Placenta
Volume 29, Supplement 2 , Pages 115-120, October 2008

Morphological Selection of Gametes

G.EN.E.R.A. Centre for Reproductive Medicine, Valle Giulia Clinic, Via G. De Notaris 2, 00197 Rome, Italy

Accepted 5 August 2008. published online 02 September 2008.

Article Outline

Abstract 

Methods of selecting gametes before the intracytoplasmic sperm injection (ICSI) technique are of paramount importance, especially where for religious, ethical or legal reasons the production of supernumerary embryos has to be avoided. In these circumstances, in fact, the research has to be focused on the identification of early markers of embryo quality at the oocyte and sperm stage before fertilisation. Oocyte quality can be influenced by several factors such as controlled ovarian hyperstimulation protocols, pharmaceutical preparations and perifollicular vascularisation. Several intracytoplasmic and extracytoplasmic abnormalities have been described, but whether these abnormalities might be predictive of oocyte competence is controversial and the selection methods proposed are still poorly effective. Recently, we have observed that oocyte morphological abnormalities might be indicators of oocyte competence. An abnormal first polar body (but not fragmented), a large perivitelline space, increased cytoplasmic granularity, and the presence of a centrally located granular area seem to have a negative effect on the oocyte potential to fertilise, cleave, and/or develop into a viable embryo. Sperm morphology can be more accurately observed at high magnification using an inverted microscope equipped with Normarski optics (1000× magnification under mineral oil) and a digital system in order to reach a final magnification of approximately 6300×. Single sperm nuclear abnormalities based on strict selection criteria seems to have a clear negative association with ICSI outcome. The possibility of observing the spermatozoa in real time at higher magnification might also be a good opportunity to study the relationship between particular sperm defects and ICSI outcome. In addition to morphology, it has been suggested that spermatozoa selection might be performed on the basis of its biochemical ability to bind to solid hyaluronic acid. However, it must be underlined that prospective randomised studies are necessary to confirm the preliminary results regarding the efficacy of the described criteria proposed, to morphologically select gametes prior to in vitro insemination.

Keywords: Gamete selection, Oocyte selection, Spermatozoa selection, IMSI, ICSI

 

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1. Introduction 

The introduction in Italy almost 4 years ago of a very restrictive law regulating assisted reproductive technology has obliged embryologists and clinicians to change some of the standardised procedures in the management of their daily work. According to this law, no more than three oocytes can be fertilised per cycle, all the embryos obtained must be transferred, embryo cryopreservation is forbidden, and no embryo selection, based on morphologic and/or genetic evaluation, is allowed before transfer [1]. This is particularly penalising in terms of success rates, since recently many studies have shown that a combination of several different morphologic criterias, each of which has been individually shown to be predictive of embryo competence, leads to more accurate embryo selection, thus improving pregnancy rates [2], [3], [4], [5], [6], [7]. With the aim of minimising the negative effects of the restrictions imposed, we had to start focusing our research on the identification of early markers of embryo quality at the oocyte and sperm stage before fertilisation. More attention has been directed towards ovarian stimulation strategies, follicle vascularisation, and oocyte and sperm selection, since the quality of the originating gametes is of utmost importance for the developmental potential of the subsequent embryo [3].

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2. Ovarian stimulation regimens and other potential markers for follicle and oocyte selection 

Several factors affect oocyte quality, such as the age of the woman, ovarian reserve, controlled ovarian stimulation protocols and pharmaceutical preparations of human gonadotrophins. While some of these factors (i.e. age and ovarian reserve) are patient-related and cannot be modified by any intervention, the others can be modified and tailored according to the patient's characteristics.

The different stimulation protocols used for controlled ovarian hyperstimulation are based on the concept of widening the “FSH window” [8] with the use of exogenous gonadotrophins from the early follicular phase to the day of human chorionic gonadotrophin (hCG) administration in order to increase the number of follicles recruited.

Over the last 30 years, easier stimulation protocols such as clomiphene citrate (CC) alone or in combination with human menopausal gonadotrophin (hMG) and urinary FSH have been gradually replaced by more complex stimulation regimens. At the beginning of the 1990s long pituitary desensitisation protocols with gonadotrophin-releasing hormone (GnRH) agonists and gonadotrophins became and still are the most widely used treatments for ovarian stimulation. These lengthy, expensive protocols allow the activity of the IVF centres to be managed more easily, enable a lower cancellation rate, raise the number of pre-ovulatory follicles, retrieve the oocytes and obtain the good quality embryos for transfer, thus leading to better pregnancy rates [9].

