Efficacy of the motile sperm organelle morphology examination (MSOME) in predicting pregnancy after intrauterine insemination

Intrauterine insemination (IUI) is a simple and non-invasive procedure that is used to treat couple subfertility. In many cases, IUI allows one to avoid more complex assisted reproduction techniques (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). On the other hand, IUI yields varied results as a consequence of the large number of variables that are involved in the treatment, including the aetiology of infertility, the age of the couple, ovarian stimulation protocols, timing and number of inseminations; this has raised doubts regarding the actual effectiveness of IUI [1]. The most common indications for IUI are cervical hostility, mild male factor, mild endometriosis, ovarian dysfunction, and unexplained infertility [2]. In regard to male infertility, there is insufficient information regarding the effectiveness of IUI, and more data are required to either recommend or advise against IUI in these situations [3]. Because one of the most common indications for IUI is mild male factor, and in view of the doubts regarding the efficacy of the procedure with respect to this indication, it would be of high clinical interest to determine the semen parameters that could serve as predictive factors.

Innovative methods for the selection of sperm in ART [4‐9] have been published, providing fresh insight into the correlation between sperm quality and clinical results. On the other hand, the value of conventional semen analysis has been the subject of debate. By analysing semen, clinicians expect to obtain a clear indication of the male's fertilisation potential, which is not provided by conventional evaluation (except in particular situations such as total teratozoospermia and globozoospermia) [10, 11]. Although none of the semen parameters (or even the functional test)--analysed either separately or jointly--can be considered definitive, morphology has been consistently shown to be the most reliable indicator of male fertility. Diverse studies that originated primarily from IVF/ICSI programmes and IUI corroborate the sensitivity of morphology as a prognostic factor [10‐20].

To test the hypothesis that subtle sperm organelle malformations [21, 22] are associated with ART results, Bartoov et al. [23, 24] developed a method of evaluating human spermatozoa in real-time at high magnification; this method is called the motile sperm organelle morphology examination (MSOME). MSOME is performed using an inverted microscope that is equipped with differential interference contrast/Nomarski optics that enable magnification exceeding 6000× [23], which is much higher than the magnification that is typically used by embryologists in selecting spermatozoa for the ICSI procedure (which ranges from 200× to 400×) and is even higher than that which is employed in routine semen examination (1000×). This method favoured the development of intracytoplasmic morphologically selected sperm injection (IMSI), which is based on sperm normality--as defined by the MSOME classification--and is aimed at improving conventional ICSI outcomes by focusing on the existence of a correlation between both sperm morphological abnormalities that can be observed at high magnification and DNA damage [24‐28]. Although MSOME was developed merely as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in the evaluation of semen quality. In the specific case of IUI, seminal evaluation using MSOME could represent an adjunct tool to predict the efficacy of the technique.

To better comprehend the diagnostic/prognostic value of analysing semen morphology using high magnification, this study aimed to evaluate the prognostic value of normal sperm morphology using MSOME classification in predicting clinical pregnancy (CP) after IUI.

Pregnancy occurred in 34 IUI cycles (CP rate per cycle: 21.8%, per patient: 30.6%). A significantly higher incidence of normal spermatozoa according to the MSOME criterion was found in the group of men in which the IUI cycles resulted in pregnancy (2.6 ± 3.1%) compared to the group that did not achieve pregnancy (1.2 ± 1.7%; P = 0.019). The laboratory and clinical parameters that were evaluated according to IUI outcome are shown in Table 1. With the exception of the percentage of normal spermatozoa, there were no significant differences between the groups.

Logistic regression revealed that the percentage of normal cells in the MSOME analysis was a determinant of the likelihood of achieving clinical pregnancy (OR: 1.28; 95% CI: 1.08 to 1.51; P = 0.003). On the other hand, logistic regression did not reveal a statistically significant (P > 0.05) association between IUI outcome (CP) and the other parameters that were analysed, including the woman's age (OR: 0.96; 95% CI: 0.88 to 1.06) and man's age (OR: 0.98; 95% CI: 0.91 to 1.05), the duration of infertility (OR: 0.81; 95% CI: 0.63 to 1.03), DNA fragmentation (OR: 0.97; 95% CI: 0.97 to 1.02), the number of follicles ≥17 mm (OR: 1.40; 95% CI: 0.53 to 5.21), and other semen characteristics, including total sperm count (OR: 1.00; 95% CI: 0.99 to 1.00), progression motility (OR:0.98; 95% CI: 0.95 to 1.00), and leukocytes (OR:1.77; 95% CI: 0.46 to 6.70). Table 2 summarises these results.

