Article Preliminary study of embryo development following assessment of male and female gametes

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1 RBMOnline - Vol 16 No Reproductive BioMedicine Online; on web 14 April 2008 Article Preliminary study of embryo development following assessment of male and female gametes H Nadir Çiray obtained his MD (1987) and Histology and Embryology (1990) degree from Ankara University. He received his PhD (1995) from Uppsala University, Sweden under the supervision of the late Professor Ulf Ulmsten. During his PhD studies, he employed electrophysiology and dye-coupling techniques for studying human myometrial gap junctional communication. His interest led him join the IVF team in Uppsala afterwards. He joined the German Hospital and Bahceci Clinic in Istanbul in 1999 as Director of the Embryology Laboratory and became an Associate Professor in His research interest focuses on identification of the best embryo to implant. Dr H Nadir Çiray H Nadir Çiray 1, Onder Coban, Asina Bayram, Ali Kizilkanat, Mustafa Bahçeci Assisted Conception Unit, German Hospital Istanbul and Bahçeci Women Health Care Centre, Siraselviler Cd. 119, 80860, Istanbul, Turkey 1 Correspondence: Tel: ; Fax: ; nadirc@superonline.com Abstract Gamete characteristics are likely to contribute to embryo development. In this preliminary study, early parameters were assessed in embryos generated following microinjection of hyaluronan-bound and unbound spermatozoa into 176 sibling oocytes, which had themselves been assessed using a polarizing light microscope imaging system. The fertilization and early cleavage rates, and day 3 embryo quality did not differ when oocytes displaying similar characteristics were inseminated with either bound or unbound sperm. Regardless of the binding characteristics of the sperm, early embryos displaying good and poor quality derived from oocytes displaying visible spindles and similar characteristics. These results indicate that early embryo developmental characteristics were independent from the hyaluronic acid binding capacity of the sperm when oocytes displayed a visible spindle. Keywords: embryo development, gamete selection, oocyte competence, sperm hyaluronan-binding, spindle visualization Introduction Embryos result from the fusion of gametes and therefore possess qualities that are reflections of the united oocyte and spermatozoon. The current strategy of assisted reproduction technology is to select the best from a cohort of embryos rather than selecting high quality gametes before insemination to form embryos with high implantation potential. The reason for not being able to select high quality gametes before insemination is inadequate parameters for their identification. Gamete quality is an undefined term that should directly correspond to the characteristics of sperm and oocyte contributing to the embryo s implantation capacity. Most probably, these characteristics are inherited and depend on the molecular, genetic and epigenetic state of the gametes (Shen et al., 1999; Barri et al., 2005; Emery and Carrell, 2006; Zini and Libman, 2006a,b) and are affected by environmental factors (Jarow, 1998; Shi et al., 2001; Belcheva et al., 2004) or ovulation induction treatment (Van Blerkom, 2000). While molecular methods have been developed for improving gamete selection (Patrizio et al., 2007), novel systems are being introduced to recognize gamete properties that remain undetected by conventional methods. For example, polarization light microscopy allows the properties of the zona pellucida and the meiotic spindle of the oocyte to be visualized (Keefe et al., 2003). These structures have been reported to play a vital role in fertilization and embryo development, and thus are critical to overall oocyte viability (Rienzi et al., 2004; Shen et al., 2006; Rama Raju et al., 2007). In spermatozoa, hyaluronic acid (HA) binding has been reported to aid identification of mature sperm with low frequency of aneuploidy (Huszar et al., 2006, 2007), and HA-coated dishes to select sperm for microinjection are now commercially available. The conclusion that these methods can be used to identify Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 gametes of high quality has been reached by assessing the endproduct, which is the embryo. However, none of the studies assessed embryo development when selection was performed on both gametes. Considering the interaction between gametes during embryo development (Ashwood-Smith and Edwards, 1996), it can be postulated that better embryo development is to be obtained when both gametes display characteristics of high quality. The aim of the present study was to test this hypothesis by assessing the early stages of the