DNA damage in prepared semen is associated with fertilisation rate in assisted reproduction technology (ART) treatment

doi:10.21203/rs.3.rs-3116263/v1

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DNA damage in prepared semen is associated with fertilisation rate in assisted reproduction technology (ART) treatment

https://doi.org/10.21203/rs.3.rs-3116263/v1

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Purpose

This study aims to determine to what extent sperm DNA damage is associated with semen quality and assisted reproduction technology (ART) treatment outcomes.

Methods

105 male partners of couples attending for infertility treatment were recruited and DNA integrity was measured by a neutral Comet assay and N7-methyldeoxyguanosine (N7-MedG) DNA levels by an immunoslotblot in sperm prepared by density gradient centrifugation for treatment use. Associations between measures of DNA damage (%tail DNA, proportion of sperm with either low (LDD) or high (HDD) levels of DNA damage, and N7-methylguanine levels), semen quality (concentration and motility) and ART outcomes (% oocytes fertilised, % embryo fragmentation, % cleavage, and birth outcome) were assessed.

Results

In the prepared sperm samples, DNA damage was significantly associated with semen quality and was lower than that in the original neat sample. % fertilisation was significantly negatively associated with N7-MedG levels, %HDD and % tail DNA and was positively associated with %LDD. % cleavage and live birth in fresh cycles were not associated with DNA damage but there was evidence that %LDD was lower, and %HDD higher, in couples with live births after a frozen embryo transfer cycle.

Conclusion

DNA damage can negatively impact on semen quality and fertilization rate but not embryo cleavage or live birth rate. These results suggest that the impact of sperm DNA damage on pregnancy outcomes appears in the early stage of embryo development.

DNA damage

DNA integrity

Comet assay

Semen quality

ART outcomes

Losses in sperm DNA integrity due, in part to single or double stranded DNA breaks have been associated with poor semen quality and adverse ART outcomes [1–3]. In somatic cells, if single strand breaks persist, highly toxic double strand breaks can be formed [4] and similarly single and double stranded DNA breaks have been reported to have different reproductive effects [3]. It is thus important to be able better characterise losses in DNA integrity and the reproductive consequences of specific types of damage. Current methods of measuring DNA integrity such as the Comet assay [5], Tunel assay [6], sperm chromatin dispersion assay/ halo assay [7], sperm chromatin structure assay [8] tend to detect a range of different types of DNA damage [3, 9]. Other assays that reportedly detect specifically single or double stranded DNA breaks have also been described [10].

The alkaline comet assay is widely used in the analysis of sperm DNA integrity [3, 5, 9] and an in vivo mammalian alkaline comet assay is used to detect gene mutations and/or clastogenicity [11]. This assay has been described as detecting single and double strand DNA breaks resulting from direct interactions with DNA, alkali-labile sites or transient strand breaks that can occur during DNA repair [5, 11]. The contribution then that each type of damage makes to the overall response is unclear. Other variants of this assay have been described such as the enzyme modified Comet assay [12] which detects strand breaks induced by enzymatic removal of DNA base damage and the neutral comet assay which has recently been described as detecting double strand breaks better than the alkaline Comet assay [3, 13]. For example, Alu1 induced DNA fragmentation was higher with the neutral Comet assay whereas H₂O₂ induced fragmentation was higher with the alkaline Comet assay [14]. Further evidence that the type of DNA damage detected by these two assays varies has been described, with age being associated with increased DNA damage as measured by the alkaline but not the neutral Comet assay [15] and cryopreservation increasing sperm DNA fragmentation as detected by the alkaline but not the neutral Comet assay [16].

Direct comparisons between the alkaline and neutral Comet assays in sperm have produced conflicting results with either DNA damage levels being comparable [14], or higher [16, 17] or lower [18, 19] using the neutral Comet assay. The relationship between DNA damage detected by the neutral Comet assay and semen quality is also currently unclear. We previously reported that DNA damage as measured by the neutral Comet assay was negatively associated with semen quality in a univariate analysis but not after adjustment for N7-MedG levels [20]. In other studies DNA damage has been negatively associated with sperm concentration [21], motility [18, 22] and morphology but other studies have reported no association with sperm concentration [15, 18, 22], motility [15] or morphology [15]. This may reflect differences in study protocols or populations and also variations in how the DNA damage was characterised [23].

