ABSTRACT
To assess whether high magnification sperm head vacuole examination (SHVE) and/or standard sperm morphology assessment can predict ICSI outcomes in terms of fertilization, embryo quality, and delivery rates, a prospective observational bicentric study was conducted in two publicly funded assisted reproductive technology (ART) units in France between January and July of 2012. A total of 111 ICSI cycles for exclusively male infertility factors were included. A Spearman’s correlation test was performed to validate SHVE reproducibility between the ART units. The normal morphology rate and SHVE performed on selected spermatozoa were respectively determined according to David’s and Vanderzwalmen’s classifications used for motile sperm organelle morphology examination (MSOME) on the day of the ICSI. Receiver Operating Characteristic (ROC) curve analysis was performed to determine thresholds associated with the occurrence of a delivery. There was an excellent correlation between the two operators (r=0.98), thus validating the study’s SHVE data. Percentages of normal morphology grade spermatozoa using the standard classification and first-best morphology grade spermatozoa determined by SHVE were not significantly associated with (i) delivery (p=0.58; 0.90 /area under curve (AUC) =0.532; 0.507), (ii) fertilization (p=0.88; 0.90), (iii) top-quality embryos (p=0.27; 0.98), and (iv) good quality embryo rates (p=0.73; 0.98), respectively. In conclusion, high magnification SHVE and standard sperm morphology assessment cannot predict clinical or biological ICSI outcomes.
Abbreviations: ART: assisted reproductive technology; HBV: hepatitis B virus; HCV: hepatitis C virus; HIV: human immunodeficiency virus; ICSI: intra-cytoplasmic sperm injection; IVF: in vitro fertilization; LNVs: large nuclear vacuoles; MSOME: motile sperm organelle morphology examination; SHVE: sperm head vacuole examination; WHO: World Health Organization
Introduction
Several authors have analyzed the possible existence of a link between sperm morphology as determined by conventional techniques according to the World Health Organization [WHO Citation2010] and outcomes of various procedures used in assisted reproductive technology (ART), i.e., intra uterine insemination (IUI) [Lee et al. Citation2002], in vitro fertilization (IVF) [Kruger et al. 1988; Coetzee et al. Citation1998], and intra-cytoplasmic sperm injection (ICSI) [De Vos et al. Citation2003]. However, the predictiveness of sperm morphology on clinical IVF [Lundin et al. Citation1997; Keegan et al. Citation2007] and ICSI outcomes [French et al. Citation2010] has since been contested. This discord highlights the need to use other morphological examination tools that may be more relevant in regard to the medical care of infertile couples. Motile sperm organelle morphology examination (MSOME) is a real-time high magnification motile sperm examination that does not involve staining and that is performed using an inverted microscope equipped with interferential contrast Normarski optics. Maximal optical magnification (100x), magnification selector (1.5x), and video-coupled magnification amount to a final video magnification of about 6,600x. This new approach to sperm morphology examination was developed by Bartoov and colleagues [Bartoov et al. Citation2002], and it is also currently used prior to performing intracytoplasmic morphologically selected sperm injection (IMSI), which consists of the intracytoplasmic injection of a spermatozoon that has been selected at high magnification. Sperm head examination during MSOME or IMSI reveals the existence of vacuoles in real-time in vital sperm. The origin of these vacuoles is, however, still an issue of debate [Kacem et al. Citation2010; Boitrelle et al. Citation2011]. Various classifications based on these observations have been proposed. As Bartoov’s MSOME classification remains quite complex for routine use, new and easier classifications have recently been described [Vanderzwalmen et al. Citation2008; Cassuto et al. Citation2009]. Initial studies evaluating the impact of these vacuoles on ICSI and IMSI outcomes showed that microinjection of spermatozoa with large vacuoles is associated with lower fertilization and pregnancy rates [Berkovitz et al. Citation2005]. Since then, few methodologically well-conducted studies have been published regarding the relevance of high magnification examination of injected spermatozoa, and controversy has persisted in this regard. Some authors have reported results favoring IMSI over ICSI in light of increased implantation and pregnancy rates and, in parallel, a decreased risk of miscarriage [Antinori et al. Citation2008; Souza Setti et al. Citation2010; Setti et al. Citation2013; Setti et al. Citation2014b; Setti et al. Citation2015]. For others, no difference was seen between ICSI and IMSI in regard to the same assessment parameters [Balaban et al. Citation2011; Teixeira et al. Citation2013; Ebner et al. Citation2014; Gatimel et al. Citation2016]. However, MSOME is considered to provide improved evaluation of semen quality [Boughali et al. Citation2006; Oliveira et al. Citation2009], and it has become a standard technique in ART for diagnostic testing, as there is no standardization to date.
