu- man cord serum (HCS). Complete hatching from the zona pellucida and in vitro outgrowth of trophectoderm cells on the petri dish surface was recorded on day 6. The formation of elongated structures consisting of an extra-embryonic and embryonic part (early egg cylinder) was recorded on days 10 and 11 (Hsu, '72). Biopsied cells were cultured overnight in CMRL 1066 supplemented with 10% FCS.

Stat is t ics A ~'-2-sample test or a Kolmogorov-Smirnov 2-

sample test was used for statistical evaluation.

RESULTS AND DISCUSSION Ninety-four percent of the 398 fertilized oocytes

developed into expanded blastocysts on day 5 of the in vitro culture. Three hundred seventy-four blastocysts were evaluated for their hatching pat- tern at 112 hr post-hCG. One hundred twenty-six embryos (34%) initiated hatching with a small number of cells at a specific site: 35% (n = 44) hatched with mural trophectoderm, 30% (n = 38) near the ICM (polar hatching), and 35% (n =

44) started shedding the ZP at an intermediate trophectoderm site. These results are in contra- diction with the studies of Perona and Wassar- man ('861, who showed that mural trophectoderm

234 M. NIJS AND A. VAN STEIRTEGHEM

TABLE 1 . Induction of hatching and its effects on subsequent development of mouse blastocysts','

Hatching from Zonular No. of Survival opening in In vitro early hatching embryos after zona pellucida egg cylinder formation region (112 hr post-hCG) puncture (116 hr post-hCG) (day 10)

Mural 18 18 10 (55%) 11 (61%) Polar 22 22 13 (59%) 13 (59%) Intermediate 19 19 8 (42%) 12 (63%)

Control 25 18 (72%)

*Day 1 is day of presence of plug; experiments were repeated 4 times. *Statistical evaluation by x2-2 sample test or Kolmogorov-Smirnov 2-sample test.

cells of the mouse blastocyst produce strypsin, a proteinase that can induce softening of the ZP at the mutal site, followed by hatching and implan- tation of the embryo in the endometrium of the uterus at the mural site. Yamazaki and Kato ('891, however, found that mouse embryos shed their ZP at a non-specific site. Contraction and re- expansion of the blastocyst, cellular projections, and rotation of the embryo could diffuse strypsin in the perivitelline space, causing the blastocyst to hatch at any site of the zona pellucida.

We tried to induce hatching by puncturing the zona pellucida at a specific site in blastocysts that showed no spontaneous hatching at 112 hr post hCG, because only 34% of the embryos initiated hatching at this moment. Fifty-nine blastocysts were punctured at a polar, mural, or intermediate trophectoderm site. They all survived the punc- turing procedure and 42% t o 59% hatched at the puncture site with a small number of cells 4 hr later. The in vitro development of these punc- tured embryos was somewhat impaired, although not significantly different from the control. Fifty- nine to 63% of the manipulated embryos reached the early egg cylinder stage on day 10, compared to 72% in the control group (Table 1).

Some manipulated embryos and biopsied cells were exposed to F'DA after the biopsy of hatching cells with a microneedle (Table 2). FDA is taken up by the cell arid hydrolysed by an esterase to yield fluorescein when cellular activity is normal (Mohr and Trourison, '80). In comparison to the control group, biopsied embryos and isolated cells from different hatching groups had a reduced fluorescent pattern indicating that biopsy of cells can impair cellular activities and/or membrane integrity of emb-ryos and biopsied cells. The in vitro development of biopsied and control em- bryos that were exposed to FDA was not different from unstained experimental and control em- bryos (data not published).

