Cytoplasmic transfer in assisted reproduction.

This report details the use of cytoplasmic transfer in human oocytes. The introduction of a small amount of ooplasm from a donor oocyte or zygote may alter the function of oocytes, with probable deficiencies. Cytoplasmic transfer from fertile donor oocytes or zygotes into compromised oocytes from patients with recurrent implantation failure after assisted reproduction has now led to the birth of nearly 30 healthy babies worldwide. Transfer of small amounts of cytoplasm probably involves mRNAs, proteins and mitochondria, as well as other factors and organelles. Even though the use of cytoplasmic transfer has been employed in several IVF clinics--and pregnancies have resulted--it is not known definitively whether the physiology of the early embryo is affected. This review outlines the experimental cytoplasmic transfer techniques and postulates the future impact in assisted reproduction.

Spontaneous and artificial changes in human ooplasmic mitochondria.

Our research has focused on promoting the development of compromised embryos by transferring presumably normal ooplasm, including mitochondria, to oocytes during intracytoplasmic insemination. Because of the enigma of mitochondrial heteroplasmy, the mixing of populations of oocyte cytoplasm has provoked considerable debate. We are currently investigating oocyte mitochondrial (mt) DNA mutations and the effects of ooplasmic transplantation on mitochondrial inheritance and mitochondrial functionality. Ageing human oocytes could accumulate mtDNA deletions, which might lead to detrimental development. Elimination of abnormal, rearranged mtDNA, such that the offspring inherit only normal mitochondria, is postulated to occur by a mtDNA 'bottleneck'. Among compromised human oocytes (n = 74) and early embryos (n = 137), investigations have shown the occurrence of deltamtDNA4977, the so-called common deletion, to be 33% among oocytes and 8% among embryos. Using a nested polymerase chain reaction (PCR) strategy of long followed by short PCR, another 23 novel mtDNA rearrangements were found: various rearrangements were present in 51% of the oocytes (n = 295) and 32% of early embryos (n = 197). The difference in the percentage of mtDNA rearrangements between oocytes and embryos was significant (P < 0.0001) and implies that there could be a process of selection as fertilized oocytes become embryos. There was no significant relationship between the percentage of human oocytes or embryos that contained mtDNA rearrangements and age. The first series of ooplasmic transfers have been performed in women with repeated implantation failure associated with slow and morphologically abnormal development of their embryos. In a total of 23 attempts in 21 women, eight healthy babies have been born and other pregnancies are ongoing. By examining the donor and recipient blood samples it is possible to distinguish differences in their mtDNA fingerprint. A small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (six out of 13), amniocytes (one out of four), placenta (two out of four), and fetal cord blood (two out of four). Ooplasmic transfer can thus result in sustained mtDNA heteroplasmy representing both the donor and recipient.

Mitochondrial DNA heteroplasmy after human ooplasmic transplantation.

OBJECTIVE: To determine the patterns of mitochondrial inheritance in embryos, fetuses, and infants after ooplasmic transplantation using the technique of mitochondrial DNA (mtDNA) fingerprinting. DESIGN: Prospective clinical study. SETTING: The IVF program at Saint Barnabas Medical Center, a nonprofit community hospital. PATIENT(S): In a total of 23 cases with recurrent implantation failure after IVF ooplasmic transplantation was performed. Thirteen embryos from two patients and amniotic cells from four patients were investigated for heteroplasmy. Placenta and fetal cord blood cells from four newborn babies/infants were also investigated. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): mtDNA fingerprinting, polymerase chain reaction, and DNA sequencing analysis. RESULT(S): In addition to the recipient maternal mitochondrial DNA, a small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (n = 6/13), amniocytes (n = 1/4), placenta (n = 2/4), and fetal cord blood (n = 2/4). Fingerprinting showed that nuclear DNA was not inherited from the donor in placenta or fetal cord blood of the babies. CONCLUSION(S): Ooplasmic transfer can result in sustained mtDNA heteroplasmy representing both donor and recipient. This was shown by mtDNA fingerprinting of embryos, amniocytes, fetal placenta, and cord blood. These results show that the donor-derived mitochondrial population persists after ooplasmic transfer and may be replicated during fetal development.