The aim of our study was to evaluate MSOME, and more precisely, sperm head vacuole examination (SHVE) and/or standard sperm morphology assessment as a predictive factor for ICSI clinical and biological outcomes. In regard to classical sperm morphology assessment, the David’s classification, which is employed widely in France, was used as it is much less restrictive than the WHO classification.
Results
The mean age of the women in the study was 35.5 years, and 38.3 years for the men. The sperm characteristics were evaluated after liquefaction. The mean sperm numeration was 15.9 ± 22.2x106, and the percentage of progressive motile sperm was 12.3 ± 4.2%. In regard to the sperm morphology, the mean percentage of typical forms was 7.9 ± 8.3%, according to David’s modified classification ().
Table 1. ICSI cycle characteristics.
SHVE accuracy and reproducibility
A total of 50 sperm photographic images were obtained using the software employed to measure the sperm head and vacuoles surface ratio (LUCIA G Image analyser®, Nikon), and 49 were independently and correctly analyzed by the two operators. These results were compared with those provided by the software, and an accuracy of 98% was determined for each operator. A blind-correlation between the two operators was established under the same SHVE conditions after analyzing 15 selected spermatozoa so as to determine interoperator reproducibility. The intraclass correlation coefficient was determined to be 0.790 for SHVE grade I and II (i.e., top quality spermatozoa) between the two operators.
Ultra-morphological and morphological description of migrating sperm
After ICSI on the selected sperm, a smear slide was made of the selected sperm for classical morphological assessment. A SHVE was performed on the same sample. The characteristics are reported in .
Table 2. Mean percentages of classical normal forms and SHVE grades following sperm selection for all spermatozoa analyzed on the day of the ICSI.
MSOME and classical normal form ‘association’ with pregnancy
The comparison of ICSI cycle characteristics in terms of the occurrence of delivery did not reveal any difference (). A Wilcoxon’s test was performed and Receiver Operating Characteristic (ROC) curves were calculated in order to determine a potential association between sperm morphology and ultra-morphology with the occurrence of delivery. No statistically significant difference between the two groups was found for the percentage of normal forms (13.1 vs. 14.2, p=0.58) and the various MSOME classes (poor and good prognosis, p>0.05) (). Also, areas under curves (AUC) of the two tests were both close to 0.5 ().
Table 3. ICSI cycles characteristics according to the occurrence of delivery.
Table 4. Sperm morphology and ultra-morphology according to the occurrence of delivery.
Figure 1. Occurrence of delivery according to sperm morphology. ROC curves were calculated in order to determine a potential association between occurrence of delivery and percentage of classical normal sperm forms (A) and percentage of motile sperm organelle morphology examination (MSOME) classes 1+2 (B). Any influence of sperm morphology on this issue was found as shown by the areas under curves (AUC) of the two tests which were both close to 0.5.
Correlation between MSOME and classical normal forms with ICSI biological outcomes
We performed a Spearman’s correlation test and we analyzed the fertilization rate and the top and good quality D2 embryo rates (since most embryo transfers occurred on Day 2). No statistically significant correlation was found for these items, both in terms of the percentage of classical normal forms (r= 0.01, -0.11, -0.03) and MSOME class 1+2 (r= -0.01, 0.03, -0.002) ()
Table 5. Spearman correlation between MSOME class 1+2 (M1+2) and classical normal forms (NF) with ICSI biological outcomes.
