Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH.

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.

Oocyte maturity and human follicular fluid prostanoids, gonadotropins, and prolactin after administration of clomiphene and pergonal.

Follicular fluid (FF) and oocytes were obtained from 130 follicles of 52 women in whom ovulation was induced with human menopausal gonadotropin (Pergonal) and clomiphene citrate. Follicular aspiration was performed 36 h after an ovulatory injection of hCG. The concentrations of LH, FSH, PRL, and the prostanoids prostaglandin E2 (PGE2), PGF2 alpha, PGI2 (as 6-oxo-PGF2 alpha), and thromboxane A2 (as TXB2) in the FF were measured by RIA and related to the degree of maturation of the oocyte-corona-cumulus complex mass (OCCC). FF obtained from follicles with immature OCCC contained significantly lower concentrations of all four prostanoids (median concentrations, picograms per mL: PGE2, 88; PGF2 alpha, 85; 6-oxo-PGF1 alpha, 40; TXB2, 50) than those with intermediate OCCC (PGE2, 175; PGF2 alpha, 325; 6-oxo-PGF1 alpha, 130; TXB2, 65) and mature OCCC (PGE2, 425; PGF2 alpha, 860; 6-oxo-PGF1 alpha, 235; TXB2, 78; all P less than 0.01). There were no significant differences between the maturity of the complexes and the concentrations of LH, FSH, or PRL. There were significant correlations between the FF concentrations of LH and FSH and those of all of the prostanoids, but not with PRL, concentrations. These results indicate that the synthesis of prostanoids in the human Graafian follicles may be modulated by gonadotropins and consolidates the view that prostanoids may play a role in human oocyte maturation and ovulation.

Corticosteroid metabolism in vitro by human placenta, fetal membranes and decidua in early and late gestation.

Corticosteroid conversion by early (7 to 10 weeks) and late (36 to 42 weeks) human placenta, fetal membranes and decidua was studied. Tritiated cortisol, cortisone, corticosterone, 11-dehydrocorticosterone (DHC) and 11-deoxycorticosterone (DOC) were incubated in vitro with homogenates of these tissues and extracts chromatographed on Sephadex LH20 columns. 11 beta-hydroxysteroid dehydrogenase activity (11 beta-HSD; EC 1.1.1.146) was present in all these tissues from as early as the seventh week of pregnancy. In the fetal membranes the enzyme activity was present in the chorion, the amnion showing no activity. Only the decidua could convert cortisone and DHC to the biologically more active cortisol and corticosterone, respectively. DOC was not transformed by any of the tissues. No 11 beta-HSD activity was found in amniotic fluid, retroplacental serum, umbilical cord plasma or cord tissue.

Inhibitors of platelet aggregation in the fetoplacental unit and myometrium with particular reference to the ADP-degrading property of placenta.

Extracts of fresh tissue from the feto-placental unit and myometrium were tested for their ability to inhibit ADP-induced platelet aggregation and to degrade ADP. Placental extracts caused rapid reversal of aggregation and degraded ADP, both effects being mimicked by HPAP. However, whereas the latter was inhibited by L-phenylalanine but not by heating to 65 degrees C for 5 minutes, the reverse was true for crude placental extracts. Umbilical cord vessels and myometrium totally inhibited platelet aggregation in a similar way to pure PGI2. Both tissues also exhibited ADP-ase activity but were much less potent in this respect than placenta. In the system used, little or no anti-aggregatory activity was detected in extracts of non-vascular cord tissue, fetal membranes or amniotic fluid, although the two latter tissues had a weak ADP-degrading effect. Thus, it appears that in contrast to myometrium and umbilical cord vessels, the major inhibitor of platelet aggregation in placenta is an ADP-ase and not PGI2. While part of the inhibitory effect of placenta may be due to HPAP, other ADP-degrading enzymes also seem to contribute to the overall anti-aggregatory property of this organ.

Does human placenta produce prostacyclin?

