Triploid pregnancy after ICSI of frozen testicular spermatozoa into cryopreserved human oocytes: Case report

C.M. Chia1, W.B. Chan, E. Quah and L.C. Cheng

Thomson Fertility Clinic, Thomson Medical Centre, 339 Thomson Road, Singapore 307677


    Abstract
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
Although freezing oocytes is ethically more acceptable than cryopreservation of embryos, variable oocyte survival, fertilization rate and possible risk of increased ploidy after cryopreservation have precluded the widespread clinical application of oocyte cryopreservation in assisted reproduction techniques. We report a triploid pregnancy from intracytoplasmic sperm injection of recombinant FSH-stimulated frozen/thawed testicular spermatozoa into cryopreserved oocytes in a hormone replacement cycle. To our knowledge, this is the first report of a pregnancy where both gametes have been frozen. It illustrates the need for further research when applying new techniques in assisted reproduction.

Key words: cryopreservation/frozen testicular spermatozoa/human oocyte/ICSI/triploid pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
We report the first case of a triploid pregnancy resulting from intracytoplasmic sperm injection (ICSI) of recombinant FSH (rFSH)-stimulated frozen testicular spermatozoa into cryopreserved human oocytes.


    Case report
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
A 33 year old married nulliparous woman sought treatment for infertility due to obstructive azoospermia in her 36 year old husband (FSH = 6.9 IU/l). Testicular sperm extraction (TESE) was carried out with the view to IVF–ICSI. The testicular biopsied samples were minced with sterile glass slides and frozen in straws according to a previously published method (Mahedevan and Trounson, 1983). Three months later, the woman commenced treatment for IVF–ICSI. Pituitary down-regulation was achieved with mid-luteal phase s.c. buserelin (500 µg/day, Hoechst, Germany) followed by ovarian stimulation (300 IU of Metrodin HP daily, Serono, Geneva, Switzerland) for 14 days. Ovulation was induced with 10 000 IU human chorionic gonadotrophin (HCG).

Thirty-eight eggs were retrieved transvaginally from 60 follicles under ultrasound guidance and deep sedation. The eggs were prepared and underwent ICSI (Palermo et al., 1992Go). Six eggs with their zonae breached at denudation were omitted. Two straws of the testicular samples, thawed rapidly at room temperature, washed and resuspended in P1 (Irvine Scientific, Santa Ana, CA, USA) supplemented with 4 mg/ml bovine serum albumin (BSA) (Sigma A-4164, St Louis, MO, USA) were incubated in 5% CO2 for 1 h before use. As previous results of the thawed testicular spermatozoa showed low survival (11%), selection of manifestly live spermatozoa was necessary to improve the outcome of ICSI. Sperm motility, although minimal, was the only suitable criterion for viability. This proved to be a monumental task as it took 4 h to retrieve eight barely motile spermatozoa for injection of eight eggs.

In view of the large number of eggs remaining to be injected (n = 24) and the extended time taken to select viable testicular spermatozoa, a decision was made to freeze all the remaining eggs close to 12 h after egg collection. The eggs were equilibrated in 1.5 mol/l propanediol in PBS containing 20% maternal serum for 10 min at room temperature (25°C). Prior to loading into straws (UH003, i.m.v., Paris), they were transferred to the same solution in the presence of 0.2mol/l sucrose up to a maximum of three eggs in each straw. After heat sealing, the straws were frozen in a Kryo 10, series III freezer (Planar, Sunbury-on-Thames, UK) using the same programme as described for embryo freezing (Lasalle et al., 1985Go) and stored at –196°C in a liquid nitrogen storage tank.