Discussion
Population
The women in the study were less than 38 years of age. This recruitment was deliberately carried out in this direction in order to avoid a female age bias. Moreover, only 111 treatments were included in our study due to local organizational difficulties between the two centers during the period of the evaluation. In regard to the day of embryo transfer, no standardization between Day 2 and Day 3 was carried out because, to date, no difference has been shown between Day 2 and Day 3 embryo transfer in terms of live birth rates [Oatway et al. Citation2004].
Methodology
The aim of our study was to evaluate the predictiveness of SHVE by high magnification and/or standard sperm morphology assessment in ICSI outcomes. Sperm morphology assessment is operator dependent and therefore very subjective. Standardization of these evaluations (classical and MSOME) is a fundamental prerequisite before using them in clinical practice. The first step in our study was hence to standardize the classical sperm morphology and SHVE assessments. In order to standardize our SHVE results, the accuracy of the operators of the two centers had to be evaluated. Their level of accuracy was found to be excellent, with an error rate of only 2%. Moreover, the reproducibility study using intraclass correlation coefficients showed a good correlation for the 1st choice spermatozoa (M1+2). This only ‘good correlation’ (close to 0.8) is explained by our sample size to determine the correlation (n=15), which decreases our statistical power. However, it allowed us to justify and continue this study and inclusions in the two centers. For the classical sperm morphology assessment, we also limited the inter-individual variability. The same operator performed the staining and reading of all of the smear slides for the study in one center. Also, we deliberately limited the inclusion to male infertility involving only ICSI. We excluded all female factors that could affect ICSI outcomes (e.g., females > 38 years of age, endometriosis, uterine and fallopian tube malformations, or infections), thereby minimizing confounding factors, even though some authors have concluded that there is no difference in clinical outcomes after IMSI or ICSI for patients with a poor ovarian response due to age or endocrine factors [Setti et al. Citation2015]. Moreover, in some predictive studies, normal cases have also been included that could present some limitations to the interpretation of the results. In our study, we deliberately excluded the normal male cases (i.e., any alteration of sperm parameters) that were undergoing conventional IVF and not ICSI.
Sperm morphology and ICSI outcomes
This is an original study that focused on the direct impact of sperm head ultra-morphology assessment on clinical and biological outcomes of ICSI. Studies reported in the literature usually describe results after microinjection at high magnification. In 2002, Bartoov and colleagues found that the morphological normalcy of sperm nuclei defined by MSOME was significantly and positively associated with both the fertilization rate and the occurrence of pregnancy [Bartoov et al. Citation2002]. In 2005, Berkovitz and colleagues reported that sperm exhibiting only large head vacuoles correlated negatively with pregnancies while correlating positively with miscarriages [Berkovitz et al. Citation2005]. The meta-analysis by Souza Setti and colleagues did not provide evidence for a difference in terms of the fertilization rate, but it did report better rates of implantation and pregnancy with IMSI [Souza Setti et al. Citation2010]. However, this meta-analysis was limited by the small number of included studies. Nonetheless, Setti et al. observed the same results in another meta-analysis regarding the use of IMSI in the setting of repeated ICSI failure and male infertility factors [Setti et al. Citation2014b]. Conversely, other authors have reported no difference between IMSI and ICSI. This is the case for De Vos et al., who reported no difference in terms of fertilization, cleavage, blastocyst formation, and clinical pregnancy rates in a randomized sibling-oocyte study [De Vos et al. Citation2013]. Moreover, the meta-analysis of the Cochrane Review found both techniques to be equivalent in terms of live births and miscarriages, thus concluding that routine use of IMSI is not warranted [Teixeira et al. Citation2013]. The same finding was highlighted in the study by Gatimel et al., who concluded that there was no improvement in clinical outcomes after IMSI [Gatimel et al. Citation2016]. Lastly, the meta-analysis by Ebner et al. indicates that there is no consensus regarding the effect of IMSI on implantation or pregnancy rates, thus leading to the conclusion that IMSI is most likely an ART procedure that should be reserved for specific cases [Ebner et al. Citation2014]. This is in line with the results of our study, since we found no significant correlation regarding fertilization and top and good quality embryo rates, especially for normal sperm forms assessed by MSOME. These results confirm other data reported in the literature [Keegan et al. Citation2007; French et al. Citation2010]. Similarly, no correlation was found between different MSOME classes according to Vanderzwalmen’s classification and the occurrence of delivery. In our study, the delivery rates were equal. This observation seems to be confirmed by the absence of an association between ‘first choice’ spermatozoa (i.e., MSOME grade 1 and 2) and deliveries, as shown on the ROC curve (AUC=0.507).
