Higher sperm H3K4me3 levels are associated with idiopathic recurrent pregnancy loss

doi:10.21203/rs.3.rs-4901487/v1

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Higher sperm H3K4me3 levels are associated with idiopathic recurrent pregnancy loss

https://doi.org/10.21203/rs.3.rs-4901487/v1

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Background:

During fertilization, spermatozoa contribute genetic and epigenetic factors such as chromatin packaged with protamines and histones; DNA methylome, micro RNAs etc. Human sperm chromatin retains 5–15% nucleosomes which can play a key role in embryonic development. Recurrent Pregnancy loss (RPL) is a condition mainly attributed to defects in embryo and placenta development. Majority of the known RPL factors are of maternal contribution while ~ 50% RPL cases are termed idiopathic (iRPL). Besides paternal genetic factors, epigenetic factors via sperm could also be responsible for iRPL. Hence, we investigated alterations in retained nucleosome content of iRPL sperm (n = 46) as compared to fertile male population (n = 40).

Results:

We measured the relative abundance of core histone H4 and Protamine-2 content along with the modified histones H4Ac, H3K4me3, H3K27me3 and H3K9me3 by flow cytometry. Enrichment of these modified histones at regulatory loci have either transcription activating or repressing roles and Protamine-2 contributes to sperm chromatin compaction. H4 and Protamine-2 levels were comparable in both groups and showed significant negative correlation. The iRPL group was found to have significantly higher levels of sperm H3K4me3 as compared to the fertile control group. The other modified histones and protamine levels showed no alterations among the two groups. Furthermore, we analyzed possible correlation of sperm DNA fragmentation index (DFI) and 5-mC content with the relative sperm core histone H4 levels. Sperm DFI was found to be significantly positively correlated with H4 MFI in both control and iRPL sperm.

Conclusion:

A fraction of the H3K4me3 enrichment is now known to resist embryonic epigenetic reprogramming; and hence, such elevated levels in the sperm would question its developmental competence requiring its implications to be explored further in RPL pathology. Also, incidence of sperm DNA fragmentation is associated with increased histone retention in both fertile and iRPL cases.

idiopathic recurrent pregnancy loss

sperm histones

H3K4me3

sperm DNA fragmentation

5-mC

Inadequate embryonic and/or placental development can lead to pregnancy loss. The loss of two or more consecutive pregnancies prior to 20th gestational week is termed as recurrent pregnancy loss (RPL). For this condition, around only 50% of the cases are diagnosed with known causative factors; whereas the remaining are termed idiopathic (iRPL). Among the known causative factors, majority are of maternal contribution such as anatomic, hematological, autoimmune, genetic, endocrinological defects and disorders [1]. The male contributions reported to be associated with RPL are higher incidences of sperm DNA fragmentation, aneuploidies, chromosomal anomalies and other genetic factors [2]. However, recently the importance of the sperm epigenome for a successful pregnancy has been highlighted. Paternal genome contributions in embryo and placenta development have been implicated by early nuclear transplant experiments and it is found to be vital mainly for placenta formation [3, 4]. The sperm epigenome carries the information for chromatin remodeling and cues for timely expression of development crucial genes. This epigenome consists of DNA methylome, histones and ncRNAs. Studies from our lab have also contributed to understanding the paternal epigenetic contribution to pregnancy related disorders by studying the sperm DNA methylome of male partners of iRPL couples. These studies have uncovered differentially methylated loci within developmental genes implicated in RPL [5, 6].

The human sperm chromatin retains 5–15% nucleosomes which can play a key role in embryonic development. At the end of spermiogenesis process, 85–90% of histones are replaced by sperm-specific proteins: protamines which make the sperm chromatin compact and majorly transcriptionally inert [7, 8, 9, 10, 11]. The retained 5–15% nucleosomes are significantly enriched at developmentally important loci such as imprinted gene clusters, microRNA clusters, HOX gene clusters and promoters of stand-alone developmental transcription and signaling factors [9, 12, 13]. A recent study by Oikawa et al., 2020 noted a conserved mechanism of paternal epigenetic code transmission to the embryo through the homogeneous retention of methylated histone H3 in sperm cells population [14]. Other recent studies have also noted methylated histones that escape epigenetic reprogramming during embryogenesis and are retained at the same loci as the sperm cells [15, 16]. Multiple studies have reported the functions of the different modifications on histones such as acetylation and methylation. Histone acetylation generally marks an open chromatin structure [17]. Methylation of specific lysine residues of the histone H3 has differential regulatory functions. H3K4me3 is known to be a transcriptionally activating mark and H3K27me3 and H3K9me3 generally have transcription repressive roles [9]. Hence, sperm histones and their loci specific enrichment on the paternal genome are crucial for developmental competence. Also, previous knowledge dictates that histone and protamine ratio is crucial for sperm chromatin compaction and a more open chromatin has a higher susceptibility to DNA fragmentation. Furthermore, histone enrichment has been reported to be regulated by differential DNA methylation [9].

