Postnatal health and welfare of offspring conceived in vitro: a case for epidemiological studies.

In vitro fertilization (IVF) has established itself as an important technique in human assisted reproduction and in livestock improvement. In both humans and livestock the possible long-term effects on health and welfare of offspring born after IVF and in vitro culture to the blastocyst stage are still largely unknown. Epidemiological studies in humans, using data collected for individuals born after normal (i.e. non-assisted) pregnancies, have provided evidence for associations between prenatal life events and adult-life disease. Due to the relatively short time that elapsed since the first IVF baby was born, comparable studies for IVF offspring are not yet possible. However, animal experiments and epidemiological studies with the available data from the livestock industry (mainly dairy cattle) may contribute to a better understanding of the risks involved.

Follicular wall maintains meiotic arrest in bovine oocytes cultured in vitro.

Cumulus oocyte complexes (COCs) were cocultured with parts of the follicular wall. Coculture conditions were such that the COCs were 1) in continuous contact with the follicular wall (FWC), 2) separated from the follicular wall at collection but in contact with it during culture (FWR), and 3) separated from the follicular wall, but cultured in its vicinity (FWNR). Oocytes cultured for 24 hr under FWC conditions maintained the germinal vesicle stage. Under FWR conditions the germinal vesicle stage was not maintained, but an arrest at metaphase I of meiosis occurred in most of the oocytes. When COCs were cultured in the vicinity of the follicular wall (FWNR), meiosis was resumed and similar numbers of oocytes progressed to metaphase II of meiosis as compared to cultures of COCs without coculture with parts of the follicular wall. When COCs were isolated from the follicular wall after 24 hr of culture and additionally cultured for another 24 hr, the oocytes showed the same capability of resuming meiosis as fresh, isolated cumulus oocyte complexes. It is concluded that maintenance of contact with the follicular wall is necessary to maintain meiotic arrest. When COCs restore a physical contact with the follicular wall during culture, an arrest at metaphase I occurs.

Bovine activin A does not affect the in vitro maturation of bovine oocytes.

The effect of recombinant bovine activin A on the in vitro maturation of bovine oocytes was investigated. Culture of cumulus enclosed bovine oocytes in the presence of activin at the concentration of 100 or 500 ng/ml did not change the proportion of oocytes in which germinal vesicle breakdown had occurred at 4 and 7 h after the onset of culture. Activin had also no effect on the progression of maturation to the M II stage. The transient inhibition of germinal vesicle breakdown by 10 mM dibutyryl cyclic AMP was not affected by the addition of activin A at the onset of culture. Radiolabeling with 35S-methionine at 4 h and at 18 h after culture in the presence or absence of activin A did not show any effect of activin either on the total incorporation of radiolabel into acid precipitable material or on the protein synthesis patterns obtained after SDS-PAGE.

Evaluation of transvaginal ultrasound-guided follicle puncture to collect oocytes and follicular fluids at consecutive times relative to the preovulatory LH surge in eCG/PG-treated cows.

Holstein-Friesian cows (n=56) were synchronized with Syncro-Mate B, and those cows (n=47) developing a normal progesterone pattern were further treated im with 3,000 I.U. eCG at Day 10 and 22.5 mg PGF2alpha 48 h later. Blood samples were collected every hour from 30 until 49 h after PG administration. Cows (n=17, 36.2%) with fewer than 8 follicles larger than 8 mm in diameter at 28 to 30 h after PG treatment and animals without an LH peak (n=7, 23%) were excluded from the study. Transvaginal ultrasound-guided puncture of the follicles was carried out two times per cow, at 30 h after PG injection (4 to 5 follicles) and again at 1 to 5 (n=6), 12 (n=8) or 22 h (n=9) after the LH peak. No differences in the concentrations of progesterone and LH were observed among the 3 groups. An average of 18 follicles per cow was punctured (total of 415 punctures, n=23); 116 cumulus-oocyte-complexes and 370 follicular fluid samples were obtained producing average recovery rates of 28.0% and 89.2%. The number of cumulus-oocyte-complexes varied between puncture times; shortly before ovulation, at 22 h after the LH peak, the recovery rate was significantly 5 times higher than immediately after the LH peak. Overall, in 75 punctures the cumulus-oocyte-complex was accompanied by a pure follicular fluid sample (3.3 per cow). In conclusion, the transvaginal ultrasound-guided puncture of preovulatory-size follicles can be used to collect follicular fluids to study changes in the microenvironment of maturing oocytes upon superovulation. However, further research is required in order to obtain an equivalent number of accompanying cumulus-oocyte-complexes.

Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer.

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC-3 by indirect immunofluorescence. During preimplantation embryo development TEC-3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage. The finding that TEC-3 is not expressed before the onset of embryonic transcription, which occurs at the eight-cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC-03 antibody showed that the TEC-3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program.

Development of a sperm zona pellucida binding assay for bull semen.

Assays based on sperm zona pellucida binding have been developed as diagnostic tests to predict the fertilisation potential of human spermatozoa. The aim of this study was to establish a similar assay for bull sperm. The results showed differences between established fertile bulls in the relative numbers of sperm cells bound to the zona pellucida of a batch of oocytes. These differences suggest that there may be a relationship between the sperm zona pellucida binding capacity and the fertility of bulls.

Morphology of preovulatory bovine follicles as related to oocyte maturation.

Thirty-three preovulatory bovine oocytes and their follicles were collected during the period of final maturation in normally cyclic cows. Cell density of the membrana granulosa, mitotic index of the membrana granulosa, and the occurrence of eosinophilic granulocytes around the basal membrane as well as the maturational stage of the oocyte were determined. Cell density decreased during the period of final maturation. Mitotic indices also decreased after an initial high level in the first hours of the final maturation. Eosinophilic granulocytes were only seen during the last hours of final maturation. The maturational stages of the oocytes were related to distinct maturational stages of the follicular wall as determined by morphological characteristics. We propose a scoring system for the maturity of the follicular wall based on cell density, presence of mitotic figures and the presence of eosinophilic granulocytes outside the vascular compartment.

Follicular and oocyte maturation in cows treated for superovulation.

The maturational stage of oocytes and their follicles was assessed at 24/26 h after the preovulatory luteinizing hormone (LH) peak by means of morphological criteria. Follicles were obtained from cows treated for superovulation (PMSG/PG) with additional anti-PMSG to neutralize the residual PMSG. Follicular fluid was also recovered and analyzed for progesterone and estradiol levels. Seventy-two percent of the oocytes were at the Metaphase II (M(II)) stage of meiosis, whereas only 28% of the follicular walls were at the proper maturational stage; assessed on morphological characteristics, 78% of the follicles were progesterone-dominated. Earlier maturational stages of oocytes and follicles were also present, including those that are restricted to periods shortly after the LH peak in the normally cyclic cow. It is concluded that upon treatment for superovulation not all oocytes and follicles mature synchronously, and that not all oocytes mature in harmony with their follicles.