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature.

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.

Determination of Duffy genotypes in three populations of African descent using PCR and sequence-specific oligonucleotides.

The expression of the Duffy Antigen/Receptor for Chemokines (DARC) on red blood cells (RBC) has been commonly determined using hemagglutination tests. Because the vast majority of African individuals are Duffy-negative, screening for DARC expression on RBC is a valuable tool to assess Caucasian admixture in populations of African descent. Furthermore, blood group antigens have been frequently tested as potential risk factors for complex diseases. We established a dot-blotting protocol using sequence-specific oligonucleotides (SSOs) for the DARC-46T ("Duffy-positive") and -46C ("Duffy-negative") alleles. With this method, but not with serological methods, Duffy-positive individuals can be further characterized as homozygous or heterozygous for the dominant Duffy-positive allele, allowing more precise estimation of allele frequencies and admixture in heterogeneous populations. In unrelated African American (n = 235), Afro-Caribbean (n = 90) and Colombian (n = 93) subjects, the frequency of the -46T allele was 21.7%, 12.2% and 74.7%, respectively. The percentage of Duffy-positive individuals (homozygous or heterozygous for the -46T allele) in each population was in accordance with published frequencies. As expected, the -46C allele was not detected in 20 Caucasian subjects. This sensitive and specific method allows for the rapid and inexpensive screening of large samples for Duffy genotypes using small quantities of genomic DNA.

Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs.

The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved in-vitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours. Eighty-seven human blastomeres from abnormal or arrested embryos were fused with bovine oocytes in a preclinical study. Fusion efficiency was 100%. In 21 of the hybrid cells, no trace of human chromatin was found. Of the remaining 66, 64 (97%) yielded chromosomes suitable for analysis. The method was used to karyotype embryos from two patients with maternal translocations. One embryo which was judged to be karyotypically normal was replaced in the first patient, resulting in one pregnancy with a normal fetus. None of the second patient's embryos was diagnosed as normal, and hence none was transferred. The results of the present study demonstrated that the ooplasmic factors which induce and maintain metaphase in bovine oocytes can force transferred human blastomere nuclei into premature metaphase, providing the basis for a rapid method of karyotyping blastomeres from preimplantation embryos and, by implication, cells from other sources.

Genome-wide screen for atopy susceptibility alleles in the Hutterites.

A genome-wide screen for loci influencing positive skin prick tests (SPT) to airborne allergens was conducted in the Hutterites, a founder population of European ancestry. Positive SPT to 14 standardized allergens was measured in 370 subjects in our primary sample and 324 subjects in a replication sample. Evidence for linkage to positive SPT was assessed using the transmission disequilibrium test (TDT) with 337 autosomal markers (average spacing 9.13 cM, SD = 7.8 cM). Three loci showed the strongest overall evidence of linkage to atopy, with at least one allele-specific and a locus-specific p< 1 x 10(-4). This study provides evidence for at least three atopy-susceptibility loci in the Hutterites on chromosomes 1, 6 and 16.

Culture and quality control of embryos.

Improvement of embryo quality during in-vitro culture can be achieved by understanding and controlling the requirements of gametes and embryos. The most obvious route is to alter culture media, but standardization could be influenced by diverse environmental factors. Abnormal embryos from patients with multiple failures probably do not benefit from standardization and require specialized therapy, that is if their physiology is not already irreversibly jeopardized during gametogenesis. This paper describes the adverse environmental factors present in laboratory air and released by common products used by laboratories. Assays and results of the air determinations in several laboratories are reported, as well as potential counter measures. The possibility of altering the immediate environment of the nucleus of the egg by ooplasmic transplantation is also considered, and the first attempts resulting in two ongoing pregnancies are reported.