The ROC curve was created only for the percentage of normal sperm morphology s by MSOME (which was the only variable that differed significantly between the groups in the previous analysis). The ROC curve (Figure 2) had an area under the curve of 0.63 (95% CI: 0.52-0.78) (see Methods), indicating that the percentage of normal forms as analysed by MSOME had reasonable prognostic potency for predicting pregnancy after IUI. Setting the threshold at 2% offered the optimal compromise between specificity (50%) and sensitivity (79%) and between positive predictive value (40%) and negative predictive value (85%). At this cut-off level, the efficacy of the percentage of normal sperm morphology by MSOME in predicting pregnancy was 65%.

The rationale for the IUI procedure is to maximise the number of motile spermatozoa at the site of fertilisation to maximise the likelihood of achieving pregnancy. Although IUI is used widely in the treatment of couple subfertility, its true efficacy is yet to be determined, particularly when the technique is employed due to male subfertility [1]. According to Merviel et al. [37], the most important predictive factors for pregnancy in IUI cycles are the recruitment of two preovulatory follicles that are > 16 mm in a woman of age ≤30 years and a concentration ≥5 million motile spermatozoa and teratospermia ≤70% after the preparation of semen by a discontinuous concentration gradient. Another study reported that IUI that was performed for male factor subfertility has a low possibility of success when the woman's age is > 35 years, the number of motile spermatozoa that are inseminated is < 5 million, or normal sperm morphology--as defined by the criteria of the World Health Organization--is < 30% [38].

On the other hand, Dorjpurev et al. [39] showed that with a motility rate of ≥30% and a motile sperm concentration of ≥10 million/ml, IUI can be a useful tool for treating male subfertility. Kamath et al. [40] reported that a duration of infertility of less than 5 years and a total motile spermatozoa count of more than 10 million are related to a better prognosis in IUI, and they found a trend toward a higher pregnancy rate with endometrial thickness > 6 mm. Wainer et al. [41] reported that a minimum of 5 million motile spermatozoa should be inseminated only when the normal morphology of the sperm after preparation is < 30%, as the quantity compensates--at least in part--for poor seminal quality; they further noted that if these parameters cannot be reached, in vitro fertilization should be recommended. Other clinical parameters have also been evaluated, including the comparison of recombinant FSH and highly purified FSH for ovarian stimulation [42], the use of GnRH antagonists to avoid a premature LH surge [43, 44], the association of chromatin condensation in spermatozoa with conventional semen parameters [45], and the use of a soft versus firm catheter for the insemination procedure [46], all of which were designed to identify predictive factors of the likelihood of achieving pregnancy in IUI.

Unfortunately, MSOME is not typically applied beyond its use in sperm selection for the ICSI procedure. In fact, to the best of our knowledge, the present study is the first to analyse MSOME as a prognostic factor for IUI; thus, our results cannot be compared with other results. However, our data are in agreement with other studies that used other morphological sperm evaluation criteria. In fact, morphological sperm alterations seem to confer a significantly poorer outcome in IUI [37, 47, 48] and are reported to be a more relevant semen parameters than sperm concentration and sperm motility [49]. Karabinus and Gelety [50] stated that pregnancy rates per cycle after IUI were not different when the percentage of morphologically normal sperm (strict criteria) in raw semen was 5%, 5-9%, 10-19%, 20-29%, or ≥30% (with rates of 6.55 ± 3.9,13.6 ± 3.2, 8.8 ± 2.4, 7.1 ± 2.5, and 9.7 ± 3.3%, respectively). Thus, the authors concluded that IUI appears to be a successful treatment modality for male factor infertility even in cases in which the percentage of morphologically normal sperm in raw semen is quite low.

On the other hand, the morphological criteria for assessing spermatozoa quality according to parameters of the World Health Organization were recently modified (with a normal morphology cut-off value of > 4% in the 2010 WHO manual) [51], demonstrating that there is still a controversy regarding the definition of a morphologically normal spermatozoon. It would be of great clinical value to establish a laboratory parameter that could reliably and more efficiently predict the outcome of IUI to both improve the outcome and optimise the indications of the technique. Recently, our group suggested that MSOME should be included among the routine criteria for semen analysis, given that it is a much stricter criterion of sperm morphology classification than the Tygerberg criterion [27]. In addition, we also demonstrated that MSOME appears reliable for analysing semen [52].