No studies, to our knowledge, have directly quantified the sperm histone/protamine profile along with the different modified variations in the case of iRPL. To investigate whether any histone retention and modification discrepancies could lead to early embryo loss, we studied the levels of the core histone and protamine content, and modified histones in sperm of male partners experiencing iRPL with respect to the fertile control population. This study reports the flow cytometry technique to quantify histones in the human sperm for the first time. This study uncovers the sperm histone/histone modifications status in male partners of iRPL cases, thus contributing to the knowledge of embryo development and implantation in humans.

2.1 Study cohorts and participant enrolment

This multicentric study involved participant enrollment from the medical institutions: i. ICMR-National Institute for Research in Reproductive and Child Health, Parel; ii. Nowrosjee Wadia Maternity Hospital (NWMH), Parel, Mumbai. The study cohorts include; I. Control group consisting of apparently healthy fertile couples; and B. Case group consisting of iRPL couples having 2 or more 1st trimester pregnancy losses. Enrollment was based on set inclusion and exclusion criteria for both groups (Table 1). Recruitment of control group participants was from Family Welfare Clinic, ICMR-NIRRCH and Department of Gynaecology and Obstetrics, NWMH. Case group participants were recruited from Infertility clinic, ICMR-NIRRCH and Department of Gynaecology and Obstetrics, NWMH. With a global prevalence of RPL at 2% [1], sample size was calculated using margin of error at 5% and confidence level of 95%, and the recommended sample size was 31. From September 2018 to February 2022, 48 control group and 58 case couples were screened. A total of 40 control and 46 case couples were then enrolled in this study.

Table 1

Inclusion and exclusion criteria for participant enrollment
	Inclusion	Exclusion
Control (Apparently healthy fertile couples)	• Couples having a live child within last two years from time of recruitment • Age: Male- 21 to 45 years Female- 18 to 35 years	• Any past history of male/female partner infertility or miscarriage • Poor semen parameters as per WHO 2010 standards
Case (iRPL couples)	• Couples with the female partner experiencing ≥ 2 consecutive first trimester pregnancy losses • Age: Male- 21 to 45 years Female- 18 to 35 years	• Known contributory female factors of RPL: abnormal hormone levels (e.g. TSH, LH, FSH), anatomical uterine abnormalities; autoimmune disorders (e.g. Lupus, Anticardiolipin antibodies); Thrombophilia; Infections (e.g. HIV, CMV, PID) • Known genetic factors in both partners • Secondary infertility

2.2 Semen collection and processing

Semen samples from male partners were obtained with sexual abstinence period of 3–5 days. Basic semen parameters such sperm concentration, motility and morphology were analysed as per the World Health Organization (WHO) criteria, 2010 [18]. Neubauer haemocytometer was used to calculate sperm concentration. Sperm motility was measured by using computer-assisted semen analysis (CASA) instrument (Hamilton Thorne Biosciences, USA), respectively. 10 ul semen was smeared on glass slides and fixed and sperm morphology was assessed using papanicolaou staining method. The semen samples were centrifuged at 8000 rpm for 10 mins and seminal plasma was removed. The sperm pellets were then subjected to subsequent Phosphate Buffer Saline (PBS) washes. 2 million sperm cells were aliquoted in 1.5 ml conical bottom vials and centrifuged. The pellets were then stored at -80℃ for further use.

2.3 Sperm DNA Fragmentation and 5-mC estimation

On day of sample collection, smears on glass slides were made with 10 µl semen samples and fixed in Carnoy’s Fixative (Methanol:Acetic acid − 2:1) for 1 hr at -20℃ and air-dried. After giving the prepared sections with PBS, they were treated with Permeabilization solution (0.1% sodium citrate, 0.1% Trion X-100 in PBS) for 20 mins at room temperature again followed by PBS washes. From the In situ Cell Death Detection kit (Roche, Germany), assay mix consisting of 95 µl streptavidin conjugated fluorescein (FITC)-labelled dUTT and 5 µl terminal deoxynucleotidyl transferase (TdT) enzyme (total 100 µl per section) was added. The sections were then incubated at 37℃ in a humidified chamber under dark conditions for 1 hr. After giving a PBS wash, DAPI solution (nucleus stain) was added onto the sections and incubated in dark conditions at room temperature. After final PBS wash, sections were mounted with cover slip using ProLong Diamond Antifade Mountant (Invitrogen, USA) and sealed. The slides were viewed under Axio fluorescence microscope (Zeiss, Germany) using 100x oil immersion lens. Minimum 200 sperms were counted per sample. The sperm showing fluorescence under FITC laser were considered positive for DNA fragmentation.