In regard to classical sperm morphology assessment, the David’s classification was used because it is much less restrictive than the WHO classification, and also because it is widely used in France. No association between the percentage of normal sperm forms after migration and delivery was discernible from the ROC curve (AUC = 0.532).
In our study, neither classical sperm morphology assessment nor high magnification SHVE were predictive of ICSI clinical and biological outcomes. However, our results could also be criticized because we mainly only considered male factors, while some female parameters such as age or ovarian function were not taken into account to any significant extent, yet these could nonetheless impact on ICSI issues. We compared the ICSI cycle characteristics in delivery and no delivery groups, and we noted no difference, even in terms of the age of the women and ovarian function. Moreover, regardless of the technique used, sperm morphology assessment was not predictive of delivery or no delivery as clinical outcomes of ICSI. In line with this observation, the study by Setti et al. resulted in similar findings, as they found that implantation, pregnancy, and miscarriage rates were similar after ICSI or IMSI in low and normo-responders, without any influence of age or ovarian deficiency [Setti et al. Citation2015].
This controversy is partly due to a lack of consensus regarding the evaluation of sperm morphology at high magnification, with determination of a pathological threshold, and difficulty in deciding on the definition of the origin of sperm head vacuoles. Thus, Setti et al. showed that the incidence of sperm with large nuclear vacuoles (LNVs) occupying >13.0 % of sperm heads was negatively correlated with blastocyst formation and implantation rates [Setti et al. Citation2014a].
Other authors evaluated different thresholds defining large vacuoles, for example 25% [Boitrelle et al. Citation2011] and 50% [Franco et al. Citation2008] of the sperm head area, with a negative impact on clinical outcomes of ICSI compared to IMSI. In their study, Perdrix et al. [Citation2011] considered the same threshold >13%. Our results contradict the findings reported by these authors. In this context, we would believe that Vanderzwalmen’s MSOME classification, used in our study, is not sufficiently discriminating, since the threshold is 4%. The fact remains that the absence of a clear definition of sperm head vacuole origin and systematization leads to these contradictory conclusions. This confirms the lack of consensus to date regarding the discriminating threshold of the sperm head vacuoles volume assessed by MSOME.
MSOME is increasingly used by French IVF centers, although it still suffers from an obvious lack in terms of being used for evaluation. This is largely because the evaluation is based on assessment of sperm head vacuoles. In light of this, we wished to investigate the potential of sperm head examination at high magnification to predict clinical and biological outcomes of ICSI. Based on a methodologically reliable study that standardized the SHVE methodology, we report here on the absence of predictivity of sperm head examination at high magnification for the occurrence of delivery. In accordance with the controversy described in the literature, we report the same conclusion regarding the use of the percentage of normal sperm forms assessed by the standard morphology test of selected spermatozoa.
Thus, our results indicate that classical sperm morphology and ultra-morphology should not be performed in order to predict ICSI clinical and biological outcomes. Our results must, however, be confirmed in more randomized clinical trials and in larger cohorts.
