Ervin Macas

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 oocytes 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.

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.

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

AbstractPurpose: 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.

Vitrification of human single pronuclear oocytes following two approaches to polar body biopsy

The purpose of the present study was to investigate whether the size of the opening in the zona pellucida (ZP) of human single pronuclear (1PN) oocytes made by laser and partial zona dissection (PZD) techniques might interfere with the survival and subsequent development to blastocyst stage upon vitrification and warming. Moreover, the viability of these blastocysts was evaluated by comparing their total cell number (TCN) to the TCN of blastocysts developed from control non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using laser-assisted and PZD techniques, respectively. The size of ZP opening made by laser and PZD techniques did not interfere with survival (94.8% and 95.4%) or development to the blastocyst stage (27.8% and 26.1%). However, the TCN of laser-derived blastocysts was significantly lower than the TCN of blastocysts developed from non-vitrified control 1PN oocytes (48.7 ± 3.4 versus 70.8 ± 7.1, P < 0.028). The vitrification protocol used here is thus revealed to be an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the viability of blastocysts developed from laser-treated 1PN oocytes seems to be negatively affected by this method of biopsy.

High survival and developmental rates of vitrified mouse zygotes following polar body biopsy

The effect of two different methods of polar body biopsy followed by vitrification on the survival and development to blastocyst stage of mouse zygotes was examined. Prior to vitrification, a total of 119 and 124 mouse zygotes were subjected to polar body biopsy using either laser-assisted or partial zona dissection (PZD) techniques, respectively. Vitrification was also applied to 122 zona-intact zygotes that served as a control group. Following vitrification, no differences in the rate of zygote survival (95.8%, 91.9% and 94.3%) or in the rate of development to expanded blastocyst stage (82.3%, 79.8% and 82.0%) were observed between the two groups of biopsied zygotes, or between the biopsied zygotes and control zygotes. The mean total number of cells comprising the blastocysts of controls (77.1 +/- 4.7) was comparable to the mean cell number recorded in the laser (66.4 +/- 4.7) and PZD (69.7 +/- 5.3) groups. Blastocysts developed from laser-treated zygotes hatched much earlier than blastocysts developed from the control and PZD groups of zygotes (P < 0.001). The data obtained in the present study demonstrate that, irrespective of the biopsy method used prior to vitrification, mouse zygotes survive and develop to blastocysts upon warming in proportions similar to those of non-biopsied zygotes.

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

PurposeOur 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. abResults 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.ConclusionMosaicism 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.

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

AbstractPurpose: 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 ≥ 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.

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 1.5M dimethyl sulfoxide, (b) slow cooling and fast thawing in 1.5M propanediol (PROH), and (c) ultrarapid freezing and fast thawing in either 3.5M DMSO or 3.0M 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<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<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<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 twocell mouse embryos after freezing and thawing. There is evidence that this is also true of human embryo cryopreservation.

Polar body biopsy for Curschmann-Steinert disease and successful pregnancy following embryo vitrification

This report describes the first successful case of preimplantation genetic diagnosis (PGD) for myotonic dystrophy type Curschmann-Steinert (DM1) using polar body biopsy with vitrification. A 39-year-old woman with expansion of a CTG trinucleotide repeat in the DMPK gene was included into the study centres PGD programme. After intracytoplasmic sperm injection, a total of 13 fertilized oocytes were successfully biopsied for the first and second polar body. Nested multiplex polymerase chain reaction was used to amplify the CTG repeat region in DMPK along with two linked polymorphic markers. Six pronuclear stage (PN) oocytes were diagnosed as unaffected and four as affected by the CTG expansion, while analysis of the remaining PN oocytes was inconclusive. Three normal PN oocytes were left in culture to develop to cleavage-stage embryos and the remaining three were vitrified by applying the Cryotop method. On the following day, only one embryo was transferred into the patients uterus and the remaining two were vitrified because of the progressive threat of ovarian hyperstimulation syndrome. Since the fresh cycle did not result in a pregnancy, 6 months later the two vitrified cleavage-stage embryos were warmed and transferred back to the patient. A clinical pregnancy was established and a healthy boy was born following Caesarean section in week 39 of gestation.