Human ova and embryo morphology

By Anette Gabrielsen and Svend Lindenberg,
Ciconia Fertility Clinic Ildervej 5, DK- 8270 Højbjerg Denmark and KAS Herlev Fertility Clinic,
Copenhagen University DK-2730 Denmark

It is important to evaluate and categorise human oocytes, fertilised ova and cleaved embryos during advanced fertility treatments due to the fact, that different developmental stages can be utilised for different procedures etc. Furthermore, a thorough insight into the development and stages of the embryos is necessary both for the technician, embryologist and the clinician during the treatment for several reasons:

to communicate better in between the professions and to the patients

to optimise the treatment, as the doctor get knowledge about the nuclear stages of the oocytes after pick-up thus optimizing the ovarian hyperstimulation protocol.

to compare data in between clinics and perform a continuos registration for optimising the selection for embryos able to implant.

The in vitro development of human ova is routinely done through an inverted microscope using Hoffmanns modulations and x 200 magnification. To our knowledge the staging of cumulus oocyt complexes is very unprecise , thus only good information about oocytes can be drawn from denuded oocytes used during the ICSI procedure. In the following we present a series of images illustrating the different stages of development in vitro of human embryos. We are scoring the embryos according to Van Abbel (Hum Reprod. 3, 1988).

Figure 1.

An image of a GV-ova, (Germinal vesicle - stage). No polar body but a germinal vesicle is seen in the cytoplasm.

Figure 2.

An image of a Metaphase I oocyt. The Germinal Vesicle has broken down.

Figure 3.

An image of a Metaphase II oocyt. Here the first polarbody is expelled into the perivitelline space .

Figure 4.

A fertilised ova with 2 normal pronuclei and 2 polarbodies.

Figure 5.

A fertilised ova with only 1 pronuclei. This indicate a failure in fertilisation. However in our experience, very often a second look at the embryo after 4 to 6 hours reveal the formation of the other pronuclei. These embryos seems to cleave normally.

Figure 6.

An unfertilised ova with 3 pronuclei, indication a failure in fertilisation. This might indicate, that a surnumerous spermatozoa has penetrated into the egg. However, the third pronuclei might represent a non extruded second polarbody. These ova can cleave normally, but of course always having chromosome aberration. These ova will always be destroyed.

Figure 7.

An image illustrating a 2-cell embryo. This embryo would be classified according to the Van Abbel´s classification as a 1.0 two cell embryo. No fragment is seen and the blastomers are of equal size.

Figure 8.

An image of a 2 - cell embryo without fragments but uneven size of the blastomeres. This embryo would be classified according to Van Abbel as a 2.0 two cell embryo.

Figure 9.

An image of a 2- cell embryo classified by Van Abbel as being a 2.1 embryo due to the fragments seen. (Less than 10% fragments within the embryo)

Figure 10.

An image of a 2- cell embryo classified by Van Abbel as being 2.2, due to 10% - 20% fragmentation.

Figure 11

An image of a 2- cell embryo classified by Van Abbel as being 3.1 due to 20% - 50% fragmentation.

Figure 12

An image of a 2- cell embryo classified by Van Abbel as being 3.2, due to more than 50% fragmentation.

Figure 13.

An image of a embryo classified by Van Abbel as being 4.0, due to only fragments present.

Figure 14.

An image of a 4- cell embryo classified by Van Abbel as being 1.0 without fragmentation.

Figure 15

An image of a 4- cell embryo classified by Van Abbel as being 2.2 due to 10% - 20% fragmentation.

Figure 16.

An image of a 6-cell embryo classified by Van Abbel as being 1.0. without fragment.

Figure 17.

An image of a 8- cell embryo classified by Van Abbel as being 2.2, due to 10% - 20% fragmentation.

Figure 18.

An image of an abnormal embryo having 3 pronuclei in one blastomere and one large fragment.

Figure 19.

An image of a 4- cell embryo classified by Van Abbel as being a 4 cell, score 2.2, due to 10% - 20%. The ova is oval possible due to high vacuum pressure during the oocyte pick-up procedure. The oocyte was already oval at oocyte retrieval.

Figure 20.

An image of a 2- cell embryo classified by Van Abbel as being 2.1, due to less than 10% fragmentation. The ova also exhibits cytoplasmic vacuole. Embryos having vacuole are not chosen for transfer.

Figure 21.

An image of a 2 cell embryo with cytoplasmic vacuole and an abnormal zona pellucida.

Figure 22.

An image of af a oocyte with an abnormal thin zona pellucida.

Figure 23.

An image of a 2-cell embryo 1.0 with an abnormal thick zona pellucida.

Figure 24. and 24a

An image of an abnormal seize ova (large) . These ova will often be abnormal fertilised.

Figure 25.

An image of a atretic oocyt.

We normally do not inject sperm during the ICSI procedures in these types of ova. Please compare this with the image of a normal seize ova

Figure 26.

An image of an empty zona pellucida. These are often seen when to high vacuum is used during ovum pick-up.

Further classifications of the embryos

It has been suggested, that not alone the stage of the embryo and the degree of fragmentation is important for selection of embryos.

Also the following has to be taken into account:

1)	the thickness and variability of the thickness of the zona pellucida
2)	the type and distribution of the fragments.
3)	the timing of the cleavages of the embryo.

Ad. 1.	Several authors have suggested that not only is zona hardening due to the in vitro conditions a problem for the embryo. A thin or variable thinning of the zona pellucida might be associated with a better pregnancy chance. This is probably because the trophoblastic cell can better penetrate a thin zona during hatching then a thick hard zona.
Ad 2.	Some authors have proposed, that an even distribution of fragments in an embryo might disturb several junctions between all blastomeres and hereby jeopardise the function of the embryo. If fragments are assembled in one area this disturbance of the embryo is not that severe.
Ad 3.	From animal studies it has been shown that the fastest normal dividing embryo also possesses the best chance of giving offspring.

Conclusion

In our hands the best embryos for transfer will be the embryos with:

	The lowest score according to Van Abbel
	The embryo has cleaved normally according to the time of transfer (i.e. 4-cells for transfer at day 2 post fertilisation)
	A variable thickness of the zona pellucida
	If fragments are present, than choose embryos with localised fragments.

All images has been taken through a Nicon Inverted microscope and stored in a FQC - system provided by Fercom, Denmark.

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SL - 22 Sep 2002