However, several complications and risks are reported with the use of these long protocols; the most significant one is without any doubt the ovarian hyperstimulation syndrome (OHSS) [10].

Recently, milder stimulation protocols have been proposed [11], [12] with a reduction in the stress, discomfort, side effects and complications that can be associated with the standard ones [13]. In addition to these possible advantages, several authors have observed better oocyte and embryo quality after milder stimulation protocols, suggesting that in patients with a normal ovarian reserve, reducing the duration and intensity of the pharmacological interventions might interfere less with natural follicle selection, obtaining oocytes with more physiological chromosome segregation behaviour during meiosis and early embryo development [12], [14].

According to these observations it might be possible that the lower the stimulation, the better oocyte quality can be obtained. Natural IVF cycles could then represent a valid method of retrieving the best quality oocytes. Furthermore, reduced costs and smaller physical (no side effects, no anaesthesia and hospital stay) and emotional (less anxiety and stress) burdens are offered to the patients. However, conflicting data come from the literature regarding the efficacy of natural IVF cycles [15], [16], [17], [18]. Moreover, with mild protocols and especially with natural IVF cycles a significantly reduced number of oocytes can be retrieved, and unfortunately data regarding the cumulative pregnancy rates obtained after mild or standard regimens are not yet available. Therefore, although these results seem very interesting, further large, prospective studies are needed to confirm these clinical data.

Besides the impact of different stimulation protocols on oocyte quality, the different pharmaceutical preparations of human gonadotrophins used to induce multiple follicular growth might also play an important role. Over the last 15 years many studies have compared the efficacy and the safety of different gonadotrophin preparations (recombinant FSH vs. urinary FSH vs. highly purified FSH vs. hMG vs. highly purified hMG), reporting conflicting clinical data [19], [20], [21], [22], [23], [24], but only very few studies have examined the effect of different gonadotrophin preparations on oocyte and embryo quality, also reporting conflicting results [25], [26], [27]. This is very likely due to the difficulties in standardising some of the important factors that may influence oocyte quality: the different timing of hCG administration, the interval between hCG and oocyte retrieval, the interval between oocyte retrieval and oocyte insemination, and also some individual factors, such as ovarian reserve, FSH–FSH receptor interaction and ethnic background.

Furthermore, some factors have been suggested to be predictive of oocyte quality, and can help to identify the most competent follicle among the cohort. These are the perifollicular vascularisation and follicular fluid anti-Müllerian hormone (FF AMH) concentrations.

Intrafollicular hypoxia correlates with insufficient perifollicular vascularisation measured using colour Doppler ultrasonography [28], [29], [30]. This condition may lead to oocyte cytoplasmic defects, disorganised chromosomes, reduced fertilisation and embryos with multinucleated blastomeres [29], [31], [32], whereas oocytes aspirated from well-vascularised and oxygenated follicles, once fertilised, generate embryos with high implantation potential [31], leading to better pregnancy rates [33], [34], [35], [36]. Unfortunately, these interesting data have not been confirmed by other studies, in which the perifollicular vascularisation was correlated to oocyte competence, either in intrauterine insemination (IUI) [37] or in in vitro fertilisation (IVF) cycles [38], [39], [40], [41].

The basal level of anti-Müllerian hormone (AMH), a member of the transforming growth factor-beta superfamily produced by the granulosa cells of ovarian follicles, is considered a unique biomarker of ovarian follicular status [42], [43], [44] and of oocyte quality [45]. In particular, low day 3 levels of AMH were associated with higher frequencies of oocytes displaying dark central granulation of the cytoplasm [45]. This specific morphological abnormality is believed to arise during the early stages of oocyte maturation and to predispose the oocyte to aneuploidy [46]. Moreover, a very recent study was able to correlate the follicle's individual ability to produce AMH with oocyte quality [47]. According to these data, it can be suggested that FF AMH concentrations might be used for oocyte selection in stimulated cycles [47].

Both perifollicular vascularisation and FF AMH could indeed be important markers for selecting the more competent oocytes, but concerns may arise regarding some of the technical aspects and further well-designed, prospective, randomised studies are needed to confirm the preliminary data.

