A treatment approach for couples with disrupted sperm DNA integrity and recurrent ART failure

Infertility affects 12–18% of couples in the USA of reproductive age [1]. Up to 15% of all infertility cases may remain unexplained, despite involving couples in which no obvious factor can be traced to either partner [2]. The reproductive treatment approach for these couples is somewhat incremental, beginning with ovulation monitoring in conjunction with timed intercourse or intrauterine insemination. If these initial treatments fail, a more invasive technique is used such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) [3]. When a specific cause for the infertility cannot be identified, a subtle male factor may be presumed. Indeed, up to 11% of men with a normal semen analysis have a noticeable sperm chromatin fragmentation (SCF), which may affect up to 5% of men with semen parameters above the 50th percentile [4, 5]. In addition, an inverse correlation between SCF and sperm motility has been shown [6]; therefore, these men are better suited for ICSI insemination in which sufficiently motile sperm cells are identified under direct visualization [7, 8]. It should be noted that a mild degree of sperm DNA fragmentation can be prevented by ooplasmic repair mechanisms [9‐11], which have been confirmed by studies assessing the ability of donor eggs to repair spermatozoa with compromised chromatin [12]. Nevertheless, this ooplasmic intervention can be defective in certain couples, particularly in cases where the woman is of advanced reproductive age.

After observing couples with unexplained infertility after a failed IUI, we designed a reproductive treatment algorithm based on a sperm DNA fragmentation assay [13]. According to this algorithm, if the male gamete chromatin integrity is confirmed, the couple can proceed to standard in vitro insemination. Conversely, if an abnormal SCF is reported, the couple is offered ICSI at the outset, utilizing ejaculated spermatozoa. ICSI would also be offered to men who, in spite of having spermatozoa with healthy chromatin, failed to achieve a pregnancy with standard in vitro insemination. Finally, for cases in which a pregnancy has not been achieved even with ICSI, men are counseled by a reproductive urologist and offered a cycle of ICSI using spermatozoa retrieved directly from the testicle [13, 14], where SCF appears to be consistently lower [15, 16].

Testicular biopsy, however, is a radical and invasive procedure that may result in scarring of the seminiferous tubules with consequent overall impairment of spermatogenesis. Moreover, the correlation between motility, particularly the progressive type, and sperm chromatin integrity prompted us to investigate alternative and more conservative procedures. Among various options, microfluidic devices have been proposed to select more suitable spermatozoa [15, 17‐20] with higher chromatin integrity and presumably greater fertilizing potential and an improved ability to support embryonic development. Since these novel devices can be sophisticated, expensive, and at times cumbersome, simpler methods have been tested. A recent report has shown the ability, and presumed clinical safety, of a chip sperm sorting device to select directly from the raw semen highly motile spermatozoa characterized by superior chromatin integrity [21].

In this investigation, SCF was assessed on aliquots of sperm specimens selected by a commercial microfluidic sperm sorting device (ZyMōt™ Multi device; DxNow, Gaithersburg, MD) and standard density gradient in comparison with raw semen. In addition, in couples with recurrent assisted reproductive technology (ART) failure and male partners with high sperm DNA fragmentation, we assessed the fertilization and implantation potential, as well as the ability to support embryonic development, of spermatozoa processed by microfluidic sperm sorting as compared with conventional density gradient centrifugation.

To test the efficacy of this novel selection method, particularly its ability to identify the highest progressively motile spermatozoa characterized by optimal chromatin integrity, we screened 48 men for SCF levels using raw semen, and after processing by DGS and MSS. Semen specimens were obtained from 23 men and SCF was assessed following 3 aliquots from each specimen: raw, density gradient, and microfluidic processing. Although the sperm concentration following MSS decreased (P < 0.0001), motility and normal morphology significantly improved (P < 0.0001). Furthermore, sperm DNA fragmentation decreased from 20.7% in the raw specimen to 1.8% after MSS (P < 0.0001; Table 1 and Fig. 2).

Encouraged by these findings, we then applied the same method to couples who underwent ICSI. This cohort included 25 couples (maternal age, 37.3 ± 6 years; paternal age, 44.4 ± 10 years). The superovulation protocol of the female partners and the couples’ age, BMI, history of infertility, ethnicity, indication to infertility, smoking and drinking habits, and the use of recreational drugs were comparable or controlled for among these 25 patients.