Detrimental effects of sodium during mouse oocyte cryopreservation.

Cryopreservation is an established way of storing embryos, but effective methods are not available for freezing eggs. Most freezing damage is caused by high solute concentration (solution effects) and intracellular ice. Sodium salts are the major components of cryopreservation media, and the main contributor to the solution effects. The present experiments examine the effect of substituting choline for sodium as the major extracellular cation in the cryopreservation of mouse eggs. The effects of serum and various cryoprotectants were also examined. Survival, fertilization, and development were inversely related to the concentration of sodium in the freezing medium. Oocytes frozen in a choline-based medium had the highest (p < 0.001) survival and development rates. The absence of serum during thawing inhibited fertilization, whereas exposure to serum or opening the zona allowed fertilization to reach the control level. Dimethyl sulfoxide was as effective as 1,2 propanediol for obtaining high survival and fertilization rates. These results support the hypothesis that the high concentration of sodium in conventional freezing media is detrimental to cells and show that choline is a promising replacement for sodium. Reducing or eliminating sodium may allow oocytes and other cells to be frozen more efficiently.

Genome-wide search for asthma susceptibility loci in a founder population. The Collaborative Study on the Genetics of Asthma.

Founder populations offer many advantages for mapping genetic traits, particularly complex traits that are likely to be genetically heterogeneous. To identify genes that influence asthma and asthma-associated phenotypes, we conducted a genome-wide screen in the Hutterites, a religious isolate of European ancestry. A primary sample of 361 individuals and a replication sample of 292 individuals were evaluated for asthma phenotypes according to a standardized protocol. A genome-wide screen has been completed using 292 autosomal and three X-Y pseudoautosomal markers. Using the semi-parametric likelihood ratio chi2 test and the transmission-disequilibrium test, we identified 12 markers in 10 regions that showed possible linkage to asthma or an associated phenotype (likelihood ratio P < 0.01). Markers in four regions (5q23-31, 12q15-24.1, 19q13 and 21q21) showed possible linkage in both the primary and replication samples and have also shown linkage to asthma phenotypes in other samples; two adjacent markers in one additional region (3p24.2-22) showing possible linkage is reported for the first time in the Hutterites. The results suggest that even in founder populations with a relatively small number of independent genomes, susceptibility alleles at many loci may influence asthma phenotypes and that these susceptibility alleles are likely to be common polymorphisms in the population.

Expression and cytogenetic localization of the human SM22 gene (TAGLN).

SM22 is a 22-kDa protein identified variously as SM22, transgelin, WS3-10, or mouse p27. Though its precise function is unknown, it is abundant in smooth muscle and so may contribute to the physiology of this widespread tissue. We found that cosmid 16b6 contains the entire 5.4-kb, five-exon human SM22 gene (HGMW-approved symbol, TAGLN), and we cytogenetically localized the gene to chromosome 11q23.2. Northern analysis of human adult tissues showed that SM22 mRNA is most prevalent in smooth muscle-containing tissues, but is also found at lower levels in heart. The human SM22 promoter contains nuclear factor-binding motifs known to regulate transcription in smooth muscle, and human SM22 promoter-luciferase reporter constructs exhibited high transcriptional activity in A7r5 or primary canine aortic smooth muscle cells, but show little activity in nonmuscle COS7 cells. In addition, human SM22 promoter activity increased by two- to threefold upon serum stimulation of nonmuscle cells.

Ooplasmic transfer in mature human oocytes.