development of embryos generated from gametes with high quality characteristics assessed using commercially available systems based on hyaluronic-acid binding (spermatozoa) and polarization light microscopy (oocytes). Materials and methods Patient preparation This preliminary study was conducted for 1 month (March 2007). The design of the study was such that cycles in which there were sufficient gametes for the experiment were included. First cycle attempts with a minimum of 12 mature (MII) oocytes and a sperm concentration of /ml were therefore included. The patients were stimulated with either agonist (three cycles) or antagonist (seven cycles) protocols, as described elsewhere (Bahceci et al., 2005), and follicular aspiration was performed 36 h after administration of human chorionic gonadotrophin (Profasi; Serono, Switzerland). Starting from the day of transfer, the luteal phase was supported by micronized vaginal progesterone. Preparation of oocytes Oocytes were separated from their cumulus cells 3 h after retrieval. Oocytes displaying a polar body were placed in a glass-bottomed culture dish (Willco Wells, The Netherlands) that contained a HEPES-buffered medium at 37 C. The dish was placed on the heated stage of an inverted microscope, which was equipped with the polarized light visualization system (CRi Oosight Imaging System; CRi, Woburn, MA, USA). The microscope was set and the background was calibrated according to the manufacturer s protocol. A holding pipette was used for rotation of the oocyte in order to optimize visualization of the polar body and the meiotic spindle. The oocyte was aligned so that the first polar body was located at the periphery and the long axis of the spindle was perpendicular to the light path. The following parameters were studied as previously described (Shen et al., 2006): spindle length, spindle retardance, spindle to polar body angle, inner zona retardance, and inner zona thickness. The data were saved in the manufacturer s image processing system for later analysis. Preparation of sperm Binding of sperm to HA-coated plates (PICSI ; Biocoat Inc, Horsham, PA) has been described earlier (Jakab et al., 2005). Briefly, ejaculated sperm, washed either by gradient or swim-up technique, were placed in prehydrated hyaluronan microdots and incubated for up to 30 min at room temperature. Sperm that were bound to the centre of the dish from the head and displaying a circular tail movement were collected by the aid of a micropipette and these were described as HA-bound sperm. Sperm that swam freely in the dish were also collected and defined as unbound sperm. Embryology procedures In each cycle, oocytes were randomly divided into two groups and injected with either HA-bound or unbound sperm. Intracytoplasmic sperm injection (ICSI) was performed blindly with respect to the localization of the spindle and by positioning the polar body at 6 or 12 o clock. Embryo culture was performed as described previously (Çiray et al., 2004). Briefly, zygotes and embryos were observed in individual 20 µl drops of Quinn s Advantage Plus Medium (SAGE In-Vitro Fertilization Inc., Trumbull, CT, USA) until day 3. Fertilization was assessed between h following ICSI. Zygotes were inspected at 26 h for early cleavage and between and h for subsequent embryo development. A good quality day 3 embryo displayed more than six cells without multinucleation and less than 20% fragmentation (Van Royen et al., 1999). All transfers were performed at day 3. After transfer, the remaining good quality embryos were frozen. Outcome parameters and statistical analysis The outcome parameters of the study were fertilization rate, day-3 embryo morphology, and embryo efficiency, which was described as the sum of embryos that were transferred and frozen. Statistical evaluation was performed using Student s t-test for continuous variables and Fisher s exact test for discontinuous proportions. Results A total of 10 cycles met the study criteria. The infertility aetiology of these cycles was: polycystic ovary syndrome (four cases), tubal (three cases), unexplained (two cases), and endometriosis (one case). The demographics and outcome of these cycles are given in Table 1. As shown in Table 2, there were no significant differences between the parameters (as assessed by the visualization system) of the oocytes injected with either HA-bound or unbound sperm. Early embryo characteristics remained similar between the groups. Table 3 shows that there was no significant difference between good and poor quality embryos with respect to the attributes of their constituent gametes (ratio of HA-bound/ unbound sperm and characteristics of the oocytes as assessed by the visualization system). 876

