A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (−); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation are human embryonic stem cells, have normal karyotypes, and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.

Primate embryonic stem cells

Pregnancy has commonly been viewed as a cooperative interaction between a mother and her fetus. The effects of natural selection on genes expressed in fetuses, however, may be opposed by the effects of natural selection on genes expressed in mothers. In this sense, a genetic conflict can be said to exist between maternal and fetal genes. Fetal genes will be selected to increase the transfer of nutrients to their fetus, and maternal genes will be selected to limit transfers in excess of some maternal optimum. Thus a process of evolutionary escalation is predicted in which fetal actions are opposed by maternal countermeasures. The phenomenon of genomic imprinting means that a similar conflict exists within fetal cells between genes that are expressed when maternally derived, and genes that are expressed when paternally derived. During implantation, fetally derived cells (trophoblast) invade the maternal endometrium and remodel the endometrial spiral arteries into low-resistance vessels that are unable to constrict. This invasion has three consequences. First, the fetus gains direct access to its mother's arterial blood. Therefore, a mother cannot reduce the nutrient content of blood reaching the placenta without reducing the nutrient supply to her own tissues. Second, the volume of blood reaching the placenta becomes largely independent of control by the local maternal vasculature. Third, the placenta is able to release hormones and other substances directly into the maternal circulation. Placental hormones, including human chorionic gonadotropin (hCG) and human placental lactogen (hPL), are predicted to manipulate maternal physiology for fetal benefit. For example, hPL is proposed to act on maternal prolactin receptors to increase maternal resistance to insulin. If unopposed, the effect of hPL would be to maintain higher blood glucose levels for longer periods after meals. This action, however, is countered by increased maternal production of insulin. Gestational diabetes develops if the mother is unable to mount an adequate response to fetal manipulation. Similarly, fetal genes are predicted to enhance the flow of maternal blood through the placenta by increasing maternal blood pressure. Preeclampsia can be interpreted as an attempt by a poorly nourished fetus to increase its supply of nutrients by increasing the resistance of its mother's peripheral circulation.

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Genetic conflicts in human pregnancy.

Computer assisted semen analyzers in andrology research and veterinary practice

Ovarian hyperstimulation syndrome (OHSS) is a rare iatrogenic complication of ovarian stimulation occurring during the luteal phase or during early pregnancy. Fortunately, the reported prevalence of the severe form of OHSS is small, ranging from 0.5 to 5%. Nevertheless, as this is an iatrogenic complication of a non-vital treatment with a potentially fatal outcome, the syndrome remains a serious problem for specialists dealing with infertility. The aim of this literature review was to determine whether it is possible to identify patients at risk, and which preventive method should be applied when an exaggerated ovarian response occurs. Data pertaining to the epidemiology and prevention of OHSS in women were searched using Medline, Current Contents and PubMed, and are summarized. Preventive strategies attempt either to limit the dose or concentration of hCG or to find a way to induce luteolysis without inducing a detrimental effect on endometrial and oocyte quality. The following particular preventive strategies were reviewed: cancelling the cycle; coasting; early unilateral ovarian follicular aspiration (EUFA); modifying the methods of ovulation triggering; administration of glucocorticoids, macromolecules and progesterone; cryopreservation of all embryos; and electrocautery or laser vaporization of one or both ovaries.

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Epidemiology and prevention of ovarian hyperstimulation syndrome (OHSS): a review

Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment.

Severe ovarian hyperstimulation syndrome using agonists of gonadotropin-releasing hormone for in vitro fertilization: a European series and a proposal for prevention.