Evidence linking DNA damage as measured by the neutral comet assay to reproductive outcomes is also contradictory. It has been suggested double strand breaks have more impact on reproductive outcomes than single strand breaks [3]. In our earlier study, DNA damage was associated with the percentage of embryos which cleaved following ICSI only [20]. Similarly, the fertilization rate, but neither the percentage of good quality embryos nor the pregnancy rate was higher in those samples with lower DNA fragmentation levels [22].

To provide further information on the associations between DNA damage as assessed by the neutral Comet assay and ART outcomes, DNA damage in prepared human sperm of men of couples undergoing ART treatment was measured and the associations between DSBs and ART treatment outcomes (% fertilization, % cleavage, and live birth) determined.

Study population

Men of couples attending the Department of Reproductive Medicine, St Mary’s Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, for ART treatment participated in this study

Couples were initially asked about their willingness to take part in ongoing research within the Department and those willing to do so provided with further information about these studies. All couples then provided written informed consent for research and this study had Local Ethics Committee Approval (Central Manchester REC ERP/91/078, HFEA research licence R0026). Inclusion criteria were therefore that (i) the participants were part of a couple attending for infertility treatment, (ii) had the ability and willingness to give informed consent and (iii) had proficiency to speak English and complete a questionnaire. After 2–5 days of recommended abstinence, male participants provided, by masturbation, a semen sample on the day of ART treatment. After liquefaction, semen volume, sperm concentration and motility were determined manually according to WHO guidelines [24]. Subsequently, 1 ml or more, as required, of the semen sample underwent density gradient centrifugation to obtain sperm for the ART treatment using SpermGrad™ and washed in SpermRinse™ (both Vitrolife, Sweden) [25]. Sperm concentration and motility were also determined manually in this prepared sample.

ART procedures

Ovulation induction and egg collection were achieved as described previously [26]. For conventional IVF, all oocytes were inseminated with ~ 1×10⁵ prepared sperm/ml in IVF culture medium (G-IVF™, Vitrolife, Sweden), incubated at 37˚C with 6% CO₂ in air. For ICSI treatment, a single motile sperm with normal morphology was microinjected into each MII oocyte. 16–18 hours after insemination or injection, oocytes were examined for the presence of two pronuclei, which is indicative of successful normal fertilization, and the fertilized oocytes were incubated in G1™ and G2™ (Vitrolife, Sweden) to cleave. 48–72 hours after insemination/injection, one or two good quality embryos were transferred into the uterus, according to Unit protocols in place at the time. Implantation of the embryo in the utero was assessed as previously described [26].

Single-cell gel electrophoresis (Comet) assay

DNA damage was assessed by a neutral Comet assay in an aliquot of the prepared sperm sample [21, 27]. Briefly, sperm were suspended in LMP agarose and applied to a microscope slide pre-coated with NMP agarose. After gel setting, sperm were lysed in buffers containing DTT and proteinase K and the sample underwent electrophoresis. The slides were neutralised, stained with SYBR gold and examined using an Olympus fluorescence microscope (OLYMPUS, U-RFL-T; Filter: FITC; Camera: Q IMAGING, RETIGA-SRV, FAST 1394; Software: QCapture Pro 6.0), images obtained and the % tail DNA was determined by using TriTeK CometScoreTM Freeware version 1.5 (TriTeK Corp, US).

N7-MedG slotblot analysis of sperm DNA

N7-MedG levels in sperm DNA were determined by immunoslotblot analysis as described previously [28, 29]. Briefly, DNA was extracted from the prepared sperm sample that remained after Comet analysis using a Qiagen Blood and cell culture DNA Midi Kit [28]. The DNA samples were sonicated and incubated in alkaline solution to induce imidazole ring opening. After neutralisation, DNA samples were heat denatured and immobilised on a nitrocellulose membrane. Membranes were incubated first with a rabbit polyclonal antibody raised against 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy) conjugated to keyhole limpet hemocyanin which specifically detected N7-MedG in mice treated with N-Nitroso-N-methylurea [30] and then a polyclonal goat anti-rabbit-horseradish peroxidase conjugate. Membranes were incubated with ECL reagent (Amersham Hyperfilm) and exposed to an X-ray film which was subsequently developed and scanned. The intensity of the bands for the samples and the standards was quantified by using ImageJ, a standard curve generated and N7-MedG levels in the samples were calculated.