3 Table 1. Cycle demographics and outcome of the study population. Parameter Value Mean age (years, range) 33.1 ± 4.0 (27 41) Mean infertility duration (years) 5.0 ± 2.8 Mean peak oestradiol (pg/ml) 3176 ± 1111 Mean no. of retrieved oocytes 21.7 ± 9.3 (217) Mean no. of MII oocytes 17.6 ± 4.2 (176) Spindle positive MII oocytes (% MII oocytes) 168 (95.5) Mean no. of two pronuclei oocytes 12.8 ± 4.4 (129) Spindle positive/negative two pronuclei oocytes 128/1 a No. of cleaved embryos 127 Mean no. of embryos transferred (total) 3.0 ± 0.7 (31) Mean no. of cryopreserved embryos (no. embryos; no. cycles) 6.0 ± 7.7 (60; 6) No. of clinical pregnancies (%) b 7 (70) No. of ongoing pregnancies c 7 No. of implantations (%) 13 (41.9) MII = metaphase II. a Oocyte displaying metaphase to anaphase transition was regarded as spindle negative. b Gestational sac with fetal heart beat 8 weeks after transfer. c 24 weeks gestation. Table 2. Oocyte characteristics and early parameters of embryos developed following injection of hyaluronan-bound and unbound sperm into sibling oocytes characterized by polarized light microscopy. Parameter Bound Unbound Total no. of MII oocytes No. of spindle positive MII oocytes (% MII oocytes) 91 (98) 77 (93) Mean spindle length ± SD (µm) 13.3 ± ± 2.3 Mean spindle retardance ± SD (nm) 1.7 ± ± 0.3 Mean spindle to polar body angle ± SD 12.3 ± ± 18.8 Mean inner zona retardance ± SD (nm) 2.2 ± ± 0.9 Mean inner zona thickness ± SD (µm) 7.7 ± ± 1.9 No. of two pronuclei oocytes (% MII oocytes) 66 (71) 62 (75) No. of early cleaved oocytes No. of zygotes displaying syngamy No. of good quality embryos a No. of embryos transferred No. transferred in positive b cycles No. of embryos frozen a Day-3 embryos which were transferred or frozen. These embryos displayed 6 blastomeres, <20% fragmentation and no multinucleation. b Cycles that resulted in clinical pregnancies. There was no significant difference between groups. MII = metaphase II. 877

4 Table 3. Characteristics of the gametes resulting in good (i.e. transferred/frozen) and poor (i.e. discarded) quality embryos, as assessed by HA-binding and polarizing light microscopy. Parameter Good quality Poor quality embryos embryos No. of embryos HA-bound/unbound sperm 45/46 20/16 No. spindle positive/negative 91/0 36/0 Mean spindle length ± SD (µm) 13.3 ± ± 2.2 Mean spindle retardance ± SD (nm) 1.7 ± ± 0.4 Mean spindle to polar body angle ± SD 11.0 ± ± 20.6 Mean inner zona retardance ± SD (nm) 2.2 ± ± 0.6 Mean inner zona thickness ± SD (µm) 7.5 ± ± Discussion As far as is known, the present report is the first study to evaluate the early development of human embryos originating from gametes that had been assessed prior to use using two novel commercial systems: PICSI (Biocoat Inc., Horsham, PA) for sperm and Oosight (CRI, Woburn, MA, USA) for oocytes. Previous studies have shown that these systems individually identified gametes of high quality. Oocytes with a visible spindle (Wang et al., 2001; Cooke et al., 2003), high spindle retardance (Shen et al., 2006; Rama Raju et al., 2007) and longer spindle (Rama Raju et al., 2007) progressed to good quality embryos. Furthermore, thickness and retardance of the inner zona of the oocyte have been reported as predictors of good subsequent embryonic development and have been associated with conception cycles (Shen et al., 2005; Montag et al., 2008). For the male gamete, binding to HA has been shown to correlate with normal frequency of aneuploidy (Jakab et al., 2005) and cellular maturity (Huszar et al., 2006). The clinical and genetic significance of utilizing HA-selected sperm for ICSI has been described (Huszar et al., 2007). In these studies, only one gamete was assessed and the interaction of the corresponding gamete was disregarded. Because of this, the impact of gamete quality on the resulting embryo may not have been accurately estimated. An example such an interaction is the repair capacity of the oocyte to compensate for sperm damage (Ashwood-Smith and Edwards, 1996). Accordingly, full term pregnancies have been reported in ICSI cycles despite high levels of sperm chromatin damage (Gandini et al., 2004). The repair capacity of the oocyte could be predicted by assessing the retardance of the inner zona layer as this parameter has been shown to correlate with oocyte competence (Shen et al., 2005; Montag et al., 2008). Conversely, successful pregnancies can possibly be obtained with oocytes possessing low repair capacity when sperm chromatin damage is minimal, which can be selected by HA-binding ability (Jakab et al., 2005; Huszar et al., 2006, 2007). The findings of the present study showed that early parameters did not differ between embryos generated from HA-bound or unbound sperm. This finding is