In vitro Fertilization Unit, Department of Obstetrics and Gynecology, Hadassah University Hospital, Ein Kerem, PO Box 12000, Jerusalem 91120, Israel
1 To whom correspondence should be addressed. e-mail: revel{at}md.huji.ac.il
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Abstract |
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Key words: embryo cryopreservation/endometrial carcinoma/fertility preservation/in vitro maturation/polycystic ovary syndrome
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Introduction |
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In vitro maturation (IVM) of human oocytes is an emerging assisted reproductive technology with great promise (Russell, 1998; Trounson et al., 1998
; Tan and Child, 2002
). This procedure was found to have the greatest success in PCOS patients. To be successful, this process must entail both nuclear and cytoplasmic maturation. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk and intra-oocyte kinasephosphatase interactions (Tsafriri et al., 1983
). Advances in immature oocyte isolation and oocyte and embryo culture conditions have increased the clinical feasibility of IVM (Cha et al., 2000
). IVM for 23 days of cumulus-enclosed oocytes, retrieved transvaginally, is routinely performed in some IVF units and yields high quality embryos (Mikkelsen and Lindenberg, 2001
).
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Case report |
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Our plan was to aspirate oocytes from the ovaries removed by laparotomy. In order to ensure available oocytes by IVM, we administered one i.m. injection of HCG (10 000 IU, Teva, Israel) 36 h prior to surgery. In accordance with the IVM protocol followed (Mikkelsen et al., 1999), no priming by FSH was used. TAH and BSO and staging were performed under combined anaesthesia through a longitudinal incision. No post-operative complications were observed. Neither frozen sections nor paraffin-embedded slides detected any myometrial invasion or metastatic disease. Accordingly, the patient was staged as having IA disease. No adjuvant therapy was required.
The ovaries were immediately transferred in phosphate-buffered saline (PBS) at 37°C to the IVF laboratory. Oocyte aspiration was performed using a 17 gauge needle (Figure 1). Seventeen oocytes were retrieved and immediately transferred to P1 medium (Irvine Scientific, Santa Ana, CA) containing 10% synthetic serum supplement (SSS) (Irvine Scientific). P1 medium supplemented with 0.075 IU/ml Gonal F (Serono, Herzliya, Israel), 0.5 IU/ml HCG (Teva) and 10% maternal serum was used for IVM.
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Table I describes the maturation of oocytes within 42 h of retrieval. Of the 17 oocytes collected, two were at the M2 (mature oocyte) stage and ICSI was performed, but no fertilization was observed. The remaining 15 oocytes were at the germinal vesicle (GV) stage and therefore underwent IVM. The following morning, three of them had matured to the M2 stage and, following ICSI with fresh sperm, we observed a 2PN and a 1PN zygote.
These zygotes were cryopreserved using a standard protocol (Testart et al., 1987). On day 2, 10 of the GV oocytes developed further, seven to M2 and three to the germinal vesicle breakdown (GVBD) stage. This enabled another ICSI attempt which was followed by the cryopreservation of an additional six cleavage stage embryos (Table I).
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Discussion |
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We report here the possibility of aspirating oocytes from ovaries that were removed by laparotomy and TAH and BSO. Furthermore, we were able to achieve IVM in 14 of 17 (82%) oocytes and to obtain eight (47%) embryos (two at the pronuclear and six at the cleavage stage) from the 17 oocytes retrieved. A high number of retrieved oocytes has indeed been reported in PCOS patients (Child et al., 2001).
To the best of our knowledge, the possibility of obtaining normal embryo development from oocytes retrieved from an unstimulated surgically removed ovary has not been reported previously. Nevertheless, many factors affect the chances of obtaining a pregnancy in this case. Most noteworthy is this patients age. However, the large number of follicles observed may be a better predictor than patient age (Nahum et al., 2001).
IVM studies in cows have indicated that morphological characteristics such as the appearance of cumulus cells, oocyte size and the time of polar body extrusion are related to the ability of oocytes to fertilize and develop to viable embryos (Younis et al., 1989). Both estradiol (Eroglu, 1993
) and FSH (Singh et al., 1993
) have been shown to delay GVBD. We therefore perform micromanipulation of GVBD oocytes and obtain pregnancies following this approach, showing that extraction of the second polar body could occur after ICSI.
The number of blastomeres of each embryo was noted. Since day 3 embryos of 4 cells can achieve pregnancies, we froze cleaving embryos containing 310 cells, although only one was an 8-cell embryo.
Embryos were graded before freezing according to the blastomere symmetry, the presence of anucleate fragments and the extent to which they filled the embryos content (Simon et al., 1998). We obtained embryos scored B (irregularity and asymmetry of the blastomeres but no fragmentation) and C (regular or irregular blastomeres with <20% of their surface filled with anucleate fragments).
Two of the eight embryos were observed to contain one pronucleus (1PN) at fertilization. 1PN embryos have been shown to be the result of normal fertilization in 7075% of cases (Staessen and Van Steirteghem, 1997), the remainder resulting from parthenogenetic activation. Moreover, 1PN embryos can develop to blastocysts (14.3%) (Plachot and Crozet, 1992
) and enable pregnancy and birth of healthy children (Gras and Trounson, 1999
). We thus decided to cryopreserve 1PN embryos. The quality and limitation of such embryos were discussed with the patient. This patient required no adjuvant chemotherapy and recovered well from surgery. She and her husband are currently coordinating embryo transfer to a surrogate gestational carrier. We believe that she has a fair chance of obtaining a successful pregnancy from the cryopreserved embryos.
While acknowledging the limitations of this approach in this 43-year-old patient, we offer this presentation to submit the possibility that IVF units interested in fertility preservation should obtain experience in IVM in order to be able to offer fertility preservation in patients in need of oophorectomy.
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References |
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Submitted on March 15, 2003; accepted on March 10, 2004.
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