After biopsy, the manipulated blastocysts were further cultured in vitro up to day 11 (Table 3). The number of embryos that shed their ZP or that were still hatchmg was recorded on day 6. In every experimenal group 8--18% of the embryos did not survive the micromanipulation procedure. Furthermore, in comparison with the control group, complete hatching from the ZP occurred later (P < .001). On days 10 and 11, embryos were evaluated for the outgrowth of trophectoderm cells on the petri dish and for the presence of the

TABLE 2. Control of viability of manipulated embryos and isolated cells determined by fluorescein diacetate staining (FDA) immediately after

bioDsv'3g,x

Zonular hatching No. of + FDA staining region embryos Embryos Groups of cells

Mural 17 14 (82%) 13 (76%) Polar 12 10 (83%) 10 (83%) Intermediate 24 18 (75%:) 20 (83%)

Control 25 25 (100%)

'Day 1 is day of presence of plug; experiments were repeated 2 tim,:s. 'Groups of cells: No. of cells ranging from 1 to 5 cells. *Statistical evaluation by x2-2 sample test or Kolmogorov-Smirnov 2-sample test,

PREIMPLANTATION BIOPSY OF MOUSE BLASTOCYSTS 235

TABLE 3. I n vitro development of manipulated embryos'

Day 10-11 Zonular No. of Day 6 hatching region embryos Dead Hatching Hatched early egg cylinder

Polar 35 3 (8%)* 25 (71%)" 7 (21%)" 14 (40%)"" Intermediate 44 5 (11%)" 32 (73%)* 7 (16'%)* 26 (59%)

Control 73 O* 19 (26%)* 54 (74%)" 51 (69%)**

'Day 1 is day of presence of plug; experiments were repeated 4 times *,**P < ,001.

Mural 35 4 (11%)* 27 (78%)* 4 (ll%)* 20 (57%)

pro-amniotic cavity and ectoplancental cone (early egg cylinder). A reduced number of em- bryos that hatched at mural and intermediate sites reached the early egg cylinder stage, al- though the initial retardation in growth was con- sistent in all experimental groups. Embryos with polar trophectoderm cells biopsied showed a significant reduction in their in vitro implanta- tion. Biopsied cells were cultured overnight and most cell groups showed mitotic division and/or accumulation of inter- or intra-cellular fluid.

The ability of blastocysts to develop to term, after removal of trophectoderm cells, was first re- ported by Rowson and Moor in sheep ('66). Gard- ner and Edwards ('68) obtained living young after sexing rabbit blastocysts by identifying sex chro- matin in small fragments of excised trophoblast. When trophectoderm cells from day 8 blastocysts of the marmoset monkey were removed after cut- ting the ZP to induce herniation at the mural site, 20% to 50% of the embryos showed hatching after 48 hr and a normal offspring was born after trans- fer of fresh and frozen/thawed biopsied embryos into recipients supplemented with hCG (Sum- mers et al., '88). Monk et al. ('88) diagnosed the Lesch-Nyhan syndrome in biopsied trophec- toderm cells obtained after spontaneous or in- duced hatching in blastocysts of an affected male and carrier female mouse embryos. Transfer into recipient females resulted in an impaired number of living young.

Our results show that esterase activities, mem- brane integrity, and further in vitro development are impaired after isolation of trophectoderm cells from mouse blastocysts. Normally, in vitro cul- tured blastocysts attach to the surface of the petri dish and mural trophoblast cells spread out to form giant cells. The blastocoelic cavity disap- pears by day 10, due to the enlargement of the ICM and due to the spreading of mural trophec- toderm cells. Polar trophoblast cells remain in ap- position with the ICM during the entire period of

growth. As the ICM continues to increase in girth, its downward growth is mechanically in- hibited by the coverslip surface and migrates up- wards, and breaks through the mural and polar trophoblast cells. Eventually it rests on top of po- lar trophoblast cells, which form the ectaplacen- tal cone. The primary endoderm-covered epiblast resting on top of this ectoplacental cone thus forms the early egg cylinder. The ectoplacental cone could be reduced in number of cells if polar trophectoderm cells are biopsied, because it is formed by the polar trophectoderm. However, if too many polar cells are isolated and/or if ICM cells are biopsied by mischance, egg cylinder for- mation could be severely hampered. This could explain the difference in impact of biopsy on in vitro development when mural, intermediate, or polar cells are isolated. Another possible explana- tion for this phenomena could be that the removal of polar trophectoderm cells exposes the ICM to the outer environment causing cells to differ- entiate into trophectoderm cells and thus reduc- ing the ICM population (Rossant and Tamura Lis, '79).

