Fertilization and Embryo Cleavage After ICSI

Pregnancy Miracle

The Pregnancy Miracle Program

Get Instant Access

After the injection procedure, oocytes are rinsed and cultured in micro-droplets covered with lightweight paraffin oil. The conditions are similar to those employed for IVF inseminated oocytes: the oocytes are kept at 37°C in an atmosphere of 5% O2, 5% CO2, and 90% N2. Injected oocytes are examined for integrity and fertilization about 16-18 hours after ICSI (67). An average damage rate of approximately 9% of the injected oocytes can be expected, irrespective of the origin of the sperm used (45). Oocytes are considered normally fertilized when two individualized or fragmented polar bodies are present together with two clearly visible pronuclei (2-PN) that contain nucleoli (Fig. 3).

The fertilization rate after ICSI is usually expressed per number of injected oocytes and ranges from 57% to 67% according to the sperm origin (45). As shown in Figure 3, abnormal fertilization may occur, reflected by one-pronuclear (1-PN) oocytes (about 3% of the injected oocytes) (45). These oocytes are likely to be parthenogenetically activated as a result of mechanical or chemical factors (68,69). The occasional finding of three-pronuclear (3-PN) oocytes (about 4%) (45) after injection of a single spermatozoon into the ooplasm is probably caused by failure of extrusion of the second polar body at the time of fertilization (69). Neither type of embryo resulting from 1-PN to 3-PN oocytes is transferred to patients.

Post-fertilization, about 90% of 2-PN oocytes obtained by ICSI enter cleavage, resulting in multicellular embryos. Cleavage characteristics of the fertilized oocytes are evaluated daily. Normally developing, good-quality embryos reach the four-cell and eight-cell stage, respectively, on day 2 and in the morning of day 3 postmicroinjection (Fig. 4). Numbers and sizes of blastomeres and the presence of anucleate cytoplasmic fragments are

Polar Body And Fragments

Figure 3 Fertilization outcome after intracytoplasmic sperm injection. (A) Oocytes are considered normally fertilized when two individualized or fragmented polar bodies are present together with two clearly visible pronuclei (2-PN) that contain nucleoli. (B) Abnormal fertilization may occur as one pronuclear (1-PN) oocyte, probably due to parthenogenic activation. (C) The occasional finding of three pronu-clear (3-PN) oocytes after injection of a single spermatozoon into the ooplasm is probably caused by non-extrusion of the second polar body at the time of fertilization.

Figure 3 Fertilization outcome after intracytoplasmic sperm injection. (A) Oocytes are considered normally fertilized when two individualized or fragmented polar bodies are present together with two clearly visible pronuclei (2-PN) that contain nucleoli. (B) Abnormal fertilization may occur as one pronuclear (1-PN) oocyte, probably due to parthenogenic activation. (C) The occasional finding of three pronu-clear (3-PN) oocytes after injection of a single spermatozoon into the ooplasm is probably caused by non-extrusion of the second polar body at the time of fertilization.

Fertilisation Polar Bodies Cleavage
(D) (E) <F)
Cleavage Fertilization

Figure 4 Embryo cleavage after intracytoplasmic sperm injection. Only embryos resulting from normally fertilized oocytes (A) will be transferred to patients. Embryo cleavage is evaluated daily. Two-cell embryos (B), four-cell embryos (C), and eight-cell embryos (D) are usually obtained on day 1 (late afternoon), on day 2, and in the morning of day 3, respectively. The blastomere number is recorded and the embryos are scored accordingly to equality of size of the blastomeres and the presence of anucleate cytoplasmic fragments. On day 4 (sometimes already on day 3), a certain degree of compaction can be observed (E). For blastocyst (F) scoring, the classification system introduced by Gardner and Schoolcraft is used. Embryo transfer is usually done on day 3 (eight-cell stage) or day 5 (blastocyst stage). Source: From Refs. 70 and 75.

Figure 4 Embryo cleavage after intracytoplasmic sperm injection. Only embryos resulting from normally fertilized oocytes (A) will be transferred to patients. Embryo cleavage is evaluated daily. Two-cell embryos (B), four-cell embryos (C), and eight-cell embryos (D) are usually obtained on day 1 (late afternoon), on day 2, and in the morning of day 3, respectively. The blastomere number is recorded and the embryos are scored accordingly to equality of size of the blastomeres and the presence of anucleate cytoplasmic fragments. On day 4 (sometimes already on day 3), a certain degree of compaction can be observed (E). For blastocyst (F) scoring, the classification system introduced by Gardner and Schoolcraft is used. Embryo transfer is usually done on day 3 (eight-cell stage) or day 5 (blastocyst stage). Source: From Refs. 70 and 75.

recorded. The cleaving embryos are scored according to equality of size of the blastomeres and proportion of anucleate fragments (70). Type A (excellent quality) embryos do not contain anuclear fragments. Type B (good quality) embryos have a maximum of 20% of the volume of the embryo filled with anucleate fragments. In type C (fair quality) embryos, anucleate fragments represent 21% to 50% of the volume of the embryo. Type D (poor quality) embryos have anucleate fragments present in more than 50% of the volume of the embryos. These embryos cannot be used for transfer to the patients. Embryos in the former three categories (type A, B, and C) are eligible for transfer.

Nowadays, most centers perform embryo transfers on day 3 or day 5 after oocyte retrieval. At that time, the embryos are expected to be at the eight-cell stage. Because the embryonic genome is fully activated after the eight-cell stage (71), it may indeed be beneficial to evaluate embryos at least until after the transition from maternal to embryonic genome, making it possible to identify those embryos with a better developmental potential. The number of embryos transferred depends on the age of the woman and on the rank of trial. In women under 37 years of age who are undergoing a first or second ICSI attempt, preference is given to transfer of only two excellent or good-quality embryos. In other cases, three or more embryos may be placed into the uterus. Higher pregnancy rates can be obtained when elective transfer of two or three embryos is possible (45,72). However, the number of embryos transferred should be limited in order to avoid multiple pregnancies.

Today, commercially available sequential culture media allows the culture of human embryos up to the blastocyst stage (day 5 or 6) (73). On day 4 (sometimes already on day 3), a certain degree of compaction can be observed. Compaction in the mammalian pre-embryo is a fundamental event that leads to the formation of the trophectoderm, the inner cell mass and the blastocoele. Full compaction (16-32-cell stage) is followed by immediate cavitation and blastocoele expansion (74). For blastocyst scoring, the classification system introduced by Gardner and Schoolcraft can be used (75). A distinction between early and expanded blastocysts is made, and the latter category is further scored, according to the quality of the inner cell mass and the trophectoderm. The possibility of prolonged human embryo culture allows for day 5 or blastocyst transfers. Preferably, expanded blastocysts with a cohesive trophectoderm and a clear inner cell mass are transferred. Possible advantages of blastocyst transfer are better embryo selection and better synchronization between embryo and endometrium, which may result in higher implantation rates per blastocyst transferred (76,77). This in turn would allow transfer of fewer embryos, thereby decreasing the number of multiple pregnancies (78).

Was this article helpful?

0 0
Get Pregnant - Cure Infertility Naturally

Get Pregnant - Cure Infertility Naturally

Far too many people struggle to fall pregnant and conceive a child naturally. This book looks at the reasons for infertility and how using a natural, holistic approach can greatly improve your chances of conceiving a child of your own without surgery and without drugs!

Get My Free Ebook


Post a comment