Sperm DNA fragmentation is a novel biomarker for early pregnancy loss
doi.org/10.1016/j.rbmo.2020.09.016
Background
It is reported that about 10-15% of couples experience spontaneous pregnancy loss, while recurrent pregnancy loss occurs in 1-2% of couples. Despite numerous studies, 50% of miscarriages remain unexplained.
The inability to identify the cause of these miscarriages makes it difficult to offer a valid treatment option for the patients. It is common knowledge that the sperm cell and egg each make a 50% contribution to the final DNA of the embryo, but it is only in recent times that some experts have identified a link between spermatozoic DNA damage and miscarriage.
Paternal genetic information is transmitted to the embryo via sperm DNA and any damage to this DNA could have a negative impact on pregnancy outcome. It is therefore essential that studies measure the extent of DNA damage thoroughly to correctly interpret the impact it can have on pregnancies.
However, to date only a few studies have used highly sensitive tests such as the Comet assay to analyse DNA damage in sperm cells.
Aim
To determine whether or not Comet assay can act as a biomarker for early pregnancy loss by comparing DNA quality between fertile men and men whose partners experienced early pregnancy loss.
Methodology
In this retrospective study, the participants were divided into 2 groups, while any men diagnosed with hereditary diseases, HIV, syphilis, hepatitis B and C, herpes, cytomegalovirus and other bacterial infections were automatically excluded from the study.
Group A, included 76 fertile men aged between 18-45 years. Group B included 217 men (202 men between 25-45 years old and 15 men over 45 years old) whose partners had suffered first-trimester miscarriages.
These 217 men were then divided into subgroups, depending on the miscarriage history of their partner:
- First miscarriage after natural conception (n = 18)
- Two or more miscarriages after natural conception (n = 147)
- First miscarriage after ART conception (n = 19)
- Two or more miscarriages after ART conception (n = 7)
- Miscarriage after biochemical pregnancy (n = 67)
All semen samples were collected after 2-5 of abstinence, while samples from men in Group B were taken within 3 months of miscarriage to ensure the sperm cells collected were inthe same spermatogenic cycle as the miscarriage.
The samples from fertile men (Group A) were cryopreserved using a fast freezing protocol and cryoprotectant, while samples from Group B participants were plunge frozen without any cryoprotectant. (Unpublished data revealed no statistically significant differences between the 2 methods).
Sperm DNA damage was then analysed using the alkaline Comet assay (single cell gel electrophoresis assay) in a temperature and humidity-controlled environment.
The following variables was then analysed and compared between each group:
- Average Comet score (ACS), which is the mean percentage fragmentation of all Comets scored
- Low Comet score (LCS), refers to the percentage of Comets with low DNA fragmentation
- High Comet score (HCS), refers to the percentage of Comets with high DNA fragmentation
This was followed by receiver operating curve analysis to evaluate the ability of each Comet variable to diagnose miscarriage.
Results
Initial analysis of population demographics and lifestyle factors, revealed no statistically significant differences between the groups, in terms of male age, alcohol use and smoking.
Unsurprisingly, analysis of sperm DNA levels showed Group A (fertile donors) had very low levels of DNA damage, with 98% of samples considered to be of good quality. On the other hand, only 16% of Group B samples had good quality DNA, while the rest had medium to high DNA damage.
| DNA Damage (%) | 0-5 | 6-10 | 11-15 | 16-20 | 21-25 | 26-30 | 31-35 | 36-40 | 41-45 | 46-50 | 51-55 | 56-60 |
| Group A (%) | 4 | 20 | 34 | 22 | 18 | 2 | 0 | 0 | 0 | 0 | 0 | 0 |
| Group B (%) | 1 | 0 | 2 | 2 | 11 | 22 | 21 | 22 | 12 | 4 | 2 | 1 |
Similarly, analysis of the 3 Comet parameters (ACS, LCS, HCS), revealed statistically significant differences between the fertile and miscarriage group overall.
| ACS (%) | LCS (%) | HCS (%) | |
| Group A – Fertile | 14.9 | 90.2 | 2.0 |
| Group B – Miscarriage | 33.3 | 47.0 | 14.5- |
| P-value | <0.001 | <0.001 | <0.001 |
All 5 Group B subcategories were also found to be significantly different to Group A (fertile donors) in terms of ACS, LCS, and HCS (P<0.001).
| ACS (%) | LCS (%) | HCS (%) | |
| Group A | |||
| – Fertile | 14.9 | 90.2 | 2.0 |
| Group B | |||
| – First miscarriage (natural) | 35.4 | 45.9 | 19.3 |
| – Two or more miscarriages (natural) | 32.7 | 47.5 | 12.7 |
| – First miscarriage (ART) | 32.2 | 51.2 | 11.9 |
| – Two or more miscarriages (ART) | 31.9 | 51.9 | 14.3 |
| – Miscarriage after biochemical | 33.1 | 47.8 | 14.6 |
Interestingly, levels of sperm DNA damage between one (first) and two or more miscarriages (recurrent) was very similar. This points to the involvement of other male or female factors in recurrent miscarriage.
Lastly, Receiver Operator Characteristic (ROC) analysis of the 2 main groups confirmed that the 3 Comet scores were able to differentiate between live births and miscarriages with high accuracy.
| Threshold value (%) | Sensitivity (%) | Specificity (%) | PPV | NPV | |
| ACS | ≥25.6 | 83.5 | 100 | 1.00 | 0.68 |
| LCS | ≤70.0 | 84.9 | 97.4 | 0.99 | 0.69 |
| HCS | ≥2.0 | 86.7 | 73.7 | 0.90 | 0.66 |
SUMMARY: CAN MISCARRIAGE BE CAUSED BY MALES
In this study, miscarriage was significantly linked to male partners with double the amount of sperm DNA damage (fragmentation). Specifically, male sperm DNA damage ≥ 25.6% (ACS) was almost certainly going to result in miscarriage for their partner (PPV = 1.00).
Limitations
- Small study size.
- Only 15 men were over 45 years old, which is usually the age when paternal age starts to impact pregnancy outcome.
Funding
This study was funded by a manufacturer of Comet assays (Examenlab Ltd).
Glossary
ART (Assisted Reproductive Technology)
Any fertility treatment in which either eggs or embryos are handled (i.e. IVF, ICSI).
Comet assay
A simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells.
Cryoprotectant
A substance used to protect cells or tissues from damage during freezing.
Cytomegalovirus
A common herpes virus.
DNA fragmentation
The separation or breaking of DNA strands into pieces.
NPV (Negative Predictive Value)
The probability of people who have a negative test result and truly do not have the disease (or outcome).
P-value
The probability that a result occurred by random chance.
Paternal
Father.
PPV (Positive Predictive Value)
The probability of people who have a positive test result and actually have the disease (or outcome).
Receiver Operator Characteristic (ROC)
A graphical plot of sensitivity vs. false positive rate (1-specificity).
Sensitivity
The proportion of people with the disease who will have a positive result.
Specificity
The proportion of people without the disease who will have a negative result.
Similar studies
Jayasena C N, et al. (2019). Reduced Testicular Steroidogenesis and Increased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage. https://doi.org/10.1373/clinchem.2018.289348
Carlini T, et al. (2017). Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss. https://doi.org/10.1016/j.rbmo.2016.09.014
Leach M, et al. (2015). Sperm DNA fragmentation abnormalities in men from couples with a history of recurrent miscarriage. https://doi.org/10.1111/ajo.12373
Robinson L, et al. (2012). The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and meta-analysis. https://doi.org/10.1093/humrep/des261
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