Duration of storage of cryopreserved human embryos.

The incidence of cell injury, embryo survival, and implantation following cryopreservation of zygotes and two- to five-cell embryos was studied in 100 patients in order to evaluate the effect of duration of storage. The incidence of individual cell survival was 58% regardless of the length of time kept in liquid nitrogen or the stage of the embryo at freezing. There were 104 of 208 (50%) thawed embryos that survived completely intact, and of those, 24 implanted successfully. Twenty-one (21%) patients had a clinical pregnancy; two of them miscarried Neither the survival of zygotes or cleaved embryos upon thawing nor the incidence of implantation was affected by the duration of cryostorage.

Cryopreservation of zygotes and early cleaved human embryos.

Zygotes and 2- to 5-cell human embryos were frozen in 1,2-propanediol and sucrose; results of the first 50 cycles (45 patients) are presented. A total of 41 zygotes (17 attempts at thawing) were thawed, resulting in six singleton clinical pregnancies (15% per embryo; 35% per cycle), of which three delivered, one aborted, and two are ongoing. Fifty-seven cleaved embryos were thawed in 33 other cycles, resulting in four singleton and one twin pregnancy (11% per embryo; 15% per cycle), of which four delivered and one is ongoing. Depending on the cell stage, 61% to 81% of embryos survived cryostorage, but 2-cell embryos did not implant. One fifth of cryoinjury was due to the formation of cracks in the zona pellucida. The incidence of implantation was not enhanced when more than one freeze/thawed embryo was replaced, most pregnancies being obtained from single embryo replacements. At least 8% more births are expected in addition to conventional in vitro fertilization methods when the current policy of replacing three fresh embryos and freezing the remainder using this technique is applied. This method will result in two to four times more pregnancies per spare embryo, compared with other cryopreservation methods using older embryos.

Transfer of immunity to Ostertagia circumcincta and IgA memory between identical sheep by lymphocytes collected from gastric lymph.

Sheep rendered immune to Ostertagia circumcincta were challenged with 50,000 larvae and lymphocytes were collected from the gastric lymph up to eight days after challenge. The cells were transferred intravenously to genetically identical worm-free sheep which, together with controls, were challenged with 50,000 larvae and killed nine days later. Cells obtained during the donors lymphoblast response to challenge transferred partial immunity, measured either as stunting or loss of worms. Significantly less immunity was transferred by cells collected either before or after this response. Thus the responding cells can mediate protective immunity to O circumcincta. On the other hand the donor sheep remained immune to their challenge infection despite being depleted of these functional cells, showing that their presence was not essential for immunity to be maintained. Comparison of the immunoglobulin A (IgA) concentrations in the gastric lymph of recipient and control sheep showed that a local IgA response had also been transferred. Enumeration of mucosal mast cells suggested that a mastocytosis had been transferred to the two recipients which were most immune to challenge.

Factors affecting survival and implantation of cryopreserved human embryos.

Five- to ten-cell embryos and expanded blastocysts from 68 patients were thawed in an attempt to establish pregnancy. Three fresh embryos had been replaced unsuccessfully in these patients. Forty-five patients had intact freeze-thawed embryos replaced and 12 became clinically pregnant. Ten of these pregnancies have now advanced beyond week 24. The preliminary results presented here demonstrate that significantly more expanded blastocysts survive cryopreservation than cleaving embryos. Faster-growing embryos did not survive better than slowly growing embryos, but the incidence of implantation was higher with faster-developing embryos. Significantly more blastocysts with a "normal" morphology survived cryostorage than those scored as "irregular" (77 versus 7%). A similar trend was observed when normal and irregular cleaving embryos were frozen, but the difference was not significant. The severity of contraction of blastocysts upon the addition of cryoprotectant increased the incidence of survival. The proportion of patients whose embryos had holes in the zona pellucida following cryopreservation was significantly higher when embryos were frozen at the cleaving stage (39%) rather than the expanded blastocyst stage (16%). Almost all embryos with damaged zonae degenerated. The proportion of cleaving embryos that survived cryostorage was inversely correlated with the number of follicles aspirated.

Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study.

The survival and implantation capacity of cryopreserved cleaving (5-cell to 10-cell) human embryos and expanded blastocysts was compared. Twice as many cleaving embryos were frozen as were expanding blastocysts because of the low developmental potential of human embryos in vitro. However, significantly more expanded blastocysts survived cryopreservation than cleaving embryos, and relatively more pregnancies were established by the replacement of thawed blastocysts than by the replacement of thawed cleaving embryos. Cleaving embryos from 26 women were thawed; 17 had thawed embryos replaced, and 4 subsequently became pregnant. Expanded blastocysts were thawed from 23 other women; 15 had thawed blastocysts replaced, and 8 subsequently became pregnant. The pregnancy of one patient in each group aborted; both patients were over 40 years of age. It is estimated that by maintaining the current policy of replacing three fresh embryos and freezing any remaining embryos when they reach blastocyst stage, the total incidence of pregnancy would increase by 3%.