Statistical analyses

Sperm DNA damage (% tail DNA) was not normally distributed and was characterised into two groups namely the % of sperm with low DNA damage (LDD sperm) with < 0.005% tail DNA and those sperm with high DNA damage (HDD sperm) with ≥ 7.5% tail DNA with cutoffs being determined prior to subsequent analysis [20]. Correlations between SLDD, SHDD and median % tail DNA and ART outcomes (% fertilization [number of fertilized oocytes (with 2 pronuclei) ×100 /number of MII oocytes used in the treatment cycle] and % cleavage (the number of fertilized oocytes developed to embryos regardless of the embryo quality×100 /number of fertilized oocytes) and live birth were determined. One-way Anova was also used to compare male age, female age and demographic data between the live birth and non-live birth groups.

Description of study population

105 couples were initially recruited but one couple was subsequently excluded as no eggs were produced following induction of ovulation. Of the remaining 104 couples, 49 underwent IVF and 55 ICSI. A further 4 couples were excluded after in vitro fertilisation as either the fertilisation failed or the embryo was not normal (Fig. 1). 95 couples underwent a fresh embryo transfer and of these 25 also underwent a frozen embryo transfer as they did not initially have a live birth (n = 24) or had a late miscarriage (n = 1). There was no difference in male and female age in those couples who did not have a live birth either through fresh embryo transfer only and those who underwent both a fresh and frozen embryo transfer (data not shown). 5 couples had all their embryos initially frozen. Demographic details of these three groups of participants (ie couples who underwent fresh transfers only, frozen transfers only and both fresh and frozen transfers) are shown in Table 1. Couples undergoing a frozen embryo transfer had a female partner who had produced more eggs that the female partner of couples undergoing a fresh embryo transfer only (p < 0.005; Table 1).

Table 1

Basic demographics, Semen quality, DNA damage in prepared sperm and treatment outcomes of study population
Variable	Value	All (n = 104)	Fresh embryo transfer only (n = 70) ^a	Fresh and frozen embryo transfer (n = 25) ^b	Frozen embryo transfer only (n = 5) ^c
Male Age	Years; mean ± SD	35.3 ± 5.8	35.4 ± 5.5	34.0 ± 6.6	37.6 ± 4.7
Female Age	Years ; mean ± SD	32.4 ± 4.3	32.8 ± 4.3	30.8 ± 4.0	32.4 ± 2.3
Treatment	IVF/ICSI; n (%ICSI)	49/55 (52.8)	34/36 (51.4)	13/12 (52.0)	1/4 (53.3)
Egg number	n; mean ± SD	8.6 ± 5.3	8.1 ± 5.2**	10.1 ± 4.4**	14.4 ± 5.3**
Semen quality	Sperm concentration (x10⁶/ml); mean ± SD	25.4 ± 31.7	26.5 ± 32.7	27.7 ± 32.9	14.0 ± 19.6
	% Progressively motile sperm; mean ± SD	62.0 ± 21.7	62.8 ± 21.4	62.1 ± 19.9	64.2 ± 26.7
	% Immotile sperm; mean ± SD	26.9 ± 22.4	26.0 ± 21.1	26.6 ± 21.6	26.2 ± 29.0
Alkylation	N7-MedG fmoles/µg DNA; mean ± SD (n)	0.26 ± 0.21 (16)	0.28 ± 0.22 (11)	0.21 ± 0.22 (5)	ND
DNA integrity	Median tail DNA (%); mean ± SD	1.1 ± 3.0	0.9 ± 2.0	1.4 ± 4.9	0.8 ± 1.0
	% Sperm with low DNA damage (LDD); mean ± SD	25.2 ± 12.2	25.5 ± 12.0	26.7 ± 13.4	22.8 ± 9.6
	% Sperm with high DNA damage (HDD); mean ± SD	13.2 ± 13.2	13.2 ± 12.9	11.0 ± 12.3	17.7 ± 16.4
Treatment outcome	% Fertilisation; mean ± SD	71.6 ± 27.1	73.4 ± 24.7	78.0 ± 23.7	66.0 ± 21.9
	% Cleavage; mean ± SD	77.9 ± 29.6	85.4 ± 22.2*	76.8 ± 25.1*	41.1 ± 38.6^*
	live birth/no live birth; n (% live birth)	39/61 (39.0) ^e	27/43 (38.6)	10/15 (40.0)	2/3 (40.0)
^a Couples underwent fresh embryo transfer only; ^b Couples underwent fresh embryo transfer and then frozen embryo transfer; ^c couples underwent frozen embryo transfer only
* p < 0.05 **p = 0.005

