Human Reproduction, Vol. 16, No. 7, 1469-1472,
July 2001
© 2001 European Society of Human Reproduction and Embryology
Pregnancy after cytoplasmic transfer in a couple suffering from idiopathic infertility: Case report
1 Centre for Reproductive Biology, Clinica Villa Del Sole, Via Manzoni, 15, 80123 Naples, 2 Dipartimento Clinico di Emergenza Ostetrica e Ginecologica e Medicina della Riproduzione, Azienda Universitaria Policlinico, Università degli Studi `Federico II', Via S. Pansini, 5, 80131 Naples, 3 Clinica Ruesch, Via Maria Cristina Di Savoia, 39, 80123 Naples and 4 Istituto di Clinica Ginecologia, II Università di Napoli, Largo Madonna delle Grazie, 80128 Naples, Italy
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Abstract |
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Couples suffering from idiopathic infertility are among the most difficult to treat in IVF cycles due to the unknown cause of failure to reproduce. This can lead to years of unsuccessful attempts to conceive and often the abandonment of treatment. In some couples, the only observable problem is the development of poor quality embryos. In this case report, we describe the successful use of cytoplasmic donation in a couple where the high level of embryo fragmentation and poor embryo development was thought to be the unique cause of failure to conceive after intracytoplasmic sperm injection (ICSI). We noted a strong reduction in embryo fragmentation and a consequent increase in embryo quality after the treatment. Transfer of four of the embryos receiving donor cytoplasm led to a pregnancy with two gestational sacs and the birth of healthy twins.
Key words: apoptosis/cytoplasmic transfer/IVF/oocyte reconstruction/preimplantation embryo
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Introduction |
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The fragmentation of human embryos is a fundamental problem in IVF clinics. The degree of fragmentation of developing embryos correlates with the pregnancy and implantation rate (Alikani et al., 1999
). A cohort of embryos from most couples usually contains a percentage of embryos with some fragmentation (Trounson and Sathananthan, 1984; Sathananthan et al., 1990); however, the selection of top quality embryos for transfer reduces the effect of this process. In cases where all of the embryos of a couple are consistently fragmented, the possibilities of the couple conceiving after IVF are low. This can mean having to recourse to oocyte donation.
A recently developed alternative to oocyte donation has been termed `cytoplasmic donation' (Cohen et al., 1997, 1998). In this technique, a fraction of cytoplasm from a donor oocyte is injected into a recipient oocyte prior to fertilization with the intracytoplasmic sperm injection (ICSI) technique. The presence of extraneous cytoplasm appears to increase the quality of the recipient oocyte to the level where the embryo produced is viable, although a measurable improvement in embryo quality does not always occur after cytoplasmic transfer. The mechanism of action of the technique is unknown.
In this case report, we describe the application of cytoplasmic donation to a couple affected by highly fragmented embryos after ICSI. The donation of cytoplasm to the couple's oocytes appeared to result in a reduction in the level of embryo fragmentation and an increase in the number of blastomeres present in the embryo on day 3. The couple had attempted ICSI 3 times without success prior to the cycle of cytoplasmic donation. With the treatment, the couple achieved a pregnancy and the birth of healthy twins.
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Case report |
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The couple began to receive assisted reproduction technology treatment in 1997 after being diagnosed as having idiopathic infertility after 7 years of unsuccessful attempts to conceive. The husband was characterized by normospermic semen characteristics, but the couple had previously attempted artificial insemination 3 times without success. For these reasons, the couple was referred to our IVF clinic in 1998. The couple attended the clinic a total of four times between 12/1998 and 11/1999. The maternal age in 11/1999 was 32 years and paternal age 35 years. The wife received similar long protocol stimulation regimes throughout the treatment, including down-regulation of the pituitary with a GnRH agonist and ovarian stimulation with exogenous FSH (Table I
). The wife responded excellently to the ovarian stimulation regime in all cycles (Table I). We applied ICSI treatment to all cycles despite the reasonable semen characteristics (Table II), due to the unknown cause of infertility.