Treatment of idiopathic infertility, cervical mucus hostility, and male infertility: artificial insemination with husband's semen or in vitro fertilization?

Couples with male infertility (n = 86), idiopathic infertility (n = 68), and cervical mucus hostility (n = 48) of a long duration were treated either by in vitro fertilization (IVF) or artificial insemination with husband's semen (AIH). The incidence of pregnancy per cycle in couples in whom the male partner was infertile was significantly (P less than 0.01) higher after IVF, compared with AIH (21% versus 5%, respectively). The differences were most apparent in couples with asthenospermia (47% versus 0%, respectively); no significant difference was found when the infertility was caused by oligospermia only (11% versus 9%, respectively). More patients with idiopathic infertility became pregnant after one cycle with IVF, compared with AIH (20% versus 8%, respectively); but, because of intragroup disparity in size, this difference was not significant. A highly significant difference (P less than 0.01) was found after one attempt with IVF, compared with AIH, in patients with cervical mucus hostility (38% versus 3%, respectively).

Prolonged storage of human spermatozoa at room temperature or in a refrigerator.

Spermatozoa from patients who underwent in vitro fertilization (IVF) therapy were prepared free from seminal plasma with the use of IVF culture medium supplemented with 8% human serum. Samples were then stored either at room temperature or in a refrigerator, and their motility and ability to penetrate zona-free hamster eggs were assessed every second day. With storage at room temperature, motility declined by less than half over the first 14 days, with some samples still active 20 days after preparation. The ability of the samples to penetrate hamster eggs was unchanged during the first 6 days of storage, and most of the samples still had positive tests after 14 days. Some spermatozoa could still penetrate after 17 days. Room-temperature-stored spermatozoa were still able to fertilize human oocytes 5 days after preparation. With storage in a refrigerator motility declined rapidly, and few sperm were motile after 14 days. However, these samples penetrated relatively more hamster eggs after 14 days' storage than room-temperature-stored samples did. Spermatozoa stored overnight in a refrigerator had significantly higher hamster egg penetration rates than spermatozoa stored overnight at room temperature. After storage overnight at room temperature, false-positive and false-negative results of hamster egg penetration tests were common in relation to IVF outcome; after refrigerated storage, no false-negative results were found.

Pregnancies following the frozen storage of expanding human blastocysts.

Human blastocysts were frozen in Earle's solution containing pyruvate and human serum, using glycerol as cryoprotectant, and stored in liquid nitrogen. Thawed blastocysts were replaced in 11 patients, which resulted in two pregnancies. One blastocyst giving a pregnancy was hatching when replaced. Three parameters appeared to be important for embryo survival and implantation: the interval between ovulation and replacement of the thawed blastocysts, satisfactory embryonic development before freezing, and the stage of blastulation when cooling began.

Cytogenetic and blood group studies of sheep/goat chimaeras.

Aggregation chimaeras were composed of quarter (or 1 cell) contributions from 4-cell blastocysts of sheep or goats, or of an 8-cell blastocyst of one species enveloped in three 8-cell blastocysts of the other. Gestation was in sheep or goat recipient females. Of the 10 living animals born, 3 were identified as interspecific chimaeras by body conformation and coat type among the 7 quarter/quarter aggregations and 1 among the 3 giant aggregates. Interspecific chimaerism was identified by cytogenetic study of umbilicus and blood lymphocytes respectively of 2 of these, one from each type of aggregate. Intraspecific sex chimaerism was found in 3 other animals; 2 were of giant aggregate origin, but the 1 of quarter/quarter origin must have acquired it by placental anastomosis with a twin conceptus. Tests using species-specific monoclonal antibodies and electrophoretic separation of haemoglobins and isoenzymes demonstrated sheep and goat erythrocytes in one giant aggregate chimaera; their relative proportions and those of the blood lymphocytes changed over a period of 31 months from approximately 60% goat and 40% sheep to more than 90% sheep. The plasma transferrins and amylases did not show similar relative changes from their predominantly goat-like character and, by implication, neither did their tissues of origin.

In vitro fertilization using cryopreserved donor semen in cases where both partners are infertile.

The incidence of pregnancy after in vitro fertilization (IVF) was studied in a group of 38 couples (55 cycles) where both partners were infertile. Cryopreserved donor semen (IVF-D) was used in all cycles. Results were compared with those in a control group of couples where the husband's semen was considered normal and only the wife was infertile. No significant differences were found between the IVF-D and control groups in the incidence of fertilization (80% versus 72%), pregnancy per cycle (33% versus 29%), and abortion (18% versus 20%), despite the considerably lower percentage of motile spermatozoa in the IVF-D group. Forty percent of patients, each treated unsuccessfully with at least 12 artificial inseminations with donor semen, became pregnant after one or two IVF-D cycles. It is concluded that IVF with frozen donor semen is a beneficial treatment for couples where both partners are infertile.

Treatment of male infertility by in vitro fertilization: factors affecting fertilization and pregnancy.