Ooplasmic transplantation aimed at restoring normal growth in developmentally compromised oocytes and embryos was evaluated in seven couples (eight cycles) with multiple implantation failures. Two approaches were investigated to transfer ooplasm from donor eggs at metaphase II (MII) stage into patient MII eggs: (i) electrofusion of a ooplasmic donor fragment into each patient egg (three cycles), and (ii) direct injection of a small amount of ooplasm from a donor egg into each patient egg (five cycles). Some donor eggs were used multiple times. Donor eggs were divided into two groups, one being used for ooplasmic extraction and the other one for egg donation. Cleaved embryos resulting from the latter were cryopreserved, where numbers and satisfactory development permitted. A second control group consisted of embryos derived from patient eggs after intracytoplasmic sperm injection without ooplasmic transfer. This was performed when sufficient number of eggs were available (n = 5). Donor eggs (n = 40) were evaluated cytogenetically after micromanipulation in order to confirm the presence of chromosomes. One egg was anuclear and the recipient embryos were not transferred. Normal fertilization was significantly higher after injection of ooplasm (63%) in comparison with fusion (23%). Pronuclear anomalies appeared enhanced after fusion with ooplasts. Embryo morphology was not improved in the three cycles with electrofusion and patients did not become pregnant. An improvement in embryo morphology was noted in two patients after injection of ooplasm and both became pregnant, but one miscarried. A third pregnancy was established in the repeat patient, without obvious embryo improvement. One baby was born and the third pregnancy is ongoing with a normal karyotype. Two other patients with male factor infertility had poor embryos after ooplasmic injection, but the donor embryo controls were also poor. The patients did not become pregnant and had no donor embryos frozen. Ooplasmic transfer at the MII stage may be promising in patients with compromised embryos; however, evaluation of ooplasmic anomalies and optimization of techniques will require further investigation prior to widescale application.

HLA-G1 protein expression is not essential for fetal survival.

HLA-G is a nonclassical, class I HLA gene that is primarily expressed by fetal cells at the maternal-fetal interface and is thought to play a key role in the induction of tolerance in pregnancy. This paper reports the identification of a single base pair deletion at position 1597 (1597delC) in exon 3 (encoding the alpha2-domain) of HLA-G on 20 of 272 (7.4 per cent) African American chromosomes, three of 102 (2.9 per cent) Hispanic chromosomes, and none of 134 Caucasian chromosomes. This relatively common frameshift mutation results in amino acid substitutions in all of the residues in the second half of exon 3 including the conserved cysteine at codon 164. An adult individual was identified who was homozygous for this 'null' allele, and a first trimester placenta that was homozygous for 1597delC had no detectable HLA-G1 protein. These data indicate that expression of HLA-G1 protein is not essential for fetal survival.

Cryopreservation of activated mouse oocytes and zygote reconstitution after thaw.

A new approach to cryopreservation of unfertilized oocytes is proposed using techniques of artificial egg activation combined with nuclear transplantation. Matured mouse oocytes were released from metaphase II arrest by brief exposure to alcohol, allowed to progress to the pronuclear stage and then frozen according to a standard freezing protocol in propandiol. After thaw the female pronuclei were enucleated and fused with a male karyoplasts that were divided from in-vivo fertilized zygotes. Reconstituted zygotes, fresh and cryopreserved culture control zygotes were cultured to the blastocyst stage and transferred to pseudopregnant recipients. The rate of blastocyst formation was 75.8, 91.6 and 44.1% respectively. A total of 110, 215 and 70 blastocysts were transferred to pseudopregnant females respectively. The implantation rates were 36.4, 72.0 and 75.7% while the rates of fetal viability at mid-gestation were 15.5 (P < 0.0001), 51.1 and 37.1% respectively.

Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro. Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2 plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30 degreesC water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.

The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm.