For 5-mC estimation, the fresh sperm pellets were treated with somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100 in sterile nuclease-free water) to remove somatic cell contamination [5] and stored as 10 million pellets. efficient somatic lysis has been previously confirmed by checking the DLK1 methylation levels by pyrosequencing [5]. These sperm pellets were then used for genomic DNA extraction using HiPurA Sperm Genomic DNA purification kit (Himedia, India). The 5-mC DNA ELISA kit (Zymo Research, USA) was used for estimation of sperm genomic DNA 5-mC content as per manufacturer’s protocol. The standards provided consisted of methylated E. coli genome in the range of 0–100%. 100 ng of standards and sample DNA solution was prepared for each well with 5-mC coating buffer and denatured in thermal cycler (MJ Research, Canada) for 98℃ for 5 mins and placed on ice immediately. The denatured DNA was then loaded onto the wells and incubated at 37℃ for 1 hr. The wells were then washed and blocked using wash buffer at 37℃ for 30 mins. Mixture of Anti-5-Methylcytosine and secondary antibody was then added into all wells and incubated at 37℃ for 1 hr. After final washing step, HRP developer was added to all wells and placed at room temperature for 30 mins. The wells were then read at 450 nm in the BioTek Synergy H1 microplate reader (Agilent, USA). The extrapolated % values of each sample using the standard graph were then multiplied with 8.24 which is the CpG density of the human genome.

2.4 Sample preparation for flow cytometry

The sample preparation protocol has been modified from Deshpande et al., 2021 [19]. The frozen 2 million sperm pellets were thawed to room temperature. 70 µl of Decondensation buffer was added to the sperm pellet and pipette mixed gently. Decondensation buffer consists of 25 mM DTT, 0.2% Triton-X 100, 200 IU/mL heparin prepared in autoclaved MilliQ-water containing protease inhibitor cocktail (PIC). The samples were incubated at room temperature for 3 minutes only. This step opens up the tightly wound sperm chromatin. Thereafter, 100 µl of 4% Paraformaldehyde solution (prepared in PBS) was added and mixed to fix the decondensed sperm cells. The tubes were then incubated at room temperature for 20 minutes. The tubes were then centrifuged at 9000 rpm for 5 minutes at 15℃. The supernatant was discarded. The pellets were given washes with 100 µl PBS containing 1% Bovine Serum Albumin and PIC. Blocking step was done by adding 100 µl Normal Goat Serum (NGS; Abcam, UK) and pipette mixing and kept for 1 hour at room temperature. The tubes were centrifuged with the previous specifications. Primary antibody mixtures (Table 2) were prepared in 10% NGS PBS solution. 50 µl of primary antibody mix was added to each sample tube. The tubes were placed for overnight shaking at 4℃. The next day, the tubes were centrifuged at 10,000 rpm for 5 minutes at 15℃. The sperm pellets were given PBS (+ PIC) washes twice. Secondary antibody (tagged with Alexafluor 488) mixture was prepared in PBS (+ PIC) at dilution 1:300 (Table 2). 100 ul of secondary antibody mix was added to the tubes and pipette mixed. The tubes were placed in the dark for 1 hour with intermittent tap mixing. The tubes were then centrifuged at 10,000 rpm for 5 min at 15℃. The cells were then counterstained with 60 µl DAPI at dilution 1:500 in the dark for 20 mins. The tubes were centrifuged and given a single PBS wash. The sperm pellet was then resuspended with 120 µl PBS and transferred to flow tubes. With every batch of this experiment, two negative control tubes were prepared. The double negative control did not contain primary and secondary antibody and DAPI. The single negative control tube contained only secondary antibody and DAPI.