DNA damage in prepared sperm

There was no difference in levels of DNA damage markers (either DNA alkylation or DNA integrity) between couples who underwent fresh or frozen embryo transfer (Table 1). Overall, % of sperm with low DNA damage (% LDD sperm) was inversely correlated with % of sperm with high DNA damage (% HDD sperm (r_s = -0.51, p < 0.001 n = 104) and with median % tail DNA (r_s = -0.93, p < 0.001 n = 104) whereas % HDD sperm was positively correlated with median % tail DNA (r_s = 0.65, p < 0.001 n = 104). N7-MedG levels in sperm DNA were not correlated with any markers of DNA integrity in prepared sperm.

Sperm concentration, % progressively motile sperm and % immotile sperm were all significantly associated with all markers of DNA integrity but not DNA alkylation (Supplementary Table 1). Sperm concentration and % progressively motile sperm were both negatively associated with % HDD sperm and % tail DNA but positively associated with %LDD sperm. In contrast, % immotile sperm was negatively associated with % LDD sperm but positively associated with % HDD sperm and median % tail DNA.

N7-MedG levels in prepared sperm were significantly lower than those in the original neat sample (Supplementary Table 2), with three prepared samples having no detectable N7-MedG although N7-MedG had been detected in the neat sample. Levels of %LDD sperm were significantly higher in the prepared samples (Supplementary Table 2) but there was no difference in % HDD or median %Tail DNA between neat and prepared sperm samples.

Associations between treatment outcomes and DNA damage in prepared sperm

In all couples, N7-MedG levels and median % tail DNA and % HDDs in prepared sperm were significantly and negatively associated with % fertilization while % LDDs in prepared sperm was positively and significantly correlated with % fertilization (Table 2). In contrast N7-MedG levels and markers of DNA integrity were not correlated with % cleavage.

Table 2

Correlation between % fertilisation and cleavage with markers of DNA damage in prepared sperm samples
DNA marker	Variable	Spearman Rho (n)
DNA marker	Variable	% fertilisation	% cleavage
Alkylation	N7-MedG (fmoles/µg DNA)	-0.50 (16) *	-0.02 (16)
Integrity	% Sperm with low DNA damage (LDD)	0.27 (104) **	0.13 (104)
Integrity	% Sperm with high DNA damage (HDD)	-0.26 (104) **	0.02 (104)
Integrity	% tail DNA (median)	-0.24 (104) *	-0.14 (104)
* p < 0.05; ** p < 0.01

Levels of markers of DNA alkylation and DNA integrity did not differ in those men whose partner did or did not have a live birth as a result of fresh embryo transfer (Table 3) or whether the treatment was by IVF or ICSI (Supplementary Table 3). In contrast, in those couples who underwent a frozen embryo transfer, % LDD was lower, but %HDD was higher, in sperm whose frozen embryo transfer resulted in live birth compared to those whose transfer did not result in live birth (Table 3), especially when the couple’s treatment was ICSI (Supplementary Table 4).

