Introduction

Fluorescence In Situ Hybridization (FISH) is one of the tests included in the advanced study of male infertility. This diagnostic technique allows the determination of the percentage of spermatozoa with chromosomal alterations in the semen sample. An abnormal test or high levels of sperm chromosomal abnormalities have been associated, at the laboratory level, with low fertilization rates1 and high rates of embryonic aneuploidy2,3 in couples undergoing In Vitro Fertilization techniques (IVF). In addition, clinically, this alteration has been directly related to recurrent pregnancy loss (RPL)4,5,6,7,8,9, recurrent implantation failure (RIF)10 and, finally, to live births with chromosomal abnormalities11,12,13,14,15.

To date, there is no specific and effective treatment that guarantees the selection and use of sperm with normal chromosome content in couples with abnormal FISH results. However, other genetic conditions, such as DNA fragmentation, can be treated. Semen samples with abnormal sperm DNA fragmentation values are treated using the magnetic activated cell sorting (MACS) technique, also known as annexin columns. This sperm selection technique has been shown to reduce the percentage of fragmented sperm and mitigate poor reproductive outcomes resulting from this alteration16,17,18.

In relation to annexin columns, seveeral studies evaluated the effect of this technique in relation to numerical chromosomal abnormalities in spermatozoa in both patients with normal19,20,21 and abnormal karyotypes22. The results showed a significant reduction in sperm aneuploidy after use in both patient groups. These data suggest a potential use for MACS in the treatment of any patient with an abnormal sperm FISH test who undergoes IVF techniques.

To corroborate this novel option, a study with two objectives was proposed. The first was to determine the prevalence of abnormal sperm FISH tests among the different indications for the analysis; the second sought to evaluate the efficacy, in laboratory terms, of using MACS in addition to conventional technique in those patients with abnormal sperm FISH results undergoing IVF.

Results

Analysis of sperm FISH results

Indications and FISH results

A total of 250 sperm FISH tests were requested during the research period with the indications listed in Table 1. Of the 250 sperm FISH requests, results were obtained in 93.6% (234/250) of them, because in the remainder the technique could not be performed due to low sperm count. The overall incidence of males with abnormal sperm FISH results was 8.5% (20/234).

Table 1 Indications for which the sperm FISH test was requested.
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Among the 27 indications deduced from the medical history which justified the request for sperm FISH, only four of them (male factor, recurrent pregnancy loss, recurrent pregnancy loss with male factor and low fertilization rate with male factor) had at least one abnormal test (Table 2).

Table 2 Indications and incidence of abnormal sperm FISH.
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The male factor, as the sole reason for the indication, had the highest incidence of abnormal tests (13.9%; 17/122). When the male factor was present together with other indications, the incidence was 6.1% (2/33). For repeated abortion as the only indication, the incidence was 2.7% (1/36), and when combined with male factor the incidence increased to 9.1% (1/11), (Table 2).

Semen characteristics and FISH results

In a second level of analysis related to abnormal sperm FISH results, we made a detailed study of the male factor. Cryptozoospermic samples, followed by oligoasthenoteratozoospermic and teratozoospermic (T) samples had the highest incidence of abnormal sperm FISH results (100%; 22.2% and; 20% respectively), (Table 3). In the oligoteratozoospermic (OT), oligoasthenoteratozoospermic (OAT), oligoasthenozoospermic (OA) and oligozoospermic (O) samples, the incidence ranged from 15 to 6.7% (Table 3). And no abnormal sperm FISH test was obtained in asthenozoospermic (A), asthenozoospermic (AT) and oligoasthenonecrozoospermic (OA) samples.

Table 3 Prevalence of patients with abnormal sperm FISH according to semen characteristics.
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As can be deduced from Table 3, among the main semen parameters analyzed (sperm count, motility and morphology), 91% (111/122) of the samples analyzed by male factor showed a low sperm count, of which 14.4% (16/111) had the FISH abnormal. In terms of morphology, teratozoospermia were present in 67% (82/122) of the tests, with 14.6% (12/82) in which sperm FISH was abnormal. Finally, 48% (59/122) of the FISH performed by male factor showed asthenozoospermia with 6.2% (6/59) being the sperm FISH abnormal.

Effect of MACS on IVF laboratory results

A total of 17 IVF-ICSI cycles were performed among the 10 couples included in the study. The clinical characteristics of the women are shown in Table 4. The age of the men ranged from 31 to 42 years (37.1 ± 4.06). Semen characteristics are shown in Table 5. None of them had urological history and only two of them had an andrological history, both males with varicocele.