We have previously demonstrated that the human placenta possesses potent platelet anti-aggregatory activity. This activity was exhibited only when aggregation was induced by adenosine diphosphate (ADP), but not when induced by adrenaline, ristocetin or collagen. We have also shown that placental extracts degrade ADP. We therefore concluded that the placenta's anti-aggregatory activity, in vitro, was not due to prostacyclin (PGI2) but to an 'ADPase'. In view of some reports claiming that the human placenta produces PGI2, we carried out a series of experiments to establish whether human placental tissue can convert [14C]-arachidonic acid [( 14C]-AA) to 6-oxo-PGF1 alpha, the stable metabolite of PGI2. Tissue from placenta and the membranes did not show any appreciable conversion of [14C]-AA into 6-oxo-PGF1 alpha. This finding was confirmed by radioimmunoassay techniques where the placenta was shown to produce spontaneously only minimal amounts of 6-oxo-PGF1 alpha. We conclude that placental tissue and the fetal membranes do not synthesize a significant amount of PGI2, certainly not enough to account for the potent platelet anti-aggregatory activity of the placenta in vitro. Placental platelet anti-aggregatory activity in vitro, is probably due entirely to ADPase activity.

Cobalamins in human pregnancy and lactation.

Samples of maternal and foetal blood and of human milk, cow's milk, and dried cow's milk have been analysed for cobalamins by chromatography and bioautography. Plasma cobalamins in late pregnancy and after delivery differed quantitatively from those in healthy non-pregnant women. Differences in the proportions of plasma cobalamins were also observed between mothers and their newborn infants. The findings suggest that methylcobalamin may be a more labile plasma form of the vitamin than 5-deoxyadenosyl cobalamin. Breast milk from healthy mothers contained more deoxyadenosylcobalamin and proportionately less methylcobalamin than their plasma. Cyanocobalamin given parenterally to lactating women was detected in the plasma, and high concentrations appeared unchanged in the milk. The B(12) in fresh cow's milk was found to be largely 5-deoxyadenosyl cobalamin whereas dried milk contained only hydroxocobalamin and a little cyanocobalamin. The nutritional implications of the findings, and their bearing on problems of cobalamin transport, are discussed.

Gamete intrafallopian transfer in women who had ectopic pregnancy previously.

Eighty-seven women who had previously had ectopic pregnancy underwent 111 gamete intrafallopian transfers (GIFT). Forty-one women had moderate or severe pelvic adhesions, eight had mild pelvic adhesions, and 18 had had the fallopian tubes surgically repaired. The remaining 20 women had no obvious pelvic abnormality except for the absence of one fallopian tube. After stimulation of superovulation, between two and 22 oocytes were retrieved and two to four were transferred into the fallopian tubes after careful assessment of their condition. Twenty-eight pregnancies were achieved (25.2% per cycle), with 23 live births (20.7% per cycle), four abortions (14.3% of all pregnancies), and one ectopic pregnancy (3.6% of all pregnancies, 0.9% per cycle). This experience shows that GIFT can safely produce a reasonable pregnancy rate in carefully selected women who have had ectopic pregnancy.

Ovum donation--a simplified approach.

To date, ovum donation (OD) has involved luteinizing hormone (LH) synchronization between recipient and donor for normally cycling women, and a complex steroid replacement regimen given on a sequential and incremental basis for women with primary or secondary ovarian failure. The authors designed a simple hormonal regimen applicable to both normally cycling women starting early in the cycle, and to those with ovarian failure. It consists of administering 2 mg estradiol (E2) valerate orally three or four times daily, augmented with either 100 mg progesterone (P) in ethyl oleate intramuscularly daily or 100 mg oral progesterone (P) orally three times daily, starting on the day preceding the recovery of the donated oocytes. Gamete intrafallopian transfer procedure was undertaken for women with patent tubes and in vitro fertilization for those with obstructed tubes. The authors report their preliminary experience with 17 women who underwent ovum donation.