Table 3

Comparison of DNA damage levels and birth outcome in fresh and frozen cycles
DNA marker	Variable	Fresh cycles (n = 95) ^a		Frozen cycles (n = 30) ^b
DNA marker	Variable	No live birth (n)	Live birth (n)	No live birth (n)	Live birth (n)
Alkylation	N7-MedG (µmol/mol dG)	0.27 ± 0.27 (9)	0.25 ± 0.13 (7)	0.23 ± 0.27 (3)	0.17 ± 0.21 (2)
Integrity	% Sperm with low DNA damage (LDD)	25.5 ± 12.0 (58)	26.3 ± 13.0 (37)	31.3 ± 12.0 (18)	18.0 ± 9.7 (12) **
Integrity	% Sperm with high DNA damage (HDD)	11.2 ± 10.3 (58)	15.0 ± 15.6 (37)	8.5 ± 4.5 (18)	17.5 ± 19.0 (12)ⱡ
Integrity	% tail DNA (median)	0.76 ± 1.81 (18)	1.51 ± 4.29 (37)	0.27 ± 0.19 (18)	2.84 ± 6.96 (12)
^a Of the 100 couples, 95 underwent a fresh embryo transfer and 5 had all their embryos frozen; ^b In addition to these 5 couples who had all their embryos initially frozen, a further 25 underwent a frozen embryo transfer after their fresh embryo transfer as 24 couples did not have a live birth and 1 had a late miscarriage.
ⱡ p = 0.063; **p < 0.01

In this study of sperm DNA damage of men attending for ART as part of a couple, the main finding was that DNA alkylation and DNA integrity (as measured by the neutral Comet assay) in prepared sperm was associated with a poor fertilisation rate but not with the cleavage rate. In fresh cycles, there was no association between DNA damage and live birth but in frozen cycles there was evidence of a positive association between DNA damage and live birth. DNA damage levels in prepared sperm were negatively associated with sperm concentration and the % of progressively motile sperm suggesting that sperm with high levels of DNA damage were more likely to be immotile. Furthermore, prepared sperm had lower levels of DNA damage than those in the original neat sperm as both DNA alkylation was lower and the % LDD sperm was higher in prepared sperm. These results are, in general, consistent with paternal DNA damage being an important factor in early pregnancy rather than late pregnancy when maternal factors might be predominant [31].

Studies using the alkaline comet assay have reported associations between DNA fragmentation and % fertilization, embryo quality, implantation rate, clinical pregnancy and live birth [32–33]. In contrast, a smaller number of small studies with the neutral Comet assay have reported inconsistent associations between DNA integrity and ART outcomes [17, 21, 22, 34]. Sperm with a high DNA fragmentation index (≥ 14%) was associated with a significantly lower fertilisation rate but not with the % of good embryos nor the pregnancy rate whereas in other studies DNA fragmentation was not associated with fertilisation rate [21, 35] but with cleavage rate [21] and implantation rate [35]. Sperm DNA fragmentation was higher in sperm from men whose partner had repeated miscarriages [17, 34]. In contrast our study found a significant negative association between DNA integrity and fertilisation rate but not with cleavage rate nor birth outcome in fresh cycles. The differences between these studies might reflect analytical and population differences but it is unclear to what extent (if any) the differences might also reflect the neutral and alkaline Comet assays measuring different types of DNA damage. The type of DNA damage detected by either the neutral or the alkaline comet assay has not been fully characterised, but it has been suggested that the neutral Comet assay detects double strand DNA breaks better than the alkaline Comet assay [3, 13]. This, though, is controversial with other authors reporting that the neutral comet assay measures single strand nicks when the DNA remains attached to the nuclear matrix [36]. Given that double strand breaks are one of the most toxic lesions occurring in human cells [4], then stronger associations might well be expected to be detected with the neutral Comet assay. Indeed, one study has suggested that DNA fragmentation as measured by the neutral Comet assay, but not the alkaline Comet assay, was associated with delays in embryo development and impaired implantation [35].

