9th Postgraduate Course for Training in Reproductive Medicine and Reproductive Biology

Embryo development in vitro

F. Urner, H. Lucas
Department of Obstetrics and Gynecology, Geneva University Hospital

Gametes

Oocytes

Oocytes are aspirated from ovarian follicles following hormonal stimulation. Most of them are mature, at the metaphase II of the second meiotic division. They are enclosed in cumulus cells which are removed by hyaluronidase treatment only for ICSI.

Spermatozoa

Ejaculated sperm are used in most cases in IVF and ICSI, but epididymal or testicular spermatozoa can also be used. Prior to insemination, spermatozoa are prepared by swim-up or gradients techniques in order to obtain a high proportion of motile and morphologically normal sperm.

In vitro fertilization

Events of fertilization

Following sperm entry into the egg, the male chromatin decondenses rapidly while the oocyte resumes meiosis and extrudes the second polar body. Male and female pronuclei become visible and migrate until they are in close apposition. A that stage, the membranes of the pronuclei disappear, the male and female chromosomes intermingle and aligne on the metaphase plate of the first division : the syngamy is achieved.

Abnormal fertilization is characterized by the presence of more than 2 pronuclei or only one pronucleus in the ooplasm.

Conventional IVF

Cumulus-enclosed oocytes are inseminated with 50'000 to 100'000 motile spermatozoa in a Petri dishes. Fertilization is assessed 14-18h after insemination by the presence of 2 pronuclei in the oocyte (Day 1)

Intracytoplasmic sperm injection (ICSI)

Using micromanipulators, one sperm is directly injected into the oocyte which is hold by the holding pipette, the first polar body being at 6 o’clock. Fertilization is assessed 14-18h after injection by the presence of 2 pronuclei in the oocyte (Day 1)

Preimplantation embryo development in vitro

Timing, morphology and characteristics of the different stages

2-4-cell :

  • Mean time for the first (2-cell) and second (4-cell) cleavages : 35 h and 45h post-insemination (Day 2)
  • The different blastomeres are easily visible and their size and shape may be regular or not. Extracellular fragmentations may be present.
  • Utilization of the stores of maternal mRNA for protein synthesis

4-8 cell :

  • Mean time for the third cleavage(8-cell) : 54 h post-insemination (Day 3)
  • Activation of the embryonic genome : utilization of embryonic mRNA for protein synthesis

8-cell :

Compaction : blastomeres form a compact sphere, thight junctions develop between the outside blastomeres to seal them and isolate the inside cells ; gap juctions form between the internal cell to allow small molecules and ions to pass between the cells.

Morula

  • Compacted embryo, 16-32-cell
  • External cells will give rise to the trophoblast cells
  • Internal cells will become the inner cell mass

Blastocyst

  • Day 5-6
  • Cavitation : the trophoblast cells secrete a fluid to create a blastocoel cavity.
  • The inner cell mass, which will give rise to the embryo, is clearly distinct from the trophoblast cells which are required for implantation.
  • The percentage of 2-cell embryos reaching the blastocyst stage in vitro is usually not very high (@ 25%).

Embryo quality

Fast cleaving embryos, with good morphology (no fragments, regular blastomeres) and able to reach the blastocysts stage are considered as good quality embryos » with a high implantation potential. This quality depends on intrinsic characteristics of the embryos but also on the cultures conditions.

Embryo culture systems

Different culture systems and media can be used.

But : the temperature is always at 37°C and the presence of 5% CO2 is needed to maintain the pH of the medium at 7.2-7.4.

Systems

  • Culture in small volume (10-50 m l) of medium under a layer of paraffin oil
  • Culture in large volume (1 ml) without oil

Media

  • Simple media
    • Basic composition : Salts, bicarbonate, energy substates (pyruvate, lactate, glucose), proteins.
  • Complex media
    • Amino-acids and vitamines are added.
  • Sequential media
    • Since the nutritive requirement of the embryos changes in function of their stage of development, culture is performed in different media according to their cleavage stage.
  • Feeder cells
    • The presence of a monolayer of feeder cell (Vero cells) can improve the development of the embryos to the blastocycst stage. Although beneficial, the mechanism of action of these cells is not well known.

Cryopreservation

Cryopresevation of embryos is performed at different stages : zygote (2 pronuclei, after DNA synthesis and before syngamy), at the early cleavage stages (2-4 cells), at the blastocyst stage.

Transfer into the uterus

2-3 embryos are transferred into the uterus, usually on Day 2 (2-4 cell) or 3 (4-8-cell). Some groups transfer embryos at the blastocyst stage.

References

  1. Atlas of the human oocyte and early conceptus, Veeck LL, (1986) , ed : CL Brown,Waverly Press, Baltimore
  2. Handbook of in vitro fertilization, (1993), eds : A Trouson and DK Gardner, CRC Press, Boca Raton
  3. Current theory and practice of ICSI (1998). eds : Devroey P, Tarlatzis BC and Van Steirteghem A, Hum Reprod 13, suppl 1
  4. Developmental Biology. Scott G (1991), Sinauer ass., Sunderland

 
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Edited by Aldo Campana,