Semen characteristics and SCF were evaluated before and after DGS and MSS processing for each patient, who therefore served as their own control (Table 2). While there was a decrease in concentration (P < 0.0001), a concurrent improvement in total motility, progressive motility, and morphology of the microfluidic sorted aliquots was seen (P < 0.0001). SCF dropped below threshold after density gradient selection (P < 0.0001) and fell dramatically after microfluidic sorting (P < 0.0001; Table 2).

Next, to compare the impact of selected spermatozoa on embryo developmental competence, we treated four couples with male partners who had a remarkably elevated SCF in their ejaculate (34.1%). The semen parameters for microfluidics as well as the SCF are depicted in Table 3. In this group of men, chromatin integrity improved dramatically (1.6%) when spermatozoa went through the microfluidic chamber (P < 0.02) (Fig. 1). There were also significant improvements in motility (P < 0.02), with a minor improvement in spermatozoa morphology. These couples (maternal age, 35.9 ± 4 years; paternal age, 40.0 ± 6 years) underwent 11 ICSI cycles with DGS and achieved 26.3% good-quality embryos with a fertilization rate of 59% that yielded a biochemical pregnancy and a pregnancy loss at 7 weeks. Once MSS spermatozoa were used in four cycles, a comparable fertilization rate (61.2%) was achieved with a trend toward a larger proportion of good-quality embryos (57.1%) that resulted in an implantation rate of 25% and a clinical pregnancy rate of 50% (Table 4).

We then included 5 couples (maternal age, 36.4 ± 5 years; paternal age, 36.7 ± 7 years) who planned to have their embryos screened by PGT-A, following ICSI. When sperm samples from the male partners in this group were processed by DGC, the motility significantly improved (28% raw vs 51% DGS; P < 0.05), while sperm morphology and SCF remained comparable. After using MSS, there was a clear and net improvement in motility (28% raw vs 99% MSS; P < 0.0001) and SCF (19.2% raw vs 1.5% MSS; P < 0.01) (Table 5). There was also a reduction in concentration, but this was not detrimental to ICSI insemination. These couples underwent 12 ICSI cycles where semen specimens were processed by DGS, resulting in a satisfactory fertilization rate (78%) and an adequate number (23/33, 69.7%) of morphologically good-quality embryos. PGT-A identified 10 euploid embryos that generated a pregnancy, which did not go to term. In the 9 cycles in which MSS was used, there was a comparable fertilization rate (72.8%) as well as number (27/38, 71%) of good-quality embryos of which half were confirmed euploid. Thus far, 4 embryos were transferred in four couples, all of which implanted and resulted in ongoing clinical pregnancies (P < 0.01; Table 6).

Finally, 16 couples that had been treated at other clinics and failed to achieve a pregnancy underwent ICSI solely with MSS at our center. In a preliminary comparison of semen parameters following DGS and MSS, motility (59.3% DGS vs 97.6% MSS; P < 0.02) and morphology (2.1% DGS vs 3.3% MSS; P < 0.05) increased dramatically while SCF (18.5% DGS vs 1.2% MSS; P < 0.02) decreased (Table 7). Of these couples, 8 underwent 13 ICSI cycles elsewhere, yielding a fertilization rate of 74.1%, but no pregnancies were reported. A total of 9 couples underwent 12 ICSI cycles with MSS and fresh embryo transfer, with a 74% fertilization rate and 83.3% (20/24) good-quality embryos. These cycles resulted in 25% (6/24) implantation and 50% (6/12; P < 0.05) clinical pregnancy rate (Table 8). The remaining 7 couples who underwent 8 ICSI cycles with MSS opted for PGT-A due to their history of having all chromosomally abnormal embryos. In these couples, a fertilization rate of 78.4% (62/79) progressed to 62.1% (23/79) good-quality embryos of which 51% (19/37; P < 0.001) were euploid. Five embryos were transferred in four patients who achieved an 80% (4/5; P < 0.01) implantation and clinical pregnancy rate (P = 0.01) (Table 8).