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3. Morphologic criteria for oocyte selection 

Immediately after oocyte retrieval, the quality of the cumulus-corona-oocyte complex is evaluated in the laboratory. In standard IVF cycles the assessment is based on the aspect of the cumulus-corona cells, and when the oocytes are denudated before the intracytoplasmic sperm injection (ICSI) procedure, the evaluation is more accurate and is based on the morphology of the oocyte cytoplasm and on the aspect of the extracytoplasmic structures, such as the zona pellucida, the first polar body and the perivitelline space. Classically, a human metaphase II (MII) oocyte is considered “normal” when under light microscopy observation it has a round clear zona pellucida, a small perivitelline space containing a single unfragmented first polar body (I PB), and a pale, moderately granular cytoplasm that does not contain inclusions [48], [49], [50], [51], [52], [53]. Whether this “normal” oocyte is more competent than one with one or more variations of the described “ideal” morphological criteria is controversial and the selection methods proposed are still poorly effective. This is particularly true of IVF cycles in which the presence of cumulus and corona cells makes morphological oocyte evaluation difficult to perform. In fact, the quality and the degree of expansion of these cells seem to be poor markers of oocyte maturity and mostly depend on the type of ovarian stimulation protocol used [54], [55], [56]. Only the presence of blood inclusions in the cumulus matrix has been associated with reduced oocyte quality [57]. On the contrary, the morphological selection of the oocytes is more reliable in ICSI cycles in which the oocyte can be easily observed after cumulus-corona cell removal. The nuclear maturity can be assessed by visualising the presence of the I PB in the perivitelline space, although recent studies using polarised light microscopy have shown that oocytes displaying a I PB may still be immature [3], [58], [59]. Only the visualisation of the meiotic spindle of the second meiotic division in the oocyte cytoplasm allows the identification of oocytes whose nuclei are not fully mature, which have higher developmental potential [60], [61]. However, nuclear maturity alone is not enough to determine the quality and the competence of an oocyte. In fact, nuclear and cytoplasmic maturation should be coordinated and completed to ensure the optimal conditions for subsequent fertilisation, and disturbances or asynchrony of these two maturation processes might result in several oocyte morphological abnormalities [62], [63], [64].

If the nuclear oocyte maturity can be assessed by the visualisation of the I PB under light microscopy and of the meiotic spindle under polarised light microscopy, cytoplasmic maturation cannot be clearly assessed. Several variations of the “ideal” cytoplasmic aspect (pale, moderately granular, without inclusions) have been described, such as increased granularity and the presence of vacuoles, refractile bodies and smooth endoplasmic reticulum (SER) clusters, but their association with oocyte competence is unclear. Similarly, an abnormal zona pellucida, a large perivitelline space and abnormal, fragmented, or degenerated polar bodies can all be observed after oocyte denudation. Perturbations in cytoplasm organisation have been associated with both developmental failure and increased rates of aneuploidies. In particular, cytoplasmic dysmorphisms that arise early in meiotic maturation (before the MI stage) seem to predispose the oocyte to aneuploidies, while cytoplasmic dysmorphisms that occur after MI seem to affect fertilisation and embryo development [46].

However, in general, the presence of morphological abnormalities does not prevent the oocytes from being fertilised by ICSI [49], [51] and the embryo quality as well as clinical pregnancy and implantation rates have been reported to be comparable after the use of “abnormal” or “normal” appearing oocytes [51], [52]. In contrast, other studies correlated I PB morphology and some cytoplasmic variations from its “ideal” aspect with fertilisation rates and embryo quality after ICSI [58], [65], [66]. Moreover, the presence of vacuoles in the oocytes seems to significantly reduce the fertilisation rate [53], [67] and some authors suggested that oocytes with “abnormal” cytoplasmic morphology may well fertilise and develop into embryos with lower implantation potential [48], [68], [69], [70]. A very recent study [71] has focused on oocytes displaying a central aggregation of SER. This particular oocyte characteristic has been associated with lower fertilisation and blastulation rates and a higher incidence of miscarriage. Furthermore, reduced birth weight was reported when ongoing pregnancies were achieved in women with SER-positive oocytes. Thus, avoidance of the insemination of these oocytes is strongly recommended [71].

Abnormal oocyte shape (ovoid oocytes with increased perivitelline shape) has also been correlated with cleavage pattern and in particular with lower rates of blastocyst formation [72]. In this case the developmental impairment of the embryos was ascribed to reduced cell-to-cell contacts.

Finally, giant oocytes, although very rarely observed after controlled ovarian hyperstimulation, should never be inseminated because it has been shown that all embryos generated from these oocytes are chromosomally abnormal, although they may have a normal cleavage and development up to the blastocyst stage [73].