IVF was attempted in more than 100 couples where the man was considered to be the cause of the infertility. 57% of all oocytes were fertilized and embryo replacement was achieved in 60-70% of cases. Pregnancies following IVF were established in cases with a long duration of infertility and different male factors such as oligospermia, teratospermia, asthenospermia and auto-immunity. Almost 50% of men with very low numbers of active spermatozoa (0.5 X 10(6)/ml motile spermatozoa) were able to establish pregnancy. No correlation was found between the percentage motility and the chance of fertilization in cases with abnormal semen, but a reduction of the incidence of fertilization was noticed in cases with extreme oligospermia (5 X 10(6)/ml) where 41% of the oocytes were fertilized. The results of the post coital hamster egg tests were inaccurate in predicting the outcome of IVF. The collection of split ejaculates, and the careful preparation of spermatozoa using sedimentation and layering methods proved to be beneficial, improving sperm motility and raising the chance of fertilization.

Resistance to Haemonchus contortus transferred between genetically histocompatible sheep by immune lymphocytes.

The pattern of faecal egg counts after infection with 10,000 Haemonchus contortus larvae was similar within but not between, four pairs of monozygous twin sheep. Transfer of whole lymph or washed lymph cells from three immune donor sheep to their identical co-twin recipients reduced the susceptibility of the recipients to challenge with 10,000 larvae as measured by faecal egg counts. Cells from a nonimmune donor did not have this effect. In a final experiment, washed cells from an immune triplet sheep, which was actively responding to a repeated challenge infection, transferred a secondary local IgA response to a second recipient triplet and resulted in a marked reduction in worm count when compared with that of a third infectivity control triplet.

The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization.

The zona-free hamster egg test was carried out using spermatozoa from 15 men which consistently failed to fertilize their wives' oocytes in vitro. Spermatozoa from nine of these men fertilized hamster eggs in vitro, indicating that positive results in this assay are an unreliable guide to human in vitro fertilization. Donor spermatozoa were needed to fertilize the wife's oocytes in three of these cases. Nevertheless, the proportion of hamster egg penetration was significantly lower compared with spermatozoa from 15 men who could fertilize their wives' oocytes in vitro. The hamster assay also failed to indicate the establishment of pregnancy.

Factors influencing the success of in vitro fertilization for alleviating human infertility.

The program for in vitro fertilization at Bourn Hall began in October 1980. Various types of infertility have been treated during this time using the natural menstrual cycle or stimulation of follicular growth with antiestrogens and gonadotrophins. Follicular growth and maturation are assayed by urinary estrogens and LH, monitored regularly during the later follicular stage. Many patients had an endogenous LH surge; others needed an injection of HCG to induce ovulation. All oocytes were recovered by laparoscopy. Wide variations occurred in the time interval between the start of the LH surge and oocyte recovery and between oocyte recovery and insemination. Embryos taken between the one- and the eight-cell stage were replaced into their mother, no standard procedure being adopted for all patients. The results of all treatments including patient's responses during the follicular and luteal phases, oocyte recovery, fertilization, cleavage, replacement, implantation, abortion, and birth and the effect of factors such as replacing two or more embryos, maternal age, and previous obstetric history are described in detail. The incidence of implantation after embryo replacement improved from 16.5% initially to 30% currently. More than 118 babies have been born, and many pregnancies are continuing.

Interspecific chimaerism between sheep and goat.

In rodents, chimaeric blastocysts produced by combining embryonic cells of two different species have been used in investigations of cell lineage and interaction during development (Mus musculus-Rattus norvegicus, M. musculus-Clethrionomys glareolus, M. musculus-Mus caroli). However, interspecific chimaerism also offers new approaches to the study of reproductive incompatibilities between species and may even allow such incompatibilities to be neutralized, thus improving the chances of successful hybridization and interspecific embryo transplantation. We report here the production of sheep-goat chimaeras by embryo manipulation and the use of interspecific chimaerism to allow successful interspecific embryo transplantation in sheep and goats.

Experimental chimaerism in sheep.

Composite sheep embryos (N = 110) were produced by aggregation of blastomeres from 2-, 4- or 8-cell embryos. Each composite embryo consisted of equal numbers of blastomeres from 2-8 parent embryos, the total cell number ranging from one quarter of the normal cell number to 8 times the normal cell number. The embryos were embedded in agar and transferred to ligated sheep oviducts to allow development up to the early blastocyst stage. Of the 101 embryos subsequently recovered, 77 had formed normally organized blastocysts and 74 of these were transferred to 51 recipients. Thirty-eight recipients went to full term, producing a total of 53 lambs. Of the 48 lambs which survived to be blood typed at 60 days of age, 36 were judged to be chimaeric on the basis of their blood type and/or on the basis of external features. The proportion of chimaeras was larger amongst the lambs produced from composite embryos of the normal number of cells or more (25/26) than amongst lambs produced from composite embryos of less than the normal cell number (11/22).