P–580 Levels of the constitutive heterochromatin (cHC) mark H3K9me3 in human sperm in relation to IVF outcome and preimplantation embryo development
Authors: Holleman K , Va. Marion E , Eleveld C , Koster W , Laven J , Poot R , Baart E 
Published: August 2021

Abstract

Study question
Can we quantify the variation in epigenetic signature of constitutive heterochromatin (cHC) in human sperm using Western blot analysis and correlate this with pregnancy outcome after IVF?

Summary answer
Variation in the quantified H3K9me3 to H3 showed an association with embryo morphokinetics and a H3k9me3/H3 ratio &lt;0,5 is negatively associated with pregnancy outcome.

What is known already
In human sperm, 5–15% of the DNA remains associated to histones. We previously showed that human mature spermatozoa still exhibit the canonical marks H3K9me3 and H4K20me3 on cHC regions and transmit these nucleosomes to the embryo after fertilization. Additionally, other groups reported an increase in the nucleosome/protamine ratio when sperm from male factor subfertility patients were compared with sperm from fertile men, indicating that incomplete chromatin remodelling during spermatid elongation may affect fertility. Thus, variations in sperm chromatin occur during human spermatogenesis that go undetected by current sperm quality tests and may have clinical significance for IVF treatment outcomes.

Study design, size, duration
An observational study of patient couples (n = 53) undergoing IVF treatment at the Erasmus MC, Rotterdam, between 2017 and 2018. Inclusion criteria were normospermia according to the WHO criteria, at least 4 oocytes harvested after ovum pickup and an embryo-transfer. After routine insemination, resulting embryos from 38 cycles were cultured in the EmbryoScopeTM and developmental timings up to the 8-cell stage were recorded. The primary outcome measure was ongoing pregnancy (ultrasound at 12 weeks of gestation).

Participants/materials, setting, methods
Surplus sperm samples from 53 IVF cycles were lysed at a concentration of 200x106 spermatozoa/ml in RIPA buffer. Lysates were loaded and separated on SDS-PAGE gels and transferred onto blotting membranes. After incubation with antibodies against H3 and H3K9me3 and fluorescent detection, signal intensity was quantified using Image Studio Lite (LI-COR Biotechnology)). Additionally, time-lapse developmental kinetics of 130 transferred and cryopreserved embryos of 38 patient couples were analyzed.

Main results and the role of chance
No significant differences were found in patient characteristics between the pregnant and non-pregnant group. However, fertilization rate was found to be a confounding factor; no pregnancies occurred below a fertilization rate of 40% and only 4 occurred below a fertilization rate of 60%. The median H3k9me3/H3 ratio in the pregnant group was 1.01 (interquartile range (IQR): 0.69–1.16) and in the non-pregnant group 0.896 (IQR: 0.54–1.06), this difference was not significant. When using logistic regression analysis, adjusted for the fertilization rate (dichotomized as ≤ 60% or &gt; 60%), we observed a positive association between the H3k9me3/H3 ratio and ongoing pregnancy (odds ratio (OR) 6.43 (95% confidence interval (CI): 1.12–36.8; p-value 0.037). More importantly, none of the samples with a ratio of 0.5 or lower resulted in an ongoing pregnancy (n = 7). After classifying sperm samples into quartiles according to the H3K9me3/H3 ratio, we observed that resulting embryos reached the 4-cell stage faster with sperm from to the lowest quartile of H3K9me3/H3 ratio, compared to the highest quartile (beta –4.4 hours, p-value 0.05). Our results point to an association between the H3k9me3/H3 ratio, embryo development and pregnancy outcome.

Limitations, reasons for caution
This initial analysis is performed with a relatively low number of samples. The OR of 6.43 has a wide 95% CI, probably due to low statistical power. Our observations await confirmation in a larger data set.
Wider implications of the findings: Semen analysis remains the cornerstone of male infertility diagnosis, despite its low prognostic value. Sperm epigenetic cHC inheritance likely has an important impact on early embryo development. Therefore, the H3k9me3/H3 ratio could serve as a parameter for sperm quality and aid in the prediction of pregnancy outcome.

Trial registration number
Not applicable