The biopsy of trophectoderm cells at the blasto- cyst stage could represent an alternative ap- proach to the isolation of blastomeres from em- bryos at earlier stages, if mural or intermediate trophectoderm cells are biopsied. The low success rate of in vitro culture of human embryos up to the blastocyst stage can be overcome (Bolton et al., '89) because of the development of uterine la- vage of in vivo fertilised human blastocysts (Bus- ter et al., '85). Still, the impact of biopsy of cells from a human blastocyst has to be investigated before being routinely applied in preimplantation diagnosis.

LITERATURE CITED Adinolfi, M., and P. Polani (1989) Prenatal diagnosis of ge-

netic disorders in pre-implantation embryos: invasive and non invasive approaches. Hum. Genet., 83:16-19.

236 M. NIJS AND A. VAN STEIRTEGHEM

Bolton, V., S. Hawes, C. Taylor, and J. Parsons (1989) Devel- opment of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates, and development to the blastocyst. J. In Vitro Fert. Embryo. Transfer, 6(11:30-35.

Buster, J., M. Bustillo, I. Rodi, S. Cohen, M. Hamilton, J. Simon, I. Thorneycroft, and J. Marshall (1985) Biologic and morphologic development of donated human ova recovered by nonsurgical lavage. Am. J. Obstet. Gynecol., 153f2): 211-217.

Gardner, R., and R. Edwards (1968) Control of sex ratio at full term in the rabbit by transferring sexed blastocyst. Nature,

Handyside, A., E. Kontogianni, K. Hardy, and R. Winston (1990) Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA-amplification. Nature, 344:768-770.

Holding, C., and M. Monk (1989) Diagnosis of beta- thalassaemia by DNA amplification in single blastomeres from mouse pre-implantation embryos. Lancet, ii:532-535.

Hsu, Y. (1972) Differentiation in vitro of mouse embryos be- yond the implantation stage. Nature, 239200-202.

Hsu, Y. (1979) In vitro development of individually cultured

21 8:346-348.

whole embryos from mouse blastocysts to early somite stage. Dev. Biol., 65:453-461.

Mohr, L., and A. Trouiison (1980) The use of fluorescein diace- tate to assess emb1,yo viability in the mouse. J. Reprod. Fertil., 58:189- 196.

Monk, M., A. Muggltkon-Harris, A. Rawlings, and D. Wit- tingham (1988) Pre-implantation diagnosis of HPRT- deficient male anc carrier female mouse embryos by trophectoderm biops:y. Hum. Reprod., 3(31:377-381.

Perona, R., and P. 'Wassarman (1986) Mouse blastocysts hatch in vitro by us tng a trypsin-like proteinase associated with cells of mural .;rophectoderm. Dev. Biol., 114:42-52.

Rossant, J., and W. 'I'amura Lis (1979) Potential of isolated mouse inner cell miiss to form trophectoderm derivates in vivo. Dev. Biol., 70:255-261.

Rowson, L., and R. Mcor (1966) Development of sheep concep- tus during the first fourteen days. J. Anat., 100:777-785.

Summers, P., J . Campbell, and M. Miller (1988) Normal in vitro development of marmoset monkey embryos after trophectoderm biopsy. Hum. Reprod., 3(3):389-393.

Yamazaki, K., and 1'. Kato (1989) Sites of zona pellucida shedding by mouse embryos other than muran trophec- toderm. J. Exp. Zoo1 ., 249:347-349.