We have previously reported in a separate study of couples undergoing ART treatment that N7-MedG levels were significantly negatively associated with the proportion of oocytes successfully fertilised irrespective of the method of fertilisation used [28]. Our current results in a separate population recruited from the same clinic several years after the initial study also shows an association between N7-MedG levels and fertilisation rate and suggest that both DNA base damage and DNA fragmentation might be key determinants of fertilisation rate and birth outcomes. We previously reported that in neat sperm [20], N7-MedG in sperm DNA was negatively associated with % LDD sperm but not % HDD sperm suggesting different exposures were associated with these markers of DNA damage, which would also suggest that to better characterise risk associated with sperm DNA damage then a holistic approach (of measuring different types of damage) might be needed to better associate damage with adverse birth outcomes rather than focussing on a single type of damage .

Whilst we found no association between DNA damage levels and birth outcome in fresh cycles, we found a positive association between DNA damage levels and birth outcome in frozen cycles. This is contrary to previous data that suggests that DNA damage would negatively impact on live birth. One possible explanation for these results is that we have introduced a form of selection bias as the population who underwent frozen cycles differed from those who had only fresh cycles. Those with frozen cycles had more eggs/embryos to freeze (Table 1), therefore this group of patients may have been ‘selected’ for having surplus good embryos, hence overcoming poor sperm quality. These embryos have also survived freezing and thawing which could be another test of viability. By contrast, women whose partners had low sperm DNA damage might have been more likely to get pregnant in the fresh cycle so taking themselves out of this group.

Although we have measured DNA damage in the corresponding neat sperm samples [20], for this study we further analysed DNA damage in samples post-preparation for IVF/ICSI, in order to relate the damage in sperm “seen by” the egg more directly to the outcomes of the treatment cycles. This is seemingly important as DNA damage levels were generally lower in the prepared sperm in this study. In contrast, a previous study with smaller numbers of men reported no differences in sperm DNA fragmentation as measured by the neutral comet in the neat ejaculate and sperm selected by a Swim-up procedure or for ICSI, though selection by either reduced DNA fragmentation as measured by the alkaline Comet assay [34].

Although this study showed a critical impact of DNA damage on fertilisation rate, we recognise that there are still some limitations with this study and in particular the relatively small number of couples that took part in it. This might potentially explain the lack of a detectable association between DNA integrity and live birth. However, there is increasing evidence that DNA damage can be an important indicator for infertility and men of infertile couples, especially with unexplained infertility [37], have been advised to check the level of DNA fragmentation in their sperm.

Funding

BA was funded by Damascus University in Syria and the British Council. The National Institute for Health Research provided support for patient recruitment.

Conflicts of interest/Competing interests

The authors have no relevant financial or non-financial interests to disclose and have no conflicts of interest to declare that are relevant to the content of this article.

Ethics approval:

This study was carried out in line with the principles of the Declaration of Helsinki and had Local Ethics Committee Approval (Central Manchester REC ERP/91/078, HFEA research licence R0026).

Consent:

Informed written consent was obtained from all individual participants included in the study.

Availability of data and material

The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

All authors contributed to the study conception and design. BA co-ordinated the study and data management was carried out by BA, ACP, HH, GH and DB. BA undertook the lab assays for DNA damage. All members of the group contributed to the collection of data for the study and discussions on the design, conduct and interpretation of the results. The first draft of the manuscript was written by BA and all authors commented on previous versions of the manuscript. All authors, bar BA who is deceased, read and approved the final manuscript.

Acknowledgments

We are indebted to the staff in Saint Mary’s Hospital, Department of Reproductive Medicine for their help and support in the process of recruiting participants for the study and collecting both the semen samples and the data regarding ART treatment outcomes. We would like to thank especially the research nurses Lucy Dwyer and Claudette Wright and the clinical embryologist Tope Adeniyi.

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No competing interests reported.

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DNA damage in prepared semen is associated with fertilisation rate in assisted reproduction technology (ART) treatment

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Study population

ART procedures

Single-cell gel electrophoresis (Comet) assay

N7-MedG slotblot analysis of sperm DNA

Statistical analyses

Results

Description of study population

DNA damage in prepared sperm

Associations between treatment outcomes and DNA damage in prepared sperm

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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