Cytoplasts with diameters of 40-45, 50-55 and 70-75 microm, derived from mouse oocytes at the germinal vesicle, metaphase II and zygote stages were incorporated into zygotes by electrofusion. Manipulated (n = 867) and culture-control (n = 1114) embryos were cultured in vitro and transferred to pseudo-pregnant recipients at the blastocyst stage. When synchronous cytoplasts measuring 40-45 and 50-55 microm in diameter were incorporated into 138 and 86 zygotes respectively, only one embryo in each group (not significant) became arrested at the 1-cell stage. A total of 124 (89.9 compared with 91. 6% for controls) and 69 embryos (80%, P < 0.001 compared with 91.6% for controls) reached the blastocyst stage respectively. In the first group, 66 out of 106 blastocysts implanted (62.2 compared with 54.9% for controls; not significant), however, only 24 (22.6 compared with 40.2% for controls, P < 0.001) were viable in comparison with controls. There were four groups of zygotes that received metaphase II cytoplasts. In the first group, 200 zygotes were fused with 40-45 microm cytoplasts. The second group of 145 zygotes were fused with cytoplasts of the same size derived from aged oocytes. In the third and fourth groups, 38 and 36 zygotes were fused with 50-55 and 70-75 microm cytoplasts respectively. In the first two groups, none of the embryos arrested at the 1-cell stage, but in the other groups the rates were 15 out of 38 (39.5%) and 36 out of 36 (100%) respectively. These zygotes remained arrested at the pronuclear stage and contained large inflated pronuclei. The blastocyst formation rates were 183 out of 200 (91.5 compared with 91.6% for controls, not significant), 109 out of 145 (75.2% lower than controls, P < 0.05) and 14 out of 38 (39.5% lower than controls, P < 0.0001) respectively. In the first two groups 109 and 25 blastocysts were transferred, of which 76 (69.7%) and 15 (60.0%) implanted. This was higher than control embryos (54.9%, P < 0.01) for the first group and similar to controls for the second group. In the first group, 60 embryos (55%) were viable on day 10 of transfer in comparison with controls (40.2%, P < 0.05) while in the second group, 11 embryos (44.0%, not significant) were viable on day 10 of transfer. Zygotes that received germinal vesicle stage cytoplasts developed poorly and the implantation rate was significantly reduced. The present study confirms the importance of the ooplasmic domain in meiotic maturation and preimplantation development. Our results suggest that implantation may be enhanced by transfer of a small amount of metaphase II cytoplasm to the mouse embryo during the 1-cell stage; however, fusion of intact zygotes with cytoplasts > 45 microm appeared largely detrimental. The mechanisms responsible for these changes are yet unknown.

Formation of male pronuclei in partitioned human oocytes.

Ninety-five metaphase II human oocytes, aged in vitro for either one day or for two days, and five fresh immature oocytes with no visible germinal vesicle nucleus were partitioned into small cytoplasts after removal of the zona pellucida and exposure to cytochalasin B. Seventy-one metaphase II and four immature oocytes were used as intact zona-free controls. The cytoplasts derived from each partitioned oocyte and all the zona-free whole oocytes were exposed to normal or subfertile donor sperm and later assessed for signs of male pronucleus development. A total of 711 fragments (an average of 8 fragments per partitioned egg) with a mean diameter of about 50 microns were produced from the 100 partitioned oocytes. When exposed to normal sperm, 76% of the 1-day-old metaphase II fragments, 67% of the 2-day-old metaphase II fragments, and 83% of the immature oocyte fragments were fertilized. The mean number of decondensing nuclei per partitioned oocyte was 11.9 for the 1-day-old metaphase II oocytes, 6.4 for the 2-day-old metaphase II oocytes, and 13 for the immature oocytes. The mean number of decondensing nuclei per a whole zona-free oocyte was 5.6 for the 1-day-old metaphase II oocytes (p < 0.05), 6.7 for the 2-day-old metaphase II oocytes (NS), and 13 for the immature oocytes. When exposed to subfertile sperm, 54% of the 1-day-old metaphase II fragments and 28% of the 2-day-old metaphase II fragments were fertilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly effective method of human oocyte activation.

Fresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium, magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.6% and 64.1%, respectively; however, the proportion of one-pronucleate oocytes in these groups was significantly lower (61.6% and 23.5%, respectively). There was no difference in the activation efficiency between the two activation modes. Twelve activated oocytes from the freshly collected group cleaved when left in culture. It is concluded that, in the human, a brief exposure to isotonic solution of mannitol with low concentrations of calcium, magnesium and chloride is a very effective activation stimulus.