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In the three cycles preceding the donation of cytoplasm, the development of all of the embryos after IVF was very poor. Embryos formed in all three cycles prior to cytoplasmic donation were characterized by a low cell number (4.26 ± 1.69 cells on day 3, mean ± SD, range 2–8, median = 4, n = 34) and a high level of fragmentation (22.4 ± 9.1% on day 3, mean ± SD, range 5–40%, median = 20%, n = 34). Four embryos were transferred in each of the two cases prior to 7/99 without achieving pregnancy. In the third cycle, assisted hatching (Cohen et al., 1992
) was performed without fragment removal and four embryos transferred without success. We considered that the major factor for the failure to achieve pregnancy in this couple was the low developmental rate and the fragmentation of the embryos. The couple therefore agreed to attempt a cycle of cytoplasmic donation. The donor of cytoplasm was from a female of 25 years undergoing IVF treatment for male factor infertility. The donor consented to the donation of a total of two oocytes for the treatment. Both donor and recipient oocytes were characterized by normal metaphase-II maturation state and morphology, without cytoplasmic inclusions. Particular care was taken to utilize cytoplasm from donor oocytes considered mature in relation to the plasma membrane response to the ICSI pipette (Palermo et al., 1996). We donated cytoplasm as outlined in previous protocols (Cohen et al., 1997, 1998) to eight oocytes. A further four oocytes were fertilized by ICSI without the donation of cytoplasm. In total, six of the oocytes receiving cytoplasm and three of the controls were observed to have fertilized 16–20 h after ICSI (Table III). Embryos were cultured to day 3. Prior to transfer, controls were characterized by a low blastomere number (3.3 ± 0.6 blastomeres on day 3, mean ± SD, range = 3–4, median = 3, n = 3, Table III) and high level of blastomere fragmentation (40 ± 10% fragmentation on day 3, mean ± SD, range = 30–50%, median = 40%, n = 3, Table III), as previously observed (see above). Embryos receiving donor cytoplasm developed well with a lower mean level of fragmentation than controls (6.2 ± 1.6 blastomeres, range = 4–8, median = 6 and 14.2 ± 9.7% fragmentation, range = 5–30% on day 3, mean ± SD, median = 12.5%, n = 6, Table III). We transferred four of the embryos receiving cytoplasm from the donor (Table III). The couple achieved a pregnancy with two gestational sacs and normal fetal heart beats. The patient gave birth in September 2000 to healthy twins weighing 3200 and 2600 g respectively.
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Discussion |
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TopAbstractIntroductionCase reportDiscussionAcknowledgementsReferences |
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In this case report, we describe the application of cytoplasmic donation to the oocytes of a couple attending the IVF clinic for idiopathic infertility with a history of poorly developing, fragmented embryos. The application of the technique appeared in the present case to reduce embryo fragmentation and increase preimplantation development, and resulted in the live birth of healthy twins. The success of the technique, where oocyte donation appeared to be the only alternative, suggests that that the technique may be a powerful alternative to oocyte donation. The technique eliminates many of the controversies surrounding oocyte donation, although it introduces new controversies (Robertson, 1999
).
Idiopathic infertility is often characterized by poor embryo quality including high levels of embryo fragmentation and slow preimplantation development. The routine use of assisted hatching and fragment removal, although successful, often fails in patients severly affected by embryo fragmentation combined with poor embryo development (Cohen et al., 1992; Alikani et al., 1993). In these cases, cytoplasmic donation appears to be a powerful alternative to the only other option presently available, donation of oocytes. It has been suggested that fragmentation of developing embryos occurs due to the activation of apoptosis in individual blastomeres (Juriscova et al., 1996). It is not currently understood how this can occur, and current evidence suggests that this process is not a response to abnormalities in chromosome segregation during meiosis or mitosis (Munné et al., 1993; Harper et al., 1995; Delhanty et al., 1997; Munné and Cohen, 1998). If embryo fragmentation is an aptotic mechanism, the expression of apoptosis in developing embryos may be due to the erroneous activation of the pathway during oogenesis or spermatogenesis (De Pol et al., 1997; Brill et al., 1999) and its consequent expression in the preimplantation embryo (Brenner et al., 1997). It is not known whether low levels of fragmentation have any physiological function during preimplantation development, although hypotheses have been proposed (Antczak and Van Blerkom 1999; Edwards and Beard, 1999).
It is not currently understood how the donation of cytoplasm from one oocyte to another can raise the potential for the recipient to form embryos of sufficient quality to implant and give rise to healthy individuals. One current hypothesis is that the transfer of mitochondria from good quality oocytes to poor permits the increased production of energy, enabling the recipient oocyte to produce sufficient metabolic products for normal development (Van Blerkom et al., 1998; Wilding et al., 2001). Although this hypothesis may be partially correct, the introduction of mitochondria may not be the only mechanism of action of donated cytoplasm. A second hypothesis is that the transfer of `good' quality cytoplasm inhibits the erroneous fragmentation by introducing inhibitory factors (proteins or RNAs). mRNAs of proteins involved in apoptosis have been found in human oocytes and embryos (Brenner et al., 1997). However, the high level of functional redundancy in the induction of apoptosis (White, 1996; Rao and White, 1997) suggests that elucidation of the function of these proteins in preimplantation human embryos will not be an easy task.
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This work was funded by grants from Ipsen, Italy to G.De Placido and Serono Pharma to B.Dale and G.De Placido. We also thank the Fondazione Nuovi Orizzonti, Naples, Italy for financial support. Vincenzo Monfrecola provided invaluable technical support.
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5 To whom all correspondence should be addressed. E-mail: dale{at}interfree.it
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Submitted on May 30, 2000; accepted on March 14, 2001.
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