in accordance to that of a recent study (Lin et al., 2007) in which sperm chromatin structure assays (SCSA) did not correlate with fertilization rates and early embryo parameters (HA binding targets sperm possessing similar biochemical properties to those reacting to the SCSA; Cayli et al., 2003). Similarly, in contrast to findings of some studies in which a threshold has been demonstrated for outcome (Virro et al., 2004; Payne et al., 2005), others reported that SCSA correlated poorly with fertilization rate and embryo quality (Larson-Cook et al., 2003) and there was no relationship between fertilization rate and DNA fragmentation (Morris et al., 2002). The benefits of utilizing the HA-bound sperm may be observed at later stages of human embryonic development. It has been proposed that factors predisposing an embryo to develop normally or arrest were largely determined at the 1-cell stage (Hardy et al., 2001). Paternal factors have been shown to affect embryos as early as the first cycle following fertilization (Tesarik et al., 2002; Marchetti et al., 2004). Early paternal contribution has been suggested to arise from the weak transcriptional activity in the human male pronucleus to promote nucleolar development or from epigenetic factors (Tesarik et al., 2002). Yet, these paternal factors may not affect HA-binding ability or may not be reflected in early developmental parameters, but subsequently, after completion of major activation of the human embryonic genome at the 8-cell stage (Braude et al., 1988) or in early and/or late pregnancy. Although there was an insignificant trend towards reduced missed abortion rates when sperm DNA stainability was increased in IVF cycles, it has been suggested that ICSI compensated for the impairment of sperm chromatin integrity, especially when only a morphologically normal sperm was selected for injection (Lin et al., 2007). In accordance, all clinical pregnancies obtained after ICSI in the present preliminary study continued until 26 weeks, indicating that the potential paternal effect of unbound sperm on late gestation could be minimal.

5 Another possible explanation for observing similar embryo development regardless of HA binding could be the oocyte factor. It has been reported that oocytes with a visible spindle yielded a higher fertilization rate and better embryo development compared to those in which a spindle was not visualized (Shen et al., 2006). The authors findings in a separate set of experiments confirmed this finding (data not shown). It is noteworthy to state that a spindle was visualized in the vast majority of oocytes in the present study and all embryos derived from spindle-positive oocytes. Therefore, as reflected in the clinical outcome, the assessed population in the present study may represent cycles with good prognosis. Furthermore, owing to the design of the study, cycles with at least 12 oocytes (oocytes from each cycle were divided in to two groups for injection with HA-bound and unbound sperm) and sperm concentration of >5 x 10 6 /ml (required for collecting the HA-bound and unbound sperm) were included, which improved the cycle characteristics. Whether the retardance of the inner zona correlates to oocyte competence (Shen et al., 2005; Montag et al., 2008) remains controversial according to the findings of the present study, as the mean inner zona retardance did not differ between good and poor embryos inseminated by HA-bound and unbound sperm (2.3 ± 0.9, 2.0 ± 0.9, 2.2 ± 0.6, 2.5 ± 0.7, respectively). In the present preliminary study, owing to the low number of cycles and non-homogenous transfers, the impact of gamete characteristics on the implantation potential of embryos was not examined. Further studies are needed to assess the longterm effects of gamete selection with these novel systems on human embryo development and the outcome of treatment. References Ashwood-Smith MJ, Edwards RG 1996 DNA repair by oocytes. Molecular Human Reproduction 2, Bahçeci M, Ulug U, Ben-Shlomo I et al Use of GnRH antagonist in controlled ovarian hyperstimulation for assisted conception in women with polycystic ovary disease: a randomized, prospective, pilot study. Journal of Reproductive Medicine 50, Barri PN, Vendrell JM, Martinez F et al Influence of spermatogenic profile and meiotic abnormalities on reproductive outcome of infertile patients. Reproductive BioMedicine Online 6, Belcheva A, Ivanova-Kicheva M, Tzvetkova P, Marinov M 2004 Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation. International Journal of Andrology 27, Braude P, Bolton V, Moore S 1988 Human gene expression first occurs between the four and eight-cell stages of preimplantation development. Nature 332, Cayli S, Jakab A, Ovari L et al Biochemical