Of the eight injected fresh eggs, two were fertilized the following day. One displayed two pronuclei while the other was abnormally fertilized. The normally fertilized egg progressed to a grade one, 4-cell stage embryo and the other to a grade one, 2-cell stage embryo on day 2. A previously published embryo scoring system was used (Dawson et al., 1993). Briefly, grade 1 embryos contained symmetrical blastomeres with no fragments, grade 2 had even blastomeres but with slight cellular debris and grade 3 had at least one degenerate cell. Embryos were assigned to grade 4 if three or more cells had completely fragmented and grade 5 if all the blastomeres were completely degenerated. On the day of egg collection and on subsequent days, the patient developed moderately severe ovarian hyperstimulation syndrome (OHSS) and the scheduled embryo transfer was cancelled. The single 4-cell embryo was frozen as described above.

Two months later, the patient was placed on a hormone replacement therapy (HRT) regime in a thaw cycle. The pituitary was down-regulated with buserelin while the endometrium was prepared with incremental doses of oestradiol valerate (Progynova; Schering AG, Germany) until adequate endometrial thickness was confirmed by ultrasound scan. Twelve eggs were thawed on the day of commencement of Cyclogest (1200 mg daily, Cox Pharmaceuticals, Barnstaple, UK) administration, using the rapid thaw method (Lasalle et al., 1985Go). Ten eggs survived and ICSI was performed on the same day with the thawed and washed testicular spermatozoa that had been incubated for 4 h with rFSH (25 IU Gonal-F; Serono) in P1 (Irvine Scientific) supplemented with 4 mg/ml BSA at 37°C in a 5% CO2 incubator (modified from Balaban et al., 1999). Overall, the grade of motility of the thawed sperm improved greatly compared to the first ICSI attempt. The 10th egg was at the germinal vesicle stage and therefore not injected. Eight eggs were fertilized, seven displaying two pronuclei and one with three pronuclei. All except one continued to cleave for the following two days and three embryos at the grade two, 4-cell stage, grade one, 7-cell stage and grade two, 8-cell stage were transferred on day 3. Serum ßHCG was 7400 IU and 31 000 IU at 4.5 and 5.5 weeks respectively. An intrauterine gestation sac, appropriate for 9 weeks gestation was confirmed by ultrasound scan (crown rump length 2.5 cm). Unfortunately, the pregnancy ended in a missed abortion. The product of conception showed a triploid karyotype XXY,t(5;6)(p?15;q?15).


    Discussion
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
None of the recent reported pregnancies resulting from ICSI of cryopreserved oocytes (including two from epididymal and testicular spermatozoa) were from frozen testicular spermatozoa (Polak et al., 1998Go; Tucker et al., 1998Go; Porcu, 1999Go). Recently it was reported (Balaban et al., 1999Go) that significantly more fresh testicular samples from non obstructive azoospermic men acquired twitching motility after 24 h incubation with rFSH at 30°C compared with testicular samples without such incubation (71.2 versus 32.9%, P = 0.001).

This is the first reported attempt to determine the benefits of incubation of frozen–thawed testicular spermatozoa with rFSH at 37°C. As the time for the identification of motile spermatozoa was considerably shortened, ICSI was carried out more efficiently in the thaw cycle. Even though the eggs were frozen 12 h after egg collection, this did not compromise the survival rate (83.3%) or fertilization rate (70%). Consequently, the unplanned freezing of the oocytes and improved motility of the frozen–thawed testicular spermatozoa have resulted in the first reported pregnancy using frozen gametes of both sexes.

