Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI.

The potential risk of transmitting chromosomally abnormal spermatozoa from infertile males into oocytes through intracytoplasmic sperm injection (ICSI) has prompted us to investigate the male pronuclei of tripronuclear zygotes (3PN) obtained after ICSI. To specify the type of anomalies, we used triple colour fluorescent in-situ hybridization (FISH) with three specific probes for chromosomes X, Y and 18. From a total of 163 paternal complements of ICSI-3PN zygotes, 90 (55.2%) had Y-chromosome signals. Eighty-three of these were normal, four had the disomy XY and three were diploid. In the remaining 73 ICSI-3PN zygotes without Y-chromosome signals, the origin of paternal pronuclei was extrapolated through chromosome constitution of the first polar body. Five anomalies were found in this group of zygotes, giving a total rate of numerical chromosome aberrations for fertilizing spermatozoa of 7.4%. In contrast to ICSI, only two disomies (1.5%) were found in the control group of IVF-3PN zygotes. Compared with the incidence of chromosome anomalies between paternal-derived pronuclei of ICSI- and IVF-3PN zygotes, the difference was statistically significant (P < 0.025). This study provides the first direct evidence of a higher incidence of numerical chromosome anomalies in sperm-fertilized human oocytes after ICSI.

MHC-genotype of progeny influenced by parental infection.

In a previous series of in vitro fertilization experiments with mice we found non-random combination of major histocompatibility complex (MHC) haplotypes in the very early embryos. Our results suggested that two selection mechanisms were operating: (i) the eggs selected specific sperm; and (ii) the second meiotic division in the eggs was influenced by the type of sperm that entered the egg. Furthermore, the proportion of MHC-heterozygous embryos varied over time, suggesting that non-random fertilization was dependent on an external factor that changed over time. As a higher frequency of heterozygous individuals correlated with an uncontrolled epidemic by MHV (mouse hepatitis virus), we suggested that MHV-infection might have influenced the outcome of fertilization. Here, we present an experiment that tests this hypothesis. We infected randomly chosen mice with MHV and sham-infected control mice five days before pairing. We recovered the two-cell embryos from the oviduct, cultured them until the blastocyst stage, and determined the genotype of each resulting blastocyst by polymerase chain reaction. We found the pattern that we expected from our previous experiments: virus-infected mice produced more MHC-heterozygous embryos than sham-infected ones. This suggests that parents are able to promote specific combinations of MHC-haplotypes during fertilization according to the presence or absence of a viral infection.

Impairment of the developmental potential of frozen-thawed human zygotes obtained after intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the effects of cryopreservation on the survival, cleavage, and morphology of embryos and on the implantation and embryonic loss rates of human zygotes obtained after ICSI compared with frozen-thawed zygotes obtained after traditional IVF. A further objective was to evaluate the same parameters in nonfrozen sibling ICSI and IVF zygotes and to compare them with corresponding frozen-thawed zygotes. DESIGN: Open, retrospective, comparative study. SETTING: University-associated assisted reproductive program. PATIENT(S): Couples with severe male factor infertility and couples undergoing IVF during the same period. INTERVENTION(S): A cohort of 408 ICSI zygotes and 299 IVF zygotes was frozen in 1,2 propanediol and sucrose using a slow-freezing protocol. Both groups of zygotes were frozen at approximately the same time after microassisted or conventional insemination. One hundred and eighty-seven ICSI and 110 IVF frozen zygotes were rapidly thawed during 44 ICSI cycles and 24 IVF cycles. Zygotes that appeared to have survived were cultured for 24 hours, and most of these embryos that were morphologically normal were transferred into patients. MAIN OUTCOME MEASURE(S): Survival rate (morphologically intact after thawing), cleavage rate and morphology of embryos, implantation rate, and the incidence of embryonic losses. RESULT(S): Except for survival rates, for which both ICSI and IVF frozen-thawed zygotes showed similar and relatively high values (87.7% and 89.1%), the outcomes of other parameters evaluated were significantly different. Thus, from a total of 128 ICSI and 68 IVF embryos transferred, 14 (10.9%) and 17 (25.0%) implanted in 44 ICSI and 24 IVF frozen-thawed cycles, respectively. This difference in implantation corresponded with the rate of cleavage and morphology of the replaced embryos; the embryos that developed from frozen-thawed IVF zygotes cleaved faster and were more regular compared to the frozen-thawed ICSI zygotes. The embryonic loss rate was 57.1% for cryopreserved ICSI zygotes and 11.8% for IVF zygotes. On the other hand, no difference in cleavage pattern, embryo morphology, implantation, and embryonic loss rates was found between nonfrozen sibling ICSI and IVF zygotes. CONCLUSION(S): The zygotes arising from ICSI cycles survived cryopreservation at a rate similar to IVF zygotes, but their ability to implant and develop further was probably affected by the cryopreservation procedure. The timing of zygote freezing was considered to be the principal reason for the lower developmental potential of frozen-thawed ICSI zygotes in the present study.