Driven by the need to identify early markers of embryo quality at the oocyte stage, before fertilisation, that represent the only option remaining in trying to minimise the negative effect of the restrictions imposed in our country, we have very recently evaluated oocyte morphologic “abnormalities” of prognostic value for further development [74]. From September 2004 to December 2005, 1191 MII oocytes from 516 couples were analysed at the Centre for Reproductive Medicine of the European Hospital. The morphological characteristics of the inseminated oocytes were evaluated at the time of ICSI and were classified as extracytoplasmic and cytoplasmic abnormalities. The most common extracytoplasmic abnormalities were fragmented I PB and large perivitelline space, which were observed in 49% and in 32% of the oocytes analysed, respectively, whereas abnormal I PB (large and/or degenerated), abnormal zona pellucida (thick and/or dark) and abnormal oocyte shape were relatively rare (4.4%, 4.3% and 1.9% of the oocytes analysed oocytes, respectively). Cytoplasmic abnormalities were observed in 62.0% of the oocytes: cytoplasmic granularity was present in 31.7%, a centrally located granular area in 5.3%, vacuoles in 3.1%, SER in 0.5%, and refractile bodies in 21.4% of the oocytes analysed. At least one selected oocyte was recorded as being morphologically “abnormal” in 92.6% of the patients involved in the present study. The fertilisation rate was significantly reduced only when the inseminated oocytes presented an abnormal I PB (large and or degenerated, while PB fragmentation was not predictive), a large perivitelline space, and vacuoles, whereas it was not affected by the other MII oocyte features. Similarly, a large perivitelline space together with cytoplasmic granularity and the presence of a centrally located granular area significantly impaired pronuclear morphology. This latter morphological feature also affected embryo quality, reducing significantly the incidence of excellent and good quality embryos. Due to the very low number of oocytes displaying SER, no relationship between embryo development and this particular aspect was found in this study [74].

The presence of a degenerated I PB in the MII oocytes may reflect an asynchrony between nuclear and cytoplasmic maturation [75], whereas a large I PB might be due to the dislocation of the meiotic spindle [25]. These conditions could explain the reduced ability of the cell to support pronuclear formation after ICSI and the poor embryo development observed.

Oocyte over-maturity at the time of ICSI may be responsible for the large perivitelline space [76], which significantly affected the fertilisation rate and pronuclear morphology. Conversely, alterations in the cytoplasmic texture may be a sign of oocyte immaturity, in particular increased cytoplasmic granularity and a centrally located granular area [76], which were associated with a poorer pronuclear stage and embryo quality.

A relative mark (weighted according to the impact that each of the following morphologic characteristics had on the fertilisation rate, pronuclear morphology and embryo quality) was given to each of the oocytes analyzed if one or more of these features were present: abnormal I PB, large perivitelline space, centrally located granular area, granular cytoplasm, and vacuoles. The oocytes that received the lowest score were expected to have the highest implantation potential.

The MII oocyte morphological score (MOMS) was correlated with the age of the woman, the basal serum FSH of the patients and the total amount of FSH administered during controlled ovarian hyperstimulation, but not with the ovarian stimulation protocol used. Similarly, it was found to be predictive of the clinical results in terms of positive hCG (P=0.04) and clinical pregnancy (P=0.05) and it was inversely correlated with the implantation potential [74].

Our study data suggest that only some oocyte morphological abnormalities at the MII stage, such as a large or degenerated I PB, a large perivitelline space, increased cytoplasmic granularity, and the presence of a centrally located granular area, might have a negative effect on the oocyte potential to fertilise, cleave, and/or develop into a viable embryo. Therefore, only these oocytes might be considered to be abnormal, whereas other MII morphologic characteristics (i.e. fragmented I PB, oval, thick, or dark zona pellucida, and refractile body) did not seem to affect the developmental potential of the deriving embryo and therefore should not be considered to be abnormal.

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4. Morphological criteria for sperm selection 

Over the last 15 years many authors have not observed any correlation between ICSI outcome and sperm sample morphology, and even with semen samples of extremely poor morphology the fertilisation and cleavage rates, as well as the clinical outcome, were comparable, with the results obtained using semen samples with better or normal sperm morphology [77], [78], [79]. These observations are biased by the selection performed by the embryologist of the best “normal-looking” motile spermatozoa prior to ICSI, which does not always reflect the quality of the spermatozoa injected. On the contrary, a clear relationship between the injected sperm morphology and fertilisation and implantation rates was found [80], suggesting chromosomal defects of the embryo that may be ascribed in these cases to the contribution of the sperm.

