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.

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 centre's 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 patient's 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.

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.

Numerical chromosome anomalies detected in paternally derived pronuclei of tripronuclear zygotes after intracytoplasmic sperm injection.

OBJECTIVE: To investigate after intracytoplasmic sperm injection (ICSI) the paternal-derived pronuclei of zygotes with three pronuclei (3PN) for numerical-chromosome anomalies by using fluorescence in situ hybridization. DESIGN: A total of 211 ICSI 3PN zygotes have been analyzed for numerical-chromosome anomalies in paternally derived pronuclei and compared with the group of 82 zygotes originated during IVF. In the ICSI group, 163 zygotes were evaluated for numerical-chromosome anomalies by using DNA probes for chromosomes 18, X, and Y, and 48 zygotes, for chromosomes 21, X, and Y. In the IVF group, 68 zygotes were evaluated for numerical-chromosome anomalies by using probes for chromosomes 18, X, and Y, and 14 zygotes, by using chromosomes 21, X, and Y. SETTING AND PATIENT(S): Tripronuclear zygotes were obtained from 74 and 176 patients participating in IVF and ICSI treatment cycles at a university hospital in Switzerland. INTERVENTION(S): To evaluate the frequency of numerical-chromosome anomalies in different populations of infertile patients, a total of 211 ICSI zygotes were divided into three groups of zygotes from men with oligozoospermia (n = 124), severe oligozoospermia (n = 53), and azoospermia (n = 34). MAIN OUTCOME MEASURE(S): Incidence of sex-chromosome aneuploidy, diploidy, and aneuploidy for chromosomes 18 or 21. RESULT(S): Overall incidence of numerical-chromosome anomalies in paternal-derived pronuclei after ICSI (9.5%) was significantly higher than the rate found in paternal-derived pronuclei of IVF zygotes (1.2%). Among ICSI zygotes, sex-chromosome aneuploidy (5.2%) and diploidy (2.8%) were two dominant numerical anomalies in paternal-derived pronuclei. In contrast, aneuploidy for autosomes 18 or 21 was not significantly different when comparing ICSI with IVF zygotes. Regarding different groups of infertile patients, the highest incidence of numerical-chromosome anomalies was found in zygotes originating from men with severe oligozoospermia (13.2%), followed by those originating from men with azoospermia (8.8%) and oligozoospermia (8.1%). CONCLUSION(S): Sex-chromosome aneuploidy and diploidy were the most frequent numerical-chromosome anomalies found in paternal pronuclei of ICSI 3PN zygotes. Surprisingly, no statistically significant difference in the incidence of numerical-chromosome anomalies was observed in the three groups of pronuclei derived from men with oligozoospermia, severe oligozoospermia, and azoospermia.

Preimplantation diagnosis in Switzerland--birth of a healthy child after polar body biopsy.

In Switzerland preimplantation genetic diagnosis is limited by law to polar body biopsy (PBB). The indications for PBB include unexplained recurrent miscarriage and improvement of the outcome of in vitro fertilisation (IVF) cycles in women at an advanced reproductive age. In this article we report the first birth of a healthy child after polar body biopsy in Switzerland in a case of unexplained miscarriage after IVF.

Do sperm cells remember?

Spontaneous alternation behavior (SAB) is the universal tendency of animals, including unicellular organisms, to alternate directional choices at consecutive left/right branchings while traversing a maze. Occurrence of SAB implies short-term memory, as a current decision is statistically dependent on previous ones. We developed a procedure to assess SAB in human spermatozoa. A total of 1302 progressively motile spermatozoa from healthy donors were observed as they entered one of two mazes, both fabricated by eximer laser ablation. The control maze was a simple T-maze (width=depth=20 microm, distance between entrance and free choice T-intersection=600 microm). The experimental maze was identical to the control maze except for a forced right-turn 600 microm before the T-intersection. We recorded individual sperm cells' left/right decisions at the T-intersections in both mazes. Of the 714 spermatozoa entering the control maze, 49.1% turned to the left (not significantly different from the chance expectation of 50.0%). Of the 588 spermatozoa entering the experimental maze, 58.6% turned left after the initial forced right turn (significant SAB; P=0.041, Wilcoxon). The statistical dependency of a directional decision on a previous one suggests a physiological 'memory' in human spermatozoa. Among the possible underlying mechanisms are refractory processes in structures responsible for flagellar beating, a postulation which deserves further scrutiny with video-monitored single-cell testing.