Material and methods
Population
This bicentric observational prospective study was conducted from January to July of 2012. It took place at two French university hospitals: Jean Verdier University Hospital in Bondy and Cochin-Port Royal University Hospital in Paris. A total of 111 ICSI cycles for exclusively male infertility (oligo-astheno-teratozoospermia) were included according to WHO [Citation2010]: sperm count < 39x106, progressive motility < 32%, and morphologically normal forms < 23% according to David’s classification and corresponding to the < 4% normal forms threshold of the WHO [Citation2010] criteria (). Oocytes were injected with selected sperm, and an intra uterine fresh embryo transfer on Day 2 or Day 3 (D2/D3) was performed for all patients. The women in question were less than 38 years of age (<38). Cryopreserved gametes (egg or sperm), gamete donors, individuals with a viral risk (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV)), and women with uterine or tubal pathologies or presenting with infection or endometriosis were not included in this study. This study was approved by the local institutional review board. Indeed, all couples treated in our center give their consent to the use of their gametes in research programs by signing an information and consent statement. Otherwise, in our study, no changes in the management of included couples have been achieved notably in the ICSI procedure.
Semen preparation
Semen samples were collected on the day of the oocyte retrieval by masturbation after an ejaculatory abstinence period that was between two and five days. After liquefaction, semen was prepared by two-layered density gradient centrifugation (90 and 45% Puresperm®, Nidacon, Sweden). Sperm motility and concentration were assessed according to World Health Organization [WHO Citation2010] criteria. Following selection on a density gradient, prepared semen samples were maintained at 37°C in a 5% CO2 atmosphere. A volume of 2 µL of the prepared semen sample was used for MSOME, and a volume of 20 µL was spread on slides for a conventional morphology assessment. The timing between the end of the sperm preparation and the beginning of ICSI did not exceed three hours.
ICSI procedure
Women mainly underwent ovarian stimulation with antagonist protocols in order to avoid ovarian hyper stimulation syndrome (OHSS). Following hormonal and ultrasound ovarian surveys, ovulation was triggered by administration of hCG (Ovitrelle®, Merck Serono, Germany). Oocyte retrieval was performed under vaginal ultrasound guidance 34h to 36h after triggering ovulation. Two h after oocyte collection, enzymatic removal of the corona cumulus complex was achieved using hyaluronidase (Hyaluronidase 80 UI®, Fertipro, Belgium). After denudation, the first polar body extrusion examination confirmed nuclear maturation of the oocytes. Conventional ICSI, was performed three h following oocyte collection. The selection of individual spermatozoon for injection was based on their motility and morphology after routine magnification at 400x (to identify the best or the least altered spermatozoon). Specific microinjection techniques, such as piezo-ICSI were not used so as to avoid introducing technical bias in the study. Moreover, this technique is not commonly used in the world, and it is not used at all in the centers where our study took place. Fertilization was assessed at 18 h post ICSI, and early cleavage at 25 h post ICSI. Embryos were cultured in a 20 µL droplet of a global medium (Global medium®, LifeGlobal, USA) under mineral oil (NidOil® Nidacon) and maintained in incubators at 37°C with a 5% CO2 atmosphere. Grading of the embryo quality was performed at D2 or D3 based on the number and the size of the blastomeres (symmetric or asymmetric), the presence or not of anucleate fragments (type A = 0%; type B ≤ 20%; type C between 21% and 50%; type D > 50%), as well as the percentage of multinucleated blastomeres. Thus, a top quality embryo at D2 was defined as an embryo with four symmetrical cells without any fragments (A4) or with less than 20% anucleate fragments (B4) and without any multinucleated blastomeres. The same criteria were applied at D3 with eight symmetrical cells (A8 or B8). Good quality embryos were defined as embryos A3 to A5, or B3 to B5 at D2 and A6 to A10 or B6 to B10 at D3, with or without symmetrical cells and with fewer than 20% multinucleated blastomeres. Intra uterine embryo transfer was performed at D2 or D3.