markers of sperm function: male fertility and sperm selection for ICSI. Reproductive BioMedicine Online 7, Çiray HN, Ulug U, Bahçeci M 2004 Transfer of early-cleaved embryos increases implantation rate in patients undergoing ovarian stimulation and ICSI-embryo transfer. Reproductive BioMedicine Online 8, Cooke S, Tyler JPP, Driscoll GL 2003 Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Human Reproduction 18, Emery BR, Carrell DT 2006 The effect of epigenetic sperm abnormalities on early embryogenesis. Asian Journal of Andrology 8, Gandini L, Lombardo F, Paoli D et al Full-term pregnancies achieved with ICSI despite high levels of chromatin damage. Human Reproduction 19, Hardy K, Spanos S, Becker D et al From cell death to embryo arrest: Mathematical models of human preimplantation embryo development. Proceedings of National Academy of Sciences 98, Huszar G, Ozkavukcu S, Jakab A et al Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertilizing potential: selection of sperm for intracytoplasmic sperm injection. Current Opinion in Obstetrics and Gynecology 18, Huszar G, Jakab A, Sakkas D et al Fertility testing and ICSI sperm selection by hyaluronic binding: clinical and genetic aspects. Reproductive BioMedicine Online 14, Jakab A, Sakkas D, Delpiano E et al Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies. Fertility and Sterility 84, Jarow JP 1998 Detection of oxidative DNA damage in human sperm and the association with cigarette smoking. Journal of Urology 159, Keefe D, Liu L, Wang W, Silva C 2003 Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reproductive BioMedicine Online 7, Larson-Cook KL, Brannian JD, Hansen KA et al Relationship between the outcomes of assisted reproduction techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertility and Sterility 80, Lin M-H, Kuo-Kuang Lee R, Li S-H et al Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertility and Sterility [Epub ahead of print Sept 26]. Marchetti F, Bishop JB, Cosentino L et al Paternally transmitted chromosomal aberrations in mouse zygotes determine their embryonic fate. Biology of Reproduction 70, Morris ID, Ilott S, Dixon L, Brison DR 2002 The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Human Reproduction 17, Montag M, Schimming T, Köster M et al Oocyte zona birefringence intensity is associated with embryonic implantation potential in ICSI cycles. Reproductive BioMedicine Online 16, Patrizio P, Fragouli E, Bianchi V et al Molecular methods for selection of the ideal oocyte. Reproductive BioMedicine Online 15, Payne JF, Raburn DJ, Couchman GM et al Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay (SCSA) and outcomes of assisted reproductive treatments. Fertility and Sterility 84, Rama Raju GA, Prakash GJ, Krishna KM, Madan K 2007 Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using Polscope imaging. Reproductive BioMedicine Online 14, Rienzi L, Martinez F, Ubaldi F et al Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Human Reproduction 18, Shen HM, Chia SE, Ong CN 1999 Evaluation of oxidative DNA damage in human sperm and its association with male infertility. Journal of Andrology 20, Shen Y, Stalf T, Mehnert C et al Light retardance by human oocyte spindle is positively related to pronuclear score after ICSI. Reproductive BioMedicine Online 12, Shen Y, Stalf T, Mehnert C et al High magnitude of light retardation by the zona pellucida is associated with conception cycles. Human Reproduction 20, Shi Q, Ko E, Barclay L, Hoang T et al Cigarette smoking and aneuploidy in human sperm. Molecular Reproduction and Development 59,

6 Tesarik J, Mendoza C, Greco E 2002 Paternal effects acting during the first cycle of human preimplantation development after ICSI. Human Reproduction 17, Van Blerkom J 2000 Intrafollicular influences on human oocyte developmental competence: perifollicular vascularity, oocyte metabolism and mitochondrial function. Human Reproduction 15 (Suppl. 2), Van Royen E, Mangelschots K, De Neubourg D et al Characterization of a top quality embryo, a step towards single embryo transfer. Human Reproduction 14, Virro MR, Larson-Cook KL, Evenson DP 2004 Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertility and Sterility 81, Wang WH, Meng L, Hackett RJ et al The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertility and Sterility 75, Zini A, Libman J 2006a Sperm DNA damage: importance in the era of assisted reproduction. Current Opinion in Urology 16, Zini A, Libman J 2006b Sperm DNA damage: clinical significance in the era of assisted reproduction. Canadian Medical Association Journal 175, Declaration: The authors report no financial or commercial conflicts of interest. Received 9 October 2007; refereed 6 November 2007; accepted 14 January