Nuclear maturity and oocyte morphology after stimulation with highly purified follicle stimulating hormone compared to human menopausal gonadotrophin.

Several studies have shown that high concentrations of luteinizing hormone (LH) in the follicular phase of stimulation can have a negative effect on oocyte quality, pregnancy rate and incidence of miscarriage. The aim of the present study was to examine the effects of highly purified follicle stimulating/hormone (FSH HP) on ovarian stimulation and particularly on nuclear maturity and morphological appearance of the oocyte in intracytoplasmic sperm injection (ICSI) therapy and to compare the results with human menopausal gonadotrophin (HMG) stimulation. For this purpose, 50 patients for ICSI (HMG: 30; FSH HP: 20) and 26 patients for in-vitro fertilization (IVF; HMG: 14, FSH HP: 12) were stimulated with either HMG or FSH HP using a short-term protocol. Patients were divided into the two groups according to the first letter of their family name. No differences were observed among the groups in relation to patient age, duration of stimulation, number of aspirated oocytes or maturity of the oocyte-cumulus complex. After removal of the cumulus-corona cells in the ICSI oocytes, a significantly higher proportion of oocytes in the FSH HP group were nuclear mature (metaphase II) than in the HMG group (FSH HP: 88.8%, HMG: 80.6%; P = 0.009). Furthermore, in the FSH HP group, significantly fewer oocytes with dark cytoplasm were observed (FSH HP: 14.4%, HMG: 22.4%; P = 0.02). Fertilization, cleavage and pregnancy rates (FSH HP 38%, HMG: 34% per retrieval) were comparable in both groups. Based on the results obtained, it can be concluded that the short-term FSH HP treatment protocol synchronizes oocyte maturation better than comparable stimulation with HMG.

The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection.

Implementation of intracytoplasmic sperm injection (ICSI) in human in-vitro fertilization (IVF) has highlighted the need for information about the risk of nuclear spindle damage caused by this procedure. For this purpose we studied the final products of oocyte meiosis at the first cleavage division of multipronuclear zygotes arising after ICSI, and compared the results with abnormally fertilized oocytes after conventional in-vitro insemination. Of 37 successfully analysed tripronuclear zygotes, 18 had three individual metaphases. Abnormal complements of 11 zygotes in this group indicated that non-disjunction occurred predominantly at the second meiotic division of the oocytes. Nine of the 37 tripronuclear zygotes exhibited two individual metaphases. Seven were abnormal and there were some indications that non-disjunction took place during oocyte meiosis. Of the 37 tripronuclear zygotes, 10 had a single metaphase and three showed an aneuploid number of chromosomes. The overall rate of aneuploidy among tripronuclear microinjected zygotes was 56.7%. In addition, seven zygotes with more than three pronuclei arising after ICSI displayed severely depleted chromosome complements. The incidence of non-disjunction in oocytes fertilized by conventional in-vitro insemination was significantly lower (20.0%, P < 0.01), since only four zygotes had an aneuploid number of chromosomes. Our findings suggest that ICSI might interfere with regular chromosome segregation at the second meiotic division of the oocytes.

Non-random fertilization in mice correlates with the MHC and something else.

One evolutionary explanation for the success of sexual reproduction assumes that sex is an advantage in the coevolutionary arms race between pathogens and hosts. Accordingly, an important criterion in mate choice and maternal selection thereafter could be the allelic specificity at polymorphic loci involved in parasite-host interactions, e.g. the MHC (major histocompatibility complex). The MHC has been found to influence mate choice and selective abortions in mice and humans. However, it could also influence the fertilization process itself, i.e. (i) the oocyte's choice for the fertilizing sperm, and (ii) the outcome of the second meiotic division after the sperm has entered the egg. We tested both hypotheses in an in vitro fertilization experiment with two inbred mouse strains congenic for their MHC. The genotypes of the resulting blastocysts were determined by polymerase chain reaction. We found nonrandom MHC combinations in the blastocysts which may result from both possible choice mechanisms. The outcome changed significantly over time, indicating that a choice for MHC combinations during fertilization may be influenced by one or several external factors.