Unfortunately, proper individual sperm morphology is difficult to assess at low magnification and only major morphologic defects can be assessed, with minor morphologic defects being mislaid in this setting. Recently, performing selection of spermatozoa at high magnification has been proposed, using an inverted microscope equipped with Normarsky optics (1000× magnification under mineral oil) and a digital system in order to reach a final magnification of approximately 6300× [81], [82]. With this system, sperm cells with morphological defects regarding six subcellular organelles (acrosome, post-acrosomal lamina, neck, mitochondria, tail and nucleus) can be excluded from micro-insemination. This technique associated with the normal ICSI procedure is named intracytoplasmic, morphologically selected sperm injection (IMSI) [81], [82].

This selection process, which involves only motile sperm, has an average range of duration between 1.5h and 5h [83] and is based on the criteria proposed by Bartoov et al. [81].

The nucleus should have an average normal length of 4.75±0.28μm and an average normal width of 3.28±0.20μm with no regional nuclear disorders and with no more than one vacuole that occupies less than 4% of the nuclear area. Smaller, larger, narrower (<2.9μm in width), wider (>3.7μm in width) and shorter (<4.2μm in length) sperm with one large or with more than one vacuole are considered abnormal. Similarly, when the acrosome and the post-acrosomal lamina are vesiculated, the sperm are excluded from selection. The neck must not be abaxial, and it must not have cytoplasmic droplets and/or disorders, whereas the tail is considered abnormal if it is coiled, broken, multi or short, and the mitochondria must not be partial or disorganised [81], [82].

The IMSI technique was originally applied in cases of severe male factor infertility (according to WHO) and in cases of repeat IVF-ICSI failures [81], [82], but soon after the indication was extended to couples in which the female partner was younger than 40 years [83], [84] and to cases of a high value of sperm DNA fragmentation [85].

So far, only a few data are available regarding IMSI outcome. In their first study, Bartoov et al. [81] reported that in cases in which less than 20% spermatozoa have morphologically normal nuclei, assessed by the selection criteria described, no pregnancies were obtained. More recently, a significantly increased fertilisation rate was not observed when morphologically selected spermatozoa were used for ICSI [82], [85], whereas the embryo quality was significantly improved according to some [82], but not all authors [85], [86]. However, all the studies so far published [82], [84], [85], [86] reported improved implantation and clinical pregnancy rates and a reduction in the abortion rates in the population of patients treated with high-magnification sperm selection. Moreover, when it is not possible to find spermatozoa with strictly defined morphologically normal nuclei at high magnification (in about 10% of the cases treated), the fertilisation rate, the embryo quality, the implantation and the pregnancy rates are severely compromised [83], [84].

Similarly, the clinical outcomes of ICSI cycles in cases of semen samples with different degrees of sperm DNA fragmentation were significantly improved by the IMSI procedure. Even if DNA damage cannot be directly evaluated in spermatozoa using the high-magnification method, the possibility of excluding spermatozoa with intranuclear small vacuoles (that may reflect a high level of sperm DNA fragmentation) seems to help to improve the results in this particular situation [85]. It seems in fact that the most important predictive criterion of spermatozoa quality is the presence of multiple or large vacuoles [83]. This observation might be explained by animal studies [87]. It has been reported that the presence of nuclear vacuoles in bovine sperm does not affect fertilisation, but increases the rate of early embryonic death. The presence of vacuoles may reflect molecular defects responsible for abnormal chromatin remodelling during sperm maturation and might render sperm cells more vulnerable to DNA damage [88].

These preliminary data seem to demonstrate that embryos obtained from the injection of a morphologically normal sperm cell, according to the selection criteria described, have improved implantation potential.