Table 4 Female baseline characteristics.
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Table 5 Sperm characteristics of the 10 males included in the clinical trial.
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Table 6 Laboratory results in patients with abnormal sperm FISH after use of MACS compared to control group.
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The fertilization rate was almost equal in both groups: 69% in the MACS group and 66.7% in the control group. However, as for the abnormal fertilization rate, we observed twice as many poorly fertilized oocytes in the control group (4.2% vs. 8.3%), although the difference was not statistically significant (Table 6).

When assessing the possible association between treatment and the different types of abnormal fertilization, the application of MACS was associated with a lower incidence of zygotes with one pronucleus than in the control group. But this was not the case for zygotes with three pronuclei, whose differences were not significant (Table 6).

More embryos were biopsied in the MACS group than in the control group (76.9% and 63.6% respectively), Table 6.

Finally, the percentage of euploid embryos was similar in both groups: 33% in the study group and 37.5% in the control group, Table 6.

Discussion

Analysis of sperm FISH results

Indications and results of FISH

Sperm FISH has become a complementary technique to the seminogram that contributes to the diagnosis of male infertility. However, the clinical indications are not clearly defined. Typically, FISH is requested for patients with RPL, abnormal semen parameters, males with abnormal karyotype and IVF failures23. In contrast, other indications such as RIF are in question.

In the present study, the incidence of abnormal sperm FISH results tests was 8.5%. This figure was lower than those found by other authors whose incidence ranged between 14.6 and 19.9%24,25. Among other reasons, the incidence of this abnormal test depends on the indications considered. For this reason, it is important to delimit the profile of patients who will benefit from this diagnostic technique. Our study is probably the one that considered the most indications when requesting FISH.

Among the indications considered, the male factor had the highest incidence of abnormal tests (13.9%). This finding coincides with those reported by other authors24,25, who also found a higher incidence in the group of patients with male factor in relation to the other indications studied. In fact, male factor was present in 95% of the abnormal sperm FISH results tests (19/20).

On the other hand, sperm chromosomal abnormalities have also been related to another of the indications studied, RPL10. In this group of patients we found an incidence of abnormal sperm FISH tests of 2.7%. However, when combined with the male factor, the incidence increased to 9.1%. These results were lower than those described by other authors, who found an incidence between 11 and 22.7%24,25,26,27. In any case, in this group of patients, sperm FISH is recognized as a useful technique that contributes to genetic diagnosis and counseling, helping in some cases to make better reproductive decisions28.

Classically, an abnormal sperm FISH results has been associated with RIF10, however, in our study none of the patients with RIF had an abnormal sperm FISH results. In this sense, a recent guideline published by the ESHRE advises against performing sperm FISH for the diagnosis of RIF29.

Semen characteristics and FISH results

Within the male factor, the highest percentage of abnormal sperm FISH results was recorded in cryptozoospermic samples. In this regard, our results are conclusive: 94% of the abnormal tests had a low sperm count. These findings support those previously published by other authors, where the sperm count was not the only one that correlated with the FISH result, but also the probability that a test was abnormal increases the more severe the oligozoospermia is24,25,30.

As has been described, the risk of an abnormal sperm FISH results is also increased in all patients with abnormal semen parameters23,31. Males with teratozoospermia have a higher rate of sperm aneuploidy than those with normal semen parameters32,33,34. In our case, we observed that 20% of the tests indicated for teratozoospermia were abnormal. In addition, 67.2% of the tests requested for male factor showed teratozoospermia, with 14.6% being abnormal.

The aforementioned correlation between seminal alterations and sperm chromosomal abnormalities can be explained in part by a malfunction of the control mechanisms during spermatogenesis, which leads to the production of unbalanced sperm. During the different stages of meiotic division, there are different checkpoints responsible for verifying the correct chromosome complement. In the event of incorrect segregation, division will stop, and the abnormal sperm will be eliminated. The arrest can occur at different stages of spermatogenesis and, therefore, its effect on sperm production will vary. Thus, a partial restriction that blocks the progression of some germ cells results in oligozoospermia, while a complete arrest leads to a total absence of sperm (azoospermia)35. In addition to these, there are other mechanisms that could also explain low sperm counts in men with high frequencies of chromosomal abnormalities in their sperm, such as dysfunction in the process of eliminating aneuploid cells, or the elimination process itself being overwhelmed by the high number of abnormal cells30.