Trophectoderm biopsy was carried out on 47 human blastocysts. A slit was made in the zona pellucida opposite the inner cell mass by micromanipulative techniques. The human blastocyst zona offered more resistance to slitting compared to that of the mouse. After 18-24 h, controlled herniation of the trophectoderm cells was observed. These cells were biopsied when the diameter of the herniation was approximately equal to that of the blastocyst. The size of the slit and the stage of embryonic development at which slitting was performed were important for successful herniation to occur. After slitting, 76% of day 5-6 blastocysts showed herniation whilst only 42% of day 7-8 blastocysts herniated. Further development of the manipulated embryos was not apparently impaired, as hatching occurred in 44% of the former and 20% of the latter, compared with 18.1% in non-manipulated controls. The biopsied cells (approximately 10-30) usually remained in a clump but 14% formed vesicles on the day after biopsy. There was, however, no evidence of adherence to the dish or formation of monolayers. These results demonstrate the feasibility of trophectoderm biopsy in human blastocysts and that sufficient extra-embryonic material can be obtained by this technique for preimplantation diagnosis of genetic disorders.

Trophectoderm biopsy in human blastocysts

Micromanipulation of human oocytes and embryos has provided new opportunities for both the treatment of infertility and the preimplantation diagnosis of genetic disease. It is important to determine whether manipulated embryos develop normally in vitro, as an indication of their suitability for transfer. However, at present there is little information on the development of non-manipulated embryos in vitro for comparison. We have therefore monitored morphological changes and human chorionic gonadotrophin (HCG) secretion in 36 non-manipulated human embryos, including 26 blastocysts and 10 cavitating morulae, daily from day 3 to day 14 of culture. Hatching was observed in 10 (38.5%) blastocysts and five of these adhered to the culture dish and appeared viable until day 14. The secretion of HCG was first detected on day 8, peaked at day 10 (51.11 +/- 8.7 mIU/ml) and then declined but was still detectable in four blastocysts on day 14. There was no overall difference in HCG secretion by hatched blastocysts and those which remained within the zona. However, those hatched blastocysts which showed adherence had significantly increased (P less than 0.05) HCG secretion. For individual blastocysts, the pattern of HCG secretion correlated well with the assessment of morphology. These data provide the basis for comparative studies of morphological changes and HCG secretion in manipulated embryos.

The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro

The effect of the strict classification of spermatozoal morphology on the outcome of in-vitro fertilization and embryo transfer treatment cycles has been assessed in a retrospective analysis of 2144 consecutive cycles. The patients all had a standardized long protocol gonadotrophin-releasing hormone analogue cycle, with luteal phase start, to achieve down-regulation. All treatment cycles where the sperm density was abnormal ( 14% normal forms, and group 2 with > or = 4%, < or = 14% normal forms). There was a statistically significantly lower chance of achieving this rate in patients of group 3 (< 4% normal forms) (P < 0.005), but 68.6% did achieve that fertilization rate or higher. There was no statistically significant difference in any of the other end points. In conclusion, a morphological classification may be appropriate as an indicator for counselling patients with regard to treatment expectations, but its use would be seem inappropriate as an index of fertilizing potential in clinical management.(ABSTRACT TRUNCATED AT 250 WORDS)

Does isolated teratozoospermia affect performance in in-vitro fertilization and embryo transfer ?

This prospective study demonstrates the feasibility and outcome of embryo transfer performed by nursing staff with medical cover available. Of 771 patients who had embryo transfer, 679 (88%) had their embryo transfer performed by a nurse. In 92 cases (12%) a doctor performed the embryo transfer, either as the first operator, or having been brought in to assist the nurse who experienced difficulty. The pregnancies per transfer for nurse transfer was 246/679 (36%) and where a doctor performed the transfer 20/68 (29%). These data show a high comparable success rate when a nurse performed the embryo transfer, and a low incidence of direct medical involvement.

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Caroline Ross

Papers

Severe ovarian hyperstimulation syndrome using agonists of gonadotropin-releasing hormone for in vitro fertilization: a European series and a proposal for prevention.

Trophectoderm biopsy in human blastocysts

The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro

Does isolated teratozoospermia affect performance in in-vitro fertilization and embryo transfer ?

Nurses performing embryo transfer: successful outcome of in-vitro fertilization.