It has also been proposed that, in addition to morphology, the selection of spermatozoa might be performed according to its ability to bind to hyaluronic acid (HA) [89]. The hypothesis behind this new technique is that only mature spermatozoa that have completed the spermiogenetic process of sperm plasma membrane remodelling, cytoplasmic extrusion and nuclear maturity are able to bind this polysaccharide that is normally present in the extracellular matrix of the cumulus oophorus surrounding the oocytes and is thus involved in the fertilisation process [90]. In agreement with this hypothesis, HA-selected sperm have been described to be less affected by DNA fragmentation [91], [92] and to display the normal range of frequencies of chromosomal disomies and diploidies, independently of the aneuploidy frequency of the initial semen [89]. To date, very few information are available on the effect of this selection procedure on ICSI outcome. One study has recently compared the standard ICSI procedure with the HA sperm selection procedure [92] in the sibling oocytes of 50 different couples. A significantly higher fertilisation rate was reported in the group with HA-selected sperm (79.4% vs. 67.7%). However, no differences were reported in the later developmental stages. We believe that further information is necessary before any conclusions regarding this new technique are drawn.

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5. Conclusions 

Methods used to select gametes before the ICSI technique is performed are of paramount importance, especially where for religious, ethical or legal reasons the production of supernumerary embryos has to be avoided. In these circumstances, in fact, the research must be focused on the identification of early markers of embryo quality at the oocyte and sperm stage before fertilisation.

Before analysing the criteria for oocyte selection, we must first bear in mind that several factors affect oocyte quality and controlled ovarian stimulation protocols constitute one of the most important ones. Recently, it has been suggested that milder stimulation approaches with reduced duration and intensity of the pharmacological interventions might interfere less with natural follicle selection, obtaining more competent oocytes. Similarly, the different pharmaceutical preparations of human gonadotrophins used to induce multiple follicular growth might also play an important role and perifollicular vascularisation may well influence oocyte quality.

Once the oocyte is retrieved, the morphological selection is more reliable in ICSI cycles where the morphology of the oocyte cytoplasm and the aspect of the extracytoplasmic structures, such as the zona pellucida, the first polar body and the perivitelline space, can be easily observed after removal of cumulus-corona cells. Several intracytoplasmic and extracytoplasmic abnormalities have been described, but whether these abnormalities might be predictive of oocyte competence is controversial and the selection methods proposed are still poorly effective. We have very recently shown that not only some cytoplasmic abnormalities, but also particular extracytoplasmic abnormalities, might be indicators of oocyte competence. In particular, some oocyte morphological abnormalities at the MII stage, such as abnormal I PB, a large perivitelline space, increased cytoplasmic granularity, and the presence of a centrally located granular area, may have a negative effect on the oocyte potential to fertilise, cleave, and/or develop into a viable embryo, whereas other MII morphologic characteristics (i.e. fragmented I PB, oval, thick, or dark zona pellucida, and refractile body) did not seem to affect the developmental potential of the deriving embryo and therefore should not be considered to be abnormal.

It must also be underlined that the presence of one single affected oocyte may reflect suboptimal follicular growth, which might impair the entire cohort [69]. Moreover, small cytoplasmic defects could be below light microscopic resolution and thus remain unnoticed during observation [71]. Because obstetric and neonatal outcomes have been negatively related to specific cytoplasmic aggregation [71], particular attention should be paid when observing the cytoplasmic texture of the oocytes.

Recently, performing selection of spermatozoa at a high magnification using an inverted microscope equipped with Normarsky optics (1000× magnification under mineral oil) and a digital system in order to reach a final magnification of approximately 6300× has been proposed. With the use of this selection system it has been shown that single sperm nuclear abnormalities based on strict selection criteria have a clearly negative association with ICSI outcome and when less than 20% of the spermatozoa have a morphologically normal nucleus there is no chance of achieving a pregnancy.

This time-consuming and expensive technique has so far only been applied in a selected population of patients with a poor prognosis due to paternal factors and characterised by repeated IVF/ICSI failures. Because the chance of picking up an abnormal sperm is higher in these cases, an additional sperm selection procedure is thus recommended. The HA selection procedure could also be combined with the high-magnification sperm selection procedure in these difficult cases. Although very few data are available on the application of this new technique, the possibility of being able to select fully mature spermatozoa, with a lower incidence of DNA defects, is interesting and deserves additional attention.

Prospective randomised studies are necessary to confirm the preliminary results regarding the efficacy of the criteria described, which have been proposed to morphologically select gametes prior to in vitro insemination.

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6. Conflict of interest 

I undersigned, Dr. Filippo Ubaldi, declare not to have any conflict of interest with regard to the manuscript entitled “Morphological selection of gametes” by Ubaldi F. and Rienzi L.

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PII: S0143-4004(08)00246-4

doi:10.1016/j.placenta.2008.08.009

Placenta
Volume 29, Supplement 2 , Pages 115-120, October 2008

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