Effect of MACS on IVF laboratory results

During fertilization, the sperm and oocyte fuse to form the zygote, from which embryo development begins. Regarding this stage, our work found no statistically significant differences after the use of MACS. These results may be due either to treatment failure or to the fact that sperm chromosomal abnormalities have no effect on fertilization. Regarding the latter, there is controversy in the literature. A limited number of publications have found a negative implication of sperm aneuploidy on fertilization rate1,36,37. However, more studies have shown the opposite24. One of these used donor oocytes, thus minimizing the maternal contribution, and concluded that an abnormal sperm FISH results does not affect the fertilization rate24.

On the other hand, the fertilization rate obtained in the control group is perfectly comparable to that achieved in other couples with and without the male factor42,43. Our data, together with those found in the literature, suggest that abnormal sperm FISH has no impact on fertilization, which is why the use of MACS in these patients is probably of no benefit.

As for the abnormal fertilization rate, in the control group was twice as high as in the MACS group. However, statistical significance was not reached (p = 0.490). Among other causes, abnormal fertilization can be due to: on the one hand, damage produced during ICSI to the cytoskeleton and meiotic plate; on the other hand, alterations produced by denudation; and finally, originating from the oocyte or spermatozoa. However, of all these, the main cause is oocyte, because, as has been described, between 5 and 10% of zygotes originated by ICSI fertilize abnormally44,45,46. This percentage is similar to that obtained in the control group. This fact, together with the previous one, indicates that an abnormal sperm FISH test does not affect abnormal fertilization.

Regarding embryo development, our data suggest that the use of MACS does not increase the percentage of biopsyable embryos in males with abnormal sperm FISH results. In our case, embryos were biopsied on day 3 of embryo development, between the 6-cell and pre-compaction stages. It is possible that any positive effects from the use of annexin MACS may not manifest at this stage because it is not until day 3 of development that the embryo genome is activated and the embryo begins to synthesise its own proteins47. Similarly, any negative effects caused by sperm chromosomal alterations on embryo quality and development would also not be measurable at the cleavage stage, but would manifest themselves at later stages48. In any case, several studies have shown no significant differences in embryo quality on the third day of development in patients with abnormal sperm FISH results compared to those with normal FISH24.

Although most embryo aneuploidies are of maternal origin, the contribution of sperm may be particularly relevant in certain cases, such as an abnormal sperm FISH results2,3. However, our results did not reduce this participation. The use of MACS did not improve embryo euploidy. A recent study found no increased incidence of embryo aneuploidy in patients with an abnormal sperm FISH results compared to those with a normal FISH25. On the other hand, the results in the control group are perfectly comparable to those found in other patients with similar clinical characteristics undergoing Preimplantation Genetic Test (PGT) cycles49,50,51. This would also support the null or scarce involvement of an abnormal sperm FISH results in embryo aneuploidy.

Finally, the capacitation techniques used in the present work have been shown to reduce the proportion of sperm with chromosomal abnormalities19,52,53. This fact, coupled with the selection of sperm with the best characteristics during ICSI32,33,34, would counteract the possible negative effect of an abnormal sperm FISH results on IVF laboratory results. Consequently, any possible beneficial effect of MACS would be masked and could justify the laboratory results obtained in both groups in our study.

Regarding the limitations, although our study employed a rigorous design and analysis, its findings are limited by sample size due to its single-centre nature, the low incidence of patients with normal karyotypes and abnormal sperm FISH test, and the discontinuation of IVF treatments for various reasons in the national public health system. Because of this, our results should be interpreted with caution; the finding of no differences with the use of MACS in the present study cannot be interpreted as definitive evidence that there is no benefit. This is even more so given that clinical outcomes were not evaluated, as our study was specifically designed to observe laboratory results. It should also be noted that our laboratory results were limited to the third day of embryonic development. Aneuploidies transmitted to the embryo by the sperm (or oocyte) are present from fertilization onwards and can be assessed on the third day of development. In fact, studies indicate that patients with abnormal sperm FISH generate a higher proportion of aneuploid embryos performing the biopsy at the cleavage stage. However, we were certainly unable to assess aspects such as the blastulation rate and the quality of the blastocysts. Nor were we able to assess mosaicism. The male factor appears to be one of the causes associated with the increased rate of mosaicism in embryos generated from IVF cycles54,55. So much so that when sperm aneuploidy exceeds 10%, mosaicism appears to be the predominant effect on the embryos generated56, although this phenomenon occurs in around 5–10% of cases.