Unexplained infertility may be attributed to a subtle male factor linked to compromised sperm DNA integrity. During ICSI, it is possible to select the most progressively motile spermatozoa that therefore have the highest chromatin integrity [6]. Moreover, it has been known for some time that once the male genome penetrates the oocyte, ooplasmic mechanisms decondense sperm chromatin and reconstitute nucleosomes by replacing protamine with histones while processing and repairing DNA strands [9, 10, 28, 29]. Therefore, a young oocyte along with the selection of the most motile spermatozoa for ICSI insemination may prevent these male genomic defects [10, 11, 29].

In the most persistent cases of male factor infertility, utilizing spermatozoa retrieved directly from the testis, presumed to retain superior chromatin integrity, has been proposed [13, 14, 30]. However, this technique is not without drawbacks, as gametes retrieved directly from the seminiferous tubules can have lower fertilization capacity, which is typical of testicular spermatozoa. Moreover, microsurgical testicular biopsy, although focused on individual tubules, is an invasive and costly procedure that may be associated with damage of the surrounding tissue with consequent scarring, requiring approximately 6 months to heal [31].

These complications led us to investigate alternative methods of identifying spermatozoa with a less impaired genome [32‐34]. Indeed, this can be achieved to some extent with a standard sperm selection technique such as DGS, which appears to lower the level of SCF [35]. This well-established method, however, does not lower SCF below the normal threshold due to its centrifugation steps that generate reactive oxygen species (ROS) and its inability to select the most progressively motile spermatozoa [36‐38]. Therefore, the known direct relationship between sperm progressive motility and chromatin integrity persuaded us to test a novel device that selects the most motile spermatozoa without having to centrifuge the specimen (Tables 1 and 2).

Our initial goal was to find a way to treat couples that had an impairment in male genomic integrity and a prior history of ART failure without resorting to invasive surgical techniques. The enhancement of motility, progression, and morphology we observed with the use of MSS was encouraging, but it was not as important as the remarkable reduction to SCF (Table 1), as has been confirmed by others [21]. These findings encouraged us to test this method of sperm sorting on 25 couples struggling with compromised SCF and persistent ART failure. The same effects of MSS on semen parameters were seen when preparing these semen samples for ICSI treatment. Intriguingly, we were able to achieve an average SCF lower than that of the 23 men we tested initially, even though this group of 25 had higher SCF compared with those men (Table 2).

In couples who had a fresh embryo replacement, MSS yielded spermatozoa with higher genomic competence as demonstrated by their ability to establish healthy pregnancies compared with cycles in which spermatozoa were processed in a standard fashion (Table 4). This was also the case for other groups that had spermatozoa selected using an apoptotic marker [39]. Interestingly, this was even true for couples who underwent PGT-A, as confirmed by the higher portion of euploid embryos that resulted in no term pregnancies (Table 6). In this cohort, MSS yielded a comparable proportion of euploid embryos and, for those couples who received a transfer by the time of this publication, all resulted in a viable pregnancy (Table 6). This remained true when we treated 16 couples with a history of recurrent implantation failure who, once treated by MSS, reported a clinical pregnancy rate of 58.8% (Table 8).

From these observations, it appears that a phenotypic selection of spermatozoa with the highest motility may yield a greater number of euploid embryos capable of resulting in a viable pregnancy. While it is difficult to comprehend how selecting spermatozoa with higher chromatin integrity would have any impact on the number of euploid embryos generated, this can only be explained by the type of nicks and breaks present within the chromatin [10, 28, 40]. When an SCF assessment was carried out with an alkaline Comet assay to detect single-strand DNA breaks and a neutral Comet assay to identify double-strand DNA breaks, the frequency of these chromatin defects was equivalent at about 50% in the fertile control as well as in oligo-astheno-terato-zoospermic patients [41]. Indeed, double-strand breaks may actually affect the chromosomal integrity of the male genome with consequent contribution to embryo aneuploidy, as has been seen in couples with idiopathic and recurrent pregnancy losses [42]. However, this will require further research utilizing an assay capable of specifically detecting this type of DNA damage, such as the neutral Comet assay.

Within the quest for an optimal spermatozoon to inject during ICSI, this study proposes a non-invasive method to recover spermatozoa with superior genomic competence for ICSI treatment in men with high SCF in their ejaculate. These findings allow us to reconsider the relevant contribution of the male genome to the normal development of the conceptus. Most importantly, this study proposes a conservative approach to treat couples with recurrent ART and/or implantation failure without resorting to testicular biopsy.