Assisted fertilization with intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the significance of intracytoplasmic sperm injection in severe male factor infertility and previous failed fertilization. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London. SUBJECTS: Sixty-nine patients with a long-standing history of infertility of which 48 had previous failed fertilization, 15 had < 1 million progressive motile sperm per ejaculate, and the remaining 6 had obstructive azoospermia. INTERVENTION: Assisted fertilization with primary intracytoplasmic sperm injection was carried out in 69 IVF. OUTCOME: Normal (two pronuclei [2PN]) fertilization and pregnancy rates. RESULTS: A total of 967, oocytes were collected and 785 were subsequently microinjected. Normal fertilization (2PN) occurred in 410 oocytes (52%) and 90.5% of those cleaved. Sixty-four patients underwent ET, with a total of 181 embryos transferred. Twenty-five patients conceived with a pregnancy rate of 39% per transfer. The implantation rate was 16% and the total pregnancy loss rate 24%. Failed fertilization after intracytoplasmic sperm injection occurred in four cases. CONCLUSIONS: Intracytoplasmic sperm injection is increasingly becoming the treatment of choice in infertile couples where assisted fertilization is indicated. The high fertilization and pregnancy rates observed with this technique, together with a low risk of abnormalities, has revolutionized treatment of male factor infertility.

Ectopic pregnancy resulting from gamete intrafallopian transfer and in vitro fertilization. Role of ultrasonography in diagnosis and treatment.

Forty-five cases of ectopic pregnancy occurred after gamete intrafallopian transfer (32 cases) or in vitro fertilization (13 cases). Ultrasonography positively identified ectopic pregnancy in 33 cases (73.4%) and suggested the presence of one in 7 cases (15.6%). There were five false-negative results (11.1%). The incidence of rare types of ectopic pregnancy after assisted fertility procedures, such as ovarian, heterotopic, cervical and ectopic pregnancy, in patients who had undergone a previous salpingectomy was increased. Ultrasound scanning was used to monitor three cases of nonviable ectopic pregnancy; all three required no further treatment. In 14 cases of viable ectopic pregnancy the gestational sac was aspirated and injected with potassium chloride and methotrexate. In seven of those cases no further treatment was needed. Patients who conceive as a result of assisted fertility procedures should be scanned four to six weeks after the procedure or sooner if they are considered at high risk of developing an ectopic pregnancy or if the condition is symptomatic.

Oocyte donation in 61 patients.

Oocyte donation was performed by gamete intrafallopian transfer in 61 women, 34 of whom were amenorrhoeic. The mean age of the donors was 32 years (range 23-38). Seventy-five treatment cycles gave 29 clinical pregnancies, of which 21 reached term, 3 continue, and 5 were lost (3 miscarriages, 1 tubal, and 1 cervical). 11 (38%) of the women who became pregnant were over 42 years old. When more than four oocytes were transferred, many of the pregnancies were multiple. 8 (38%) of the pregnancies that came to term were complicated by pre-eclamptic toxaemia. When oocytes are obtained from young women, the fertility potential is high irrespective of the recipient's age.

Experience with gestational surrogacy as a treatment for sterility resulting from hysterectomy.

A retrospective study was carried out to assess the potential of 16 hysterectomized women to achieve surrogate pregnancies. A total of 11 patients completed 16 cycles of assisted conception treatment incorporating in-vitro fertilization and gestational surrogacy. Three other women failed to respond to ovulation induction while two more patients produced few oocytes which also failed to fertilize. Six host mothers became pregnant thereby giving a pregnancy rate of 37.5% (6/16) per patient and embryo transfer and 27.3% (6/22) per cycle of treatment commenced. Two women later miscarried, three have given birth to twins and the remaining host has delivered a male infant. The commissioning mother's age was closely related to occurrence and continuation of pregnancy in the host. Hysterectomized women demonstrate varying patterns of response to assisted conception treatment but gestational surrogacy generally appears to be a feasible option especially in younger patients.