In conclusion, sperm FISH is a useful tool in the study of infertility when indicated in cases of male factor, especially with oligozoospermia and teratozoospermia, and recurrent pregnancy loss. These groups of patients with abnormal sperm FISH should be offered appropriate genetic counselling and information about the different reproductive options. On the other hand, the use of MACS in couples with abnormal sperm FISH and normal karyotype did not showed significant differences in laboratory results in our limited cohort. Studies with larger sample sizes are required, which also evaluate clinical data, in order to obtain more conclusive results and to provide definite conclusions.

Methods

Study design

A pilot quasi-randomized clinical trial was developed in the reproduction unit of the Hospital General Universitario Dr. Balmis (third level) in Alicante. The study was registered with the National Institute of Health (ClinicalTrials.gov registration: NCT06524102) on 24 July 2024. Approval for the study was obtained from Drug Research Ethics Committee (CEIm) of the Alicante Health Department. (Ref. PI2020-049) and was registered at the Instituto de Investigación Sanitaria y Biomédica de Alicante (Id.: 190767). The study complied with the deontological standards for such cases and was conducted in accordance with current Spanish legislation and ethical principles of the Helsinki Declaration.

In addition, a descriptive analysis of the results of all cases in which sperm FISH was performed during the study period, in relation to the indication for which study was requested, was carried out. Patients with an abnormal sperm FISH results test were included in the clinical trial (Fig. 1).

Fig. 1
figure 1

Patient selection flow diagram.

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As illustrated in Fig. 2, the process started with semen capacitation. After this, one aliquot formed the control group and another was processed using Magnetic Activated Cell Sorting (ART, Miltenyl Biotic GmbH, Germany), constituting the study group. Mature oocytes from each of the couples were inseminated by ICSI. All couples could have benefited from the experimental treatment, as they had the opportunity to inseminate their oocytes with sperm from both groups (50/50%), thus excluding treatment bias or asymmetry. The use of annexin columns is considered safe and without contraindications as no side effects have been reported.

Fig. 2
figure 2

Design of the quasi-randomized clinical trial and outcomes. All couples benefited from the experimental treatment since they had the opportunity to inseminate their oocytes with sperm from both groups, thus excluding bias or asymmetry of treatment.

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Participants

Couples with no previous healthy children in common with indication for IVF-ICSI with preimplantation genetic testing due to abnormal sperm FISH were selected. Women between 18 and 40 years old and men between 18 and 55 years old. Couples with previous voluntary sterilization, one partner with positive serology or abnormal karyotype, and documented medical contraindication for infertility treatment or pregnancy were not included. Participants were informed of the risks, benefits, discomforts and rights by the principal investigator. Prior to inclusion in the study, all participants gave their approval and consent signing the informed consent form.

Randomization

Mature oocytes from each couple were quasi-randomized to both study groups. At the time of insemination, they were alternatively allocated in a 1:1 ratio, without following any sequence and without regard to any morphological parameters, to be microinjected with spermatozoa treated with the annexin columns and with those not treated with this technique. In cases with an odd number, the excess oocyte was assigned alternately to each of the groups, thus avoiding imbalances in their sizes. It is important to note that this method is free of any selection bias by guaranteeing the similarity of the groups, since the genetic nature of the oocytes and spermatozoa used, which could influence the possible effect of the intervention, is unknown.

Procedures

Sperm FISH test

Semen samples were obtained by masturbation after 3 days of sexual abstinence. The samples were washed three times with phosphate buffered saline (PBS). The sediment obtained was resuspended in 3 ml of pre-warmed hypotonic solution and incubated at 37 °C for 15 min. After incubation, the samples were centrifuged, 1.5 ml of Carnoy solution was added to the resulting pellet and incubated for 5 min at 4 °C. The Carnoy fixation step was repeated three times.

A drop of the sample was deposited on a slide and allowed to air dry. The slides were treated with Saline-sodium citrate buffer (SSC) for 5 min, transferred to a staining jar with dithiethriol (DTT) and incubated at 37 °C for 8 min. The sample was then dehydrated with 70%, 90% and 100% ethanol for min at each step.

For FISH analysis, centromeric probes for chromosomes 13, 18, 21 and X, Y (Devyser) were used. In the dark, 1.5 µl of the corresponding probe was added. The slides were denatured (92 °C for 2 min) and subsequently hybridized (37 °C overnight). Washes were then performed to remove any unbound DNA probes. Slides were immersed in SSC and detergent solution (NP-40) at 73 °C for 2 min. Finally, the slides were drained and stained with DAPI.