With 1,2 propanediol as the cryoprotectant, some of the reported undesirable consequences of oocyte freezing include parthenogenetic activation (human, mouse), cooling of the meiotic spindle (mouse), premature zona hardening (rabbit, human) and decreased fertilization (mouse) (Schalkoff et al., 1989Go; Shaw and Trounson, 1989Go; Vincent et al., 1989Go; Imoedehme and Sigue, 1992Go; Eroglu et al., 1998Go). However, not all of these effects were observed in human oocytes. For example parthenogenetic activation by simple exposure to 1,2-propanediol was not observed (Gook et al., 1995aGo) although the cryopreservation procedure did increase the parthenogenetic activation and ploidy in human oocytes in some reports (Al-Hasani et al., 1987Go; Gook et al., 1995aGo; Kazem et al., 1995Go) but not in others (Mandelbaum et al., 1988Go). Applying ICSI to the thawed human oocytes, reasonable fertilization and blastocyst rates (50 and 43% respectively) were obtained in human oocytes after cryopreservation (Gook et al.,1995bGo) the same group also showed normal karyotypes and absence of stray chromosomes in 60% of the human oocytes that survived freezing (Gook et al., 1994Go). Recently, a report of a live birth after vitrification, thawing and ICSI of human oocytes offers a novel alternative to oocyte freezing (11 out of 17 oocytes survived), although this approach requires confirmation by a larger study (Kuleshova et al., 1999Go).

In this case report, the high serum ßHCG concentrations were consistent with a triploid pregnancy (Jauniaux et al., 1997Go). One possible explanation of how a triploid XXY pregnancy could arise from clearly 2PN zygotes with two polar bodies, is the asynchronous appearance of the third pronucleus. It could have been missed as the fertilization check was performed at only one time point, 16–18 h after injection. At the time of fertilization check, the unequal size of the two pronuclei was noted. Retrospectively, the appearance of dysmorphic pronuclei did portend the presence of chromosomal anomalies that were confirmed by the subsequent karyotype result. Increased incidence of chromosomal abnormalities in zygotes with dysmorphic pronuclei compared with zygotes with evenly sized pronuclei was previously reported (Sadowy et al., 1998Go; Manor et al., 1999Go). Interestingly, the earlier authors noted that women displaying dysmorphic pronuclei had a significantly greater average number of oocytes retrieved, as in the present case. It is unlikely that the triploidy resulted from dispermic fertilization (which would result in XXX or XYY complements) or polyspermy of two spermatozoa since only one spermatozoa was injected into each egg in the ICSI procedure. The more likely explanation for the triploidy is fertilization of a digynic egg. The presence of two polar bodies observed in the digynic egg could have arisen from fragmentation of the first polar body rather than extrusion of the second polar body.

Polyploidy can result from a number of factors, namely ageing of eggs, the cryopreservation technique and suboptimal egg quality following ICSI of human oocytes. Increased incidence of triploidy (5.7%) in aged unfertilized eggs of at least 20 h old has been reported (Morton et al., 1997Go). In contrast, polyploidy (14%) after ICSI of non-aged but cryopreserved eggs has been reported (Gook et al., 1995bGo) suggesting that cryoinjury may be a factor. While we could not exclude ageing (12 h in our case) and cryoinjury as contributory factors, we note that in our patient, a 3PN zygote was obtained even after ICSI of her fresh eggs, suggesting egg quality as a major cause of triploidy in her frozen egg.

Although the number of births recorded by this technique is still small, it is unequivocal that cryopreservation of eggs combined with the ICSI technique could offer young women undergoing chemotherapy or radiotherapy treatment a feasible option in preserving their fertility. Moreover, the freezing of eggs would also avoid the ethical and legal controversies associated with embryo cryopreservation. However, further refinement of techniques is necessary to reduce any adverse consequences of oocyte freezing. At the time of submission of this paper for publication, the patient is again pregnant with twin sacs (fetal hearts present), in the second thaw cycle from the same batch of frozen gametes.


    Acknowledgments
 
The authors would like to thank Dr K.P.Tay for performing the testicular biopsy and the nursing staff, F.Y.Lee, Z.Kamis, and L.Hung for providing excellent counselling and nursing support to the couple.


    Notes
 
1 To whom correspondence should be addressed at Thomson Fertility Clinic, Thomson Medical Centre, 339 Thomson Road, Singapore 307677. E-mail: cmchia{at}tmc-sin.com.sg Back


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Submitted on January 19, 2000; accepted on May 19, 2000.