Normal forms (NF) after selection
Classical sperm morphology was evaluated according to David’s modified classification [David et al. Citation1975; Auger and Eustache Citation2000]. A smear slide was made using the prepared semen sample from every patient in both centers (109 patients). The smear slides were stained by the Papanicolaou method in one of the two centers and then fixed to avoid any changes over time. These slides were then stored in a sealed container at room temperature. Sperm morphology was then examined by microscopy at 400x. A single experienced operator performed the staining and the assessment, and they were blinded to the other assessed parameters. The percentage of normal sperm forms was determined within seven days of ICSI. For two patients, the sperm morphology assessment was not performed due to extreme oligozoospermia.
SHVE procedure
Following sperm selection and the ICSI procedure, SHVE was performed according to the adapted MSOME classification used by Vanderzwalmen et al. [Citation2008] on the specimens derived from the 111 patients in our study. Inverted microscopes (Ti Eclipse®, Nikon, Japan) equipped with interferential contrast Nomarski optics were used at each center. Maximal optical magnification (100x), magnification selector (1.5x), and video-coupled magnification led to a final video magnification of approximately 6,600x. Less than two hours after the ICSI procedure, 2 µL of semen preparation were placed in a 5 µL droplet of polyvinylpyrolidone (PVP) in a glass bottom dish with a thickness of 0.17 mm (Will-Co dish®) and covered with mineral oil (NidOil® Nidacon). For each patient, one hundred spermatozoa were analyzed and classified according to the four grades of Vanderzwalmen’s MSOME classification based on the presence or size of vacuoles: grade I, no vacuoles; grade II, ≤2 small vacuoles; grade III, ≥1 large vacuole; grade IV, large vacuoles with other abnormalities [Vanderzwalmen et al. Citation2008]. In our study, grade IV was divided into two subclasses: IVA (morphological abnormality of the sperm nucleus without any vacuoles or with ≤2 small vacuoles) and 4B (morphological abnormality of the sperm nucleus with ≥2 small vacuoles or large vacuoles).
SHVE standardization
The Vanderzwalmen’s MSOME classification is very amenable for routine use. However, SHVE remains a subjective operator-dependent examination, largely in terms of evaluation of vacuole size, especially around the threshold of 4% of the head volume that allows for differentiation between class two and class three spermatozoa.
This study took place at two IVF centers, so standardization of the SHVE grading was necessary before commencing with the evaluation. We first determined our level of accuracy: the operator of each center correlated their reading with software using the LUCIA G Image analyser® (Nikon) which allows images of sperm to be taken at high magnification, as well as measurement of the sperm head and vacuole surfaces and determination of their ratio. The same sperm image was analyzed by each operator, and grading of the sperm morphology at high magnification according to Vanderzwalmen’s classification was performed separately by each operator on a total of 50 sperm images. A blind-correlation between the two operators was then established under the same conditions using 15 selected spermatozoa to determine the inter-operator reproducibility. Each sperm was isolated in a microdrop prior to the analysis at high magnification. This method guaranteed that the same sperm was sampled by both of the operators.
Statistical analysis
Results were described in terms of the actual values, percentages, means, and standard deviations, or as medians and quartiles (for asymmetric distributions). Groups defined by delivery outcomes were compared by using the Wilcoxon test. The ROC curve and AUC were calculated to assess the predictiveness of standard morphology and SHVE. Moreover, Spearman’s correlation tests were used to compare SHVE and the percentage of NF with the different biological outcomes of ICSI.
Declaration of interest
The authors report no conflict of interest.
Additional information
Notes on contributors
Khaled Pocate-Cheriet
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Ilan Heilikman
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Raphael Porcher
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Virginie Barraud-Lange
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Nathalie Sermondade
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Charlene Herbemont
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Jean Philippe Wolf
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
Christophe Sifer
Performed the experiments and wrote the manuscript: KPC, IH; Analyzed data: RP; Assisted with conventional morphology assessments: VBL; Assisted with the MSOME: NS, CH; Designed the study, revised the manuscript, and approved the final draft: CS, JPW.
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