A total of 1000 spermatozoa were analyzed in each sample and were classified as abnormal when the number of spermatozoa with abnormalities in at least one of the chromosomes was significantly (p-values less than 0.05) higher than that observed in the control group. The control group consisted of fertile individuals with normal karyotype and semen parameters.

Sperm capacitation

Semen samples were obtained by masturbation after 2 days of sexual abstinence, and were processed during the first hour after collection.

Samples were capacitated according to the density gradient method (Sydney IVF; COOK Medical) and subsequently swim-up. Equal parts discontinuous density gradients were used. The volume of the layers was adjusted according to the semen characteristics of each sample.

The 80% layer, the 40% layer and finally the semen sample were placed in a conical bottom tube. Centrifugation was performed at 592 g for 25 min. After centrifugation, the cell pellet was recovered and transferred to a new tube.

The sample was washed with 3 ml of growth media (FERTILISATION, GEMS) at 323 g for 10 min. The supernatant was discarded and 250 µl of growth media was added, avoiding resuspending the cell button. Samples were incubated with 6% CO2 at 37 °C until use.

Annexin columns (MACS)

MACS were performed according to the manufacturer’s protocol. After capacitation, the sperm concentration was adjusted to be between 0.1 × 106 and 5 × 107 sperm/ml. The cell suspension was incubated for 20 min with annexin V magnetic labeled microbeads (MACS ART Annexin V Reagent). After incubation, the cell suspension was loaded onto a separation column (MACS ART MS Column) placed in a magnetic field (MACS ART Separation Unit). The eluted cell fraction was collected in a conical bottom tube. Subsequently, the column was washed with 500 µl of MACS ART Binding Buffer and the eluted fraction was also collected in the same tube. Finally, the sperm suspension was washed and reserved until use in an incubator at 37 °C at 6% CO2.

Embryo biopsy

The biopsy was performed on the third day of embryo development between the 6-cell and pre-compaction stages. The obtained cells were analyzed by massive sequencing using the Ion ReproSeq PGS Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Bioinformatics analysis was performed with the Ion Reporter v5.0 platform (Thermo Fisher Scientific).

Outcomes

The primary outcome was embryo euploid rate, defined as embryos without chromosomal alterations in relation to the total number of those that were informative. The outcome was obtained on the fifth day of embryo development. Secondary outcomes included fertilization rate, defined as number of correctly fertilized zygotes among the total number of mature oocytes inseminated; abnormal fertilization rate, defined as number of abnormal fertilized zygotes among the total number of mature oocytes inseminated; and biopsiable embryo rate, defined as number of biopsied embryos versus the total number of correctly fertilized zygotes. Fertilization assessment was performed between 16 and 22 h after insemination. Biopsiable embryos were determined between 67 and 69 h after insemination.

The total incidence of abnormal sperm FISH was described as the number of abnormal tests out of the total that were requested. The incidence of abnormal sperm FISH tests was also calculated for each indication for which the test was requested.

Sample size

To date, the efficacy of MACS on laboratory results in patients with abnormal sperm FISH has not been evaluated; therefore, we estimated the sample size based on previous studies on the implications of this alteration in relation to embryo euploidy. The percentage of embryo euploid in the group of patients with abnormal sperm FISH results reported by some authors2,3 is 42% compared to 66% in patients with normal sperm FISH.

To estimate the sample size it was assumed that MACS can reverse the harmful effect of abnormal sperm FISH results on embryo euploidy, equating the results to the patient population with normal FISH. Based on this premise, considering that the frequency of unaltered embryos in the intervention group woud be approximately 66%, and a frequency of euploid embryos in the control group of approximately 42%, assuming an alpha error of 0.05 and a beta risk of 0.2 (statistical power of 80%), it was estimated that the number of oocytes to be studied is 67 in each group, making a total of 134 oocytes to be included in the study. A total of 143 oocytes were finally included; 71 in the intervention group (MACS) and 72 in the control group.

Statistical analysis

To study the possible effect of MACS on the different response variables studied, mixed logistic regression models were fitted, introducing a random intercept for each patient, as well as a random intercept for each cycle within each patient (nested design). In the case of the fertilization analysis, since it consists of three categories, it fitted with a multinomial regression model. The significance threshold was set at α = 0.05 for all analyses, and 95% confidence intervals were estimated for the different parameters of interest. All analysis was performed using R software (version 4.3.2) and the lme4 package (version 1.1–35.1).