Table 2

List of primary and secondary antibodies used for flow cytometry.
Primary Antibodies (raised in rabbit)	Dilution used	Vendor (Cat No.)
Anti-Histone H4 antibody – ChIP Grade	1:50	Abcam (ab10158)
Anti-Protamine 2 antibody	1:50	Abcam (ab190791)
Anti-acetyl-Histone Antibody	1:200	Millipore (06-866)
Anti-Histone H3 (tri methyl K4) antibody-ChIP Grade	1:100	Abcam (ab8580)
Anti-Timethyl-Histone H3 (Lys27) antibody	1:100	Millipore (07-449)
Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade	1:50	Abcam (ab8898)
Secondary Antibody
Goat Anti-Rabbit IgG H&L (AlexaFluor 488)	1:300	Abcam (ab97051)

2.5 Gating and sample analysis by flow cytometry

Total 20,000 ungated events were recorded on FACS Aria SORP (Becton Dickinson) with the FACS Diva 6.1.3 software (Becton Dickinson). Initially, the double negative tube was inserted to set the DAPI positive gate. First gating P1 was done with the graph as Side Scatter vs. Forward Scatter. The selected population was then viewed with the DAPI filter. The second tube inserted for analysis was the single negative control. The homogenous DAPI positive population P2 was selected as intact cells. This population was further screened on as FITC graph. P3 gate was set as FITC negative. In case of the sample, P4 gate was applied for FITC positive cells. The Mean Fluorescence Intensity (MFI), an arbitrary value, was recorded for each sample (Supplementary Fig. 1). With each batch, the voltage was adjusted to have the negative FITC peak having MFI ± 10% of the first batch MFI. After having the same negative FITC gate, the samples were analyzed. Final analysis was done using FlowJo v.10.8.1 (BD Biosciences). To check reproducibility, the intra-day and inter-day assays were done for selected samples (n = 3) for each antibody and the Coefficient of Variation (CV) % were calculated.

2.6 Statistical analysis

Statistical analysis of each histone and protamine MFI of the study population was done using non-parametric t test with Welch’s correction and correlation analysis was done by calculating the two-tailed non-parametric Spearman’s co-efficient ‘r’ with 95% CI using GraphPad Prism v.8 (Dotmatics, USA). All statistical significance was set at p value ≤ 0.05.

3.1 Semen analysis and sperm parameters

The sperm concentration and normal morphology % of both groups were found to be within the normal range as per WHO (2010) reference values with no statistical significance between mean values. However, 3 participants from iRPL group had motility % less than prescribed lower reference limit and, overall, sperm motility was found to be significantly lower in the iRPL group as compared to control. The mean age of male and female partners enrolled in this study was comparable (Table 3). The mean DNA fragmentation index % was calculated and no significant difference was observed between both groups (Table 3). Also, the mean sperm genomic 5-mC % of both groups showed no significant difference (Table 3).

Table 3

Participant characteristics, semen parameters, sperm DNA Fragmentation Index (DFI) % and genomic 5-mC %
Parameters	Control	iRPL	p-value
Male partners:
Age (years)	33.03 ± 4.97	34.15 ± 5.51	0.562
Sperm concentration (millions/ml)	97.5 ± 92.69	93.96 ± 60.64	0.369
Sperm motility (%)	73.78 ± 17.52	65.56 ± 16.51	0.048^*
Sperm morphology (%)	7.34 ± 2.36	7.44 ± 2.70	0.869
Sperm DFI (%)	15.13 ± 9.33	15.10 ± 8.36	0.832
Sperm genomic 5-mC (%)	28.01 ± 9.96	27.08 ± 7.27	0.837
No. of Smokers/Tobacco users	6	10
Female partners:
Age	27.39 ± 4.24	29.00 ± 3.75	0.113
No. of Smokers/Tobacco users	2	1

Control (n = 40) and iRPL (n = 46) groups. Mean ± SD, *p ≤ 0.05.

3.2 Relative levels of core histone H4 and Protamine-2 in sperm

H4 histone levels were estimated to represent the relative core histone octamer levels. There was no significant mean MFI difference observed between control and case groups (Fig. 1A). Protamine-2 levels were also estimated and, similarly, no significant difference was observed between both the groups (Fig. 1B). As majority of the sperm chromatin is compacted with Protamines and only a fraction of the genome has retained histones, we checked for any correlation between the levels. Histone levels are known to vary within individuals along with which the protamine content would vary as well. A significant negative correlation of H4 MFI vs. Protamine-2 was observed in the total study population (Fig. 1C). The Co-efficient of Variance (CV) % for both H4 and Protamine-2 estimation was calculated and averaged for intra and inter day reproducibility and was noted to be