Effect of a programmed short-term stimulation protocol on the replacement of cryopreserved embryos.

PURPOSE: Through problems in the management of embryo thawings and transfers, we investigated in a comparative study the use of the natural cycle and of a programmed short-term stimulation protocol as preparation method for the replacement of cryopreserved embryos. To date, 117 embryos have been thawed, and 60 (51%) survived with > or = 50% intact blastomeres. METHODS: In 31 cases, replacement of the frozen-thawed embryos was planned in the natural cycle (group A) and in nine cases in the stimulated cycle (group B). In group A, 16 replacements could be performed (cancellation rate 48%). Six transfers took place on weekends (37%). Two clinical pregnancies could be established (13% per replacement). In group B, there were no canceled cycles (p < 0.025). All thawings and transfers could be conducted between Monday and Friday. In one case, the single one frozen embryo was degenerated after thawing. RESULTS: Thus, eight replacements could be performed which resulted in three clinical pregnancies (38% per replacement, n.s.) and one biochemical pregnancy. CONCLUSIONS: Based on our results, we conclude that a programmed short-term protocol not only provides viable embryos with a good cryopreservation potential, but it is also a reliable preparation method for the replacement of frozen-thawed embryos.

Identification of nitric oxide synthase in human and bovine oviduct.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs. NOS activity has also been localized in the reproductive tract, although direct evidence for its presence in the human or bovine oviduct is still lacking. In the present study, four different techniques were used to identify the presence of NOS activity in human (n = 11) and bovine (n = 9) oviduct: (i) conversion of [3H]-L-arginine to [3H]-L-citrulline; (ii) production of nitrite/nitrate (NO2/NO3; stable NO metabolites); (iii) identification of NADPH-diaphorase activity; and (iv) immunostaining with antiserum to endothelial NOS. Cytosolic extracts from human ampullary segments of the Fallopian tube, obtained from post-partum patients (n = 4), converted [3H]-L-arginine to [3H]-L-citrulline (21.0 +/- 8.8 fmol/mg protein/min). This conversion rate was significantly (P < 0.05) reduced in the presence of either EDTA or N-monomethyl-L-arginine monoacetate (L-NMMA), an inhibitor of NOS activity. When bovine (n = 3) ampullary segments were incubated for 36 h in Hanks' balanced salt solution, the concentration of NO2/NO3 in the medium was increased (P < 0.05) if segments were pretreated with lipopolysaccharide (LPS; an inducer of inducible NOS), but not after treatment with LPS + L-NMMA. Additionally, epithelial cells cultured from ampullary segments showed positive staining both for NADPH-diaphorase activity and with antiserum to endothelial NOS. The results of the present study provide direct evidence for the presence of both the Ca(2+)-dependent constitutive form of NOS, as well as the inducible form of NOS activity in human and bovine oviduct. Since the oviduct plays a key role in the reproductive process, it is possible that the two forms of NOS may be involved in the physiological regulation of oviduct function.

Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure.