One of the most important alterations that can be observed with MSOME is the presence of large nuclear vacuoles (LNV; see Figure 1B). We consider LNV as those that occupy more than 50% of the nuclear area [52]. LNV are specific sperm alterations that can be observed at high magnification, and their presence has been shown to have clinical implications. Based on electron microscopy data, Bartoov et al. [23, 53] and Berkovitz et al. [32] assumed that nuclear vacuoles indicate a chromatin abnormality. Other studies confirmed the association between nuclear vacuoles at high magnification and chromatin damage. Berkovitz et al. [54] graded the severity of nuclear morphological alterations, highlighting principally the presence of large vacuoles and suggesting that vacuolisation of the sperm nucleus reflects some underlying chromosomal or DNA defect. Franco et al. [31, 55] demonstrated an association between large nuclear vacuoles and the presence of DNA fragmentation, denaturation and protamination in the spermatozoa. Garolla et al. [56] showed that the presence of nuclear vacuoles affects mitochondrial function, chromatin status, and aneuploidy rate. Using electron microscopy, Toshimori and Ito [57] associated the presence and content of nuclear vacuoles with DNA damage. In addition, the authors emphasised that IMSI/MSOME aids in identifying vacuoles. Oliveira et al. [28] observed a significant positive correlation between the percentage of sperm that contain large nuclear vacuoles and the percentage of DNA fragmentation. Gopalkrishnan et al. [58] found that the chromatin material of spermatozoa from men whose partners presented with early pregnancy loss was often found to be either compact or partially compact with irregular nuclear borders and large vacuoles. Moreover, a recent study has related LNV to the absence of acrosome reaction, which could have deleterious effects on the processes of fertilization and embryo development [59]. On the other hand, it has been clearly established that the capacity of human sperm to fertilise an oocyte and produce an embryo with a high potential for implantation and development depends on the sperm cell's DNA integrity [56]; thus, MSOME could provide important information regarding the likelihood of success with ART.

Many investigators have reported the outcome of in vitro fertilization techniques according to MSOME [24, 25, 32, 52, 60‐63], but this evaluation has not been employed previously to determine the success rate of IUI. The present study investigated the outcome of IUI compared with semen morphology as assessed with MSOME. We observed a significantly higher pregnancy rate with a higher percentage of normal cells according to the MSOME parameters. However, a logistic regression analysis failed to identify a significant correlation between treatment outcome and other characteristics such as patient age, duration of infertility, the number of follicles and other semen parameters. This is in contrast with other published reports [1, 37, 39]. The lack of consensus in the literature clearly highlights the difficulties in finding reliable prognostic parameters for determining IUI outcome.

With regard to the percentage of morphologically normal spermatozoa, we observed an optimum cut-off point of 2%. With this cut-off point, the efficacy of the exam in predicting pregnancy following IUI reached 65%, as shown by the ROC curve. Because there are doubts regarding the efficacy of IUI for male factor, MSOME could also play a role as a method for the classification of sperm morphology to offer an additional parameter for the indication of the technique. Besides the woman's age, the duration of infertility and the concentration of motile spermatozoa, which are predictive factors that are considered to be important by other investigators, our data suggest that 2% morphologically normal spermatozoa at MSOME is desirable to maximise the likelihood of pregnancy following IUI.

The accuracy with which the morphological normality of spermatozoa can be assessed depends on the resolution power of the optical magnification system. Spermatozoa that appear to be morphologically normal at 1000× magnification may in fact have various structural abnormalities that can only be detected at higher optical magnification (> 6000×). Few studies have attempted to analyse MSOME as a morphological classification method for semen. Using MSOME, Bartoov et al. [23] reported an incidence of sperm normality of 2.9 ± 0.5% (range 0-5%). In our study, we found an incidence of 1.5 ± 2.2% (0-13%). Differences with regard to the observation of nuclear vacuoles can explain the divergence found in the normality rates between MSOME and other morphological criteria. Employing the Tygerberg criteria, Bar-Chama et al. [64] analysed the number of sperm vacuoles in a series of 1,295 fresh post-processed sperm samples. They found vacuoles in only 19.5% (253) of the samples. On the other hand, MSOME revealed that the ejaculates of males who were routinely referred for ICSI exhibit an average of 30-40% of spermatozoa with vacuolated nuclei [54]. In addition, there is divergence in the quantification (i.e., normal or abnormal) of the presence of nuclear vacuoles. Using MSOME, the nuclear chromatin content is considered to be abnormal if it contained one or more vacuoles that occupied more than 4% of the nuclear area [23]. Nevertheless, other criteria (for example, the Tygerberg criteria) are much more tolerant with regard to the presence of vacuoles. A head is considered to be defective only when > 20% of its area is occupied by unstained vacuolar areas [29, 65].

On the other hand, Bartoov et al. [23] emphasised that whereas a routine morphological examination is applied to semen samples as a whole, MSOME focuses only on the fraction of motile spermatozoa. Analysing only the motile spermatozoa by MSOME can confer an additional advantage, as it will provide information regarding the sample fraction with a higher potential for achieving fertilization and development. Even though analyses using other criteria also employ high magnification, the characteristics of these procedures (i.e., fixation and staining) preclude the possibility of obtaining information for the motile portion.

In conclusion, the present study suggests that the use of MSOME for semen evaluation can be a reliable predictor of the incidence of normal forms in a sperm sample. Performing MSOME prior to the IUI procedure can represent a valuable tool to support or contraindicate this relatively inexpensive treatment and ultimately contribute to the indication of more complex ART procedures, thereby avoiding a loss of time. The present findings support the use of high-magnification microscopy both for selecting spermatozoa and as a routine method for semen analysis with potential clinical applications.