PURPOSE: Fertilization of an egg by injection of a single spermatozoon into the cytoplasm has been shown to be an effective procedure to obtain a pregnancy in human in vitro fertilization. This, as one of the most invasive micromanipulation techniques, has generated concern about inducing embryo abnormalities. The objective of this study was to obtain insight into the chromosomal constitution of zygotes proceeded after the intracytoplasmic sperm injection procedure. METHODS: For this purpose the first cleavage division of 33 single- and 16 multipronucleated zygotes proceeded after the intracytoplasmic sperm injection procedure was cytogenetically analyzed. RESULTS: Chromosome spreading permitted adequate karyotyping in 28 (84.8%) single-pronucleated zygotes. Among these, 19 (67.9%) were haploid, 5 (17.9%) hypohaploid (n = 20-22), and 4 (14.3%) hyperhaploid (n = 24-25). The overall rate of aneuploidy found here for single-pronucleated zygotes was 32.1%. In the 23 (82.1%) analyzed single-pronucleated zygotes, besides single mitotic complements, an intact sperm head or sperm nuclei structure has been found, indicating the maternal origin of these chromosomes. CONCLUSIONS: Ten digynic zygotes with three pronuclei and six zygotes with more than three pronuclei were identified after injecting a single spermatozoon, representing 3.2% of all the fertilized oocytes. Aneuploid chromosome complements were found in three of seven tripronuclear zygotes and each one exhibited a hypo-, hyper-, and haploid complement (23,X, 21,X,-A,-B, 25,X, +A,+B; 23,Y, 22,X,-D, 24,X,+D; 23,X, 22,X,-G, 24,X,+G). The absolute difference in the number of chromosomes between each of these two imbalanced corresponding haplotypes was the same and this difference was caused by the chromosomes belonging to the same groups of karyotype. Of the remaining four tripronuclear zygotes, three had triploid and one had diploid numbers of chromosomes. Furthermore, five zygotes having more than three pronuclei at the first cleavage division displayed severely depleted chromosome complements. The majority of imbalanced multipronuclear zygotes was found after delay of the microinjection, suggesting that aging of oocytes might be the reason for their abnormal chromosomal arrangements.

[Intracytoplasmic versus subzonal spermatozoa injection. A comparison of 2 different methods of micro-assisted fertilization]. / Intrazytoplasmatische (ICSI) versus subzonale Spermatozoeninjektion (SUZI). Ein Vergleich von zwei verschiedenen Methoden der mikroassistierten Fertilisierung.

In order to define the usefulness of subzonal sperm insertion (SUZI) and intracytoplasmic sperm injection (ICSI), we studied in a comparative trial 46 consecutive treatment cycles of microassisted fertilisation by SUZI and ICSI. By ICSI 9% of the oocytes in metaphase II were damaged in 26 treatment cycles, by SUZI, however, not one single egg in 20 cycles (p < 0.001). Fertilisation rate after ICSI (65%) was, particularly in cases with combined sperm defects as well, significantly higher than after SUZI (35%; p < 0.001). Additionally, after ICSI a higher transfer (100% vs. 75%; p < 0.05) and pregnancy rate could be obtained (38% vs. 10% per cycle; p < or = 0.05). At the moment, 3 healthy children are born (2 after SUZI, 1 after ICSI), 4 patients are in the 2nd and 3rd trimenon, respectively, the remaining 5 patients aborted. In conclusion, the ICSI technique yields better results than SUZI, especially in cases of very severe male subfertility.

Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity.

Endogenous nitric oxide (NO) is an important functional mediator in several physiological systems, including the reproductive system. However, when generated in excessive amounts for long periods, mainly during immunological reactions, NO is cytotoxic and cytostatic for invading microbes, as well as for the cells generating it and the tissues present around it. Since infertility associated with urogenital tract infection in males and females is also accompanied by reduced sperm motility and viability, it is possible that reduced fertility in these patients is due to NO-induced sperm toxicity. We therefore evaluated the direct effects of NO, chemically derived from S-nitroso-N-acetylpenicillamine (SNAP, 0.012-0.6 mM) and sodium nitroprusside (SNP, 0.25-2.5 mM), on the motility and viability of human spermatozoa. Furthermore, we tested whether inhibition of NO synthesis prevents sperm motility and viability by incubating washed total cells present in the semen (spermatozoa, round cells) with N-nitro-L-arginine-methyl-ester (L-NAME), a NO synthesis inhibitor. Treatment of purified spermatozoa with SNAP or SNP decreased forward progressive sperm motility and straight line velocity, and also increased the percentage of immotile spermatozoa in a concentration-dependent manner. Furthermore, the percentage of immotile spermatozoa positively correlated with the percentage of dead spermatozoa. In contrast to freshly prepared SNAP, SNAP preincubated for 48 h had no effect on the motility and viability of the spermatozoa. Furthermore, as compared to untreated controls, a significantly higher percentage of forward progressive sperm motility as well as viability (P < 0.05) was maintained in washed semen incubated with L-NAME (0.15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating nitrite/nitrate levels increase with follicular development: indirect evidence for estradiol mediated NO release.

Ovarian dysfunction as well as impaired release of endothelium-derived nitric oxide (NO) is associated with increased incidence of cardiovascular disease in postmenopausal women. Estrogen replacement therapy in postmenopausal women has a cardioprotective effect. Hence, using follicular development (FD) phase of the menstrual cycle of normal women (n = 7) and of patients undergoing in vitro fertilization (n = 16), we tested whether ovarian/sex hormones modulate NO-release. Levels of nitrite/nitrate (stable metabolites of NO), 17 beta-estradiol (E), progesterone (P), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured in serum collected during FD-phase. Serum nitrite/nitrate levels increased (p < .01) with the normal, as well as hormonally-induced FD and correlated with increases in serum E levels (r2 = 0.67; p < .01), but not with P, FSH and LH (p > .05). Post-ovulatory increase in P and LH levels decreased serum nitrite/nitrate levels (p < .05), even in presence of high E levels. In conclusion, nitrite/nitrate increase with follicular development, an effect that is potentially mediated by E induced NO release.

Endothelin production by bovine oviduct epithelial cells.

The production of immunoreactive-endothelin by bovine oviduct epithelial cells in culture was studied. The minimum amount of endothelin detected in the supernatant of cultured bovine oviduct epithelial cells after 6 h of incubation was 97.3 +/- 10 pg endothelin mg-1 protein and the amounts increased significantly (P < 0.01) with time of incubation. Pretreatment of bovine oviduct epithelial cells with the calcium ionophore A23187 increased the production of endothelin (P < 0.05). These results provide the first evidence that bovine oviduct epithelial cells produce endothelin and, therefore, may be locally involved in the muscular oviduct wall contractility and play an important role in the transport of gametes and embryos.

Endogenous nitric oxide modulates endothelin-1 induced contraction of bovine oviduct.

We recently reported that oviduct epithelial cells in culture produce endothelin (ET) and postulated that ET could play an important role in the contraction of the oviduct. In the present study using an organ chamber myograph system, we evaluated the contractile effects of ET-1 on bovine oviduct ampullary-isthmus segments. Additionally, the possibility whether the basal release/synthesis of nitric-oxide (N(O)) and/or prostacyclin modulates the contractile effect of ET-1 was investigated. ET-1 (10(-10) to 10(-7) M) contracted oviduct rings in a concentration dependent manner (P < 0.05). ET-1 (10(-7) M) induced contractions were significantly enhanced in presence of nitric oxide synthase inhibitor N-nitro-L-arginine-methyl-ester (10(-4) M), but remained unaltered in the presence of indomethacin (10(-5) M), an inhibitor for prostacyclin synthesis. In conclusion, ET-1 induced contraction of the oviduct is reduced/modulated by endogenous basal release/synthesis of N(O).

[Micro-insemination--a novel treatment method for sterility in severe male subfertility]. / Mikroinsemination--eine neue Sterilitätsbehandlungsmethode bei schwerer männlicher Subfertilität.

Microinsemination is a new method of treating sterility. It improves the chance of fertilization in cases of severe male subfertility and of unexplained infertility. Fertilization is assisted by opening the zona pellucida with a microneedle (PZD), by introducing a small number of sperms with a micropipette into the perivitelline space (SUZI) or by direct insertion of a spermatozoon into the ooplasm (ICSI). We report on the first delivery achieved by microinsemination (SUZI) at the University Hospital Zurich and, as far as we know, in Switzerland.

Chromosomal constitution of mouse blastocysts derived from oocytes inseminated by multiple sperm insertion into the perivitelline space.

PURPOSE: Our purpose was to evaluate the rate of chromosomal aberrations in mouse blastocysts obtained after microinjection of multiple spermatozoa under the zona pellucida of mature oocytes. Without detecting the appearance of pronuclei, the microinjected mouse oocytes containing two polar bodies were cultivated to the blastocyst stage and then analyzed cytogenetically. RESULTS: A chromosome study was carried out in a total of 109 blastocysts derived after microinjection of motile spermatozoa into the perivitelline space. Fifty-five blastocysts (50.5%) exhibited normal diploid chromosome complements, 30 (27.5%) showed different forms of mosaicism, and 24 (22%) exhibited haploidy caused by parthenogenetic activation. Compared to in vivo and in vitro control groups there was a significant increase in the parthenogenesis and mosaic forms of embryos produced by micromanipulation (P < 0.001). A total of 360 well-spread metaphases of 103 blastocysts was analyzed to determine whether the micromanipulation procedure increased the chance of aneuploidy. Aneuploid numbers of chromosomes were absent in all the metaphases analyzed. CONCLUSION: Mosaicism and parthenogenesis appear to be increased significantly following microinjection of multiple spermatozoa under the zona pellucida of mouse oocytes, and there was no evidence of aneuploidy.

[In-vitro fertilization--progress in a method of sterility treatment]. / In-vitro-Fertilisation--Fortschritte einer Sterilitätsbehandlungsmethode.

UNLABELLED: The first IVF-baby (in-vitro fertilization) was born in England in 1978 and was greeted enthusiastically. But, despite the enormous effort invested, pregnancy rates were disappointingly low. Although in recent years the clinical pregnancy rate has increased worldwide to 15-20% per retrieval, a vast number of people remained unaware of this improvement. To demonstrate and analyze the progress of IVF we evaluated all 196 IVF treatments effected from 1988-1991 at Zürich University Hospital. With an initial clinical pregnancy rate of only 6% per retrieval in 1988, the rate improved to 35% (p < 0.025) in 1991. The crucial factors relating to this improvement were the 1990 introduction of the programmed short-term protocol for ovarian stimulation and reevaluation of the previously used embryo freezing technique. CONCLUSION: After a difficult start, IVF has become, if accurately indicated, a method of sterility treatment with a realistic pregnancy and delivery prospect.

[Programmed stimulation for in vitro fertilization]. / Programmierte Stimulation für die in-vitro-Fertilisation.

A programmed stimulation protocol for in vitro fertilisation was evaluated in order to perform oocytes aspiration from Tuesday to Friday only. For this purpose, norethisterone acetate (Primolut-Nor) was given in a daily dose of 10 mg for 10-25 days, starting on the 2nd day of the preceding cycle and ending on a Monday. The stimulation was started on the following Friday by administering daily 0.1 mg D-Trp-6-LHRH s. c. (Decapeptyl). In group A (preliminary study) the treatment with HMG 150 IU/d (Pergonal) was initiated two days later. In group B the treatment with HMG was individualised with respect to the initial dose and the starting point. 10 ultrasonographic, needle-guided, transvaginal aspirations were performed from January to March 1990 in Group A, 29 aspirations from April to December 1990 in Group B. Indication for IVF in all patients was tubal infertility. All aspirations were possible from Tuesday to Friday. In Group A 5 of 15 cycles (33%) had to be cancelled. A total of 53 oocytes (5.3 +/- 2.3 per aspiration) were retrieved. Cleavage per oocyte took place in 62%. There was no pregnancy. In Group B, the cancellation rate could be reduced to 3% (1/30 cycles; p less than 0.025). 227 oocytes were recovered (7.8 +/- 3.1 per aspiration; p less than 0.05) and there was a higher cleavage rate (74%, n.s.). The clinical pregnancy rate (with allowance for progressive introduction of freezing) increased to 30% per stimulation cycle (9/30, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental capacities of two-cell mouse embryos frozen by three methods.

The following three methods were evaluated in order to obtain a most efficient freezing protocol for the preservation of two-cell mouse embryos: (a) slow cooling and slow thawing in 1.5 M dimethyl sulfoxide, (b) slow cooling and fast thawing in 1.5 M propanediol (PROH), and (c) ultrarapid freezing and fast thawing in either 3.5 M DMSO or 3.0 M PROH. In the slow-cooling procedures (a and b) ice nucleation (seeding) was induced manually or automatically. With method a, only a slight difference, 51.8% for manual and 58.9% for automatic seeding, was observed in survival rates, while the development to blastocysts was significantly affected: 35.4% with manual and less than 10% with automatic induction (P less than 0.001). Method b gave high survival (86.2%) and developmental rates (69.0%) with manual seeding compared with automatic seeding (20.7 and 9.8%, respectively; P less than 0.001). Using protocol c, higher survival and developmental rates were obtained with DMSO (84.8 and 55.9%) than with PROH (39.8 and 19.4%, P less than 0.001). These results demonstrate that inducing nucleation manually is superior to the use of a highly sophisticated autoseeding system and that method b with manual seeding is most effective in preserving the developmental capacity of two-cell mouse embryos after freezing and thawing. There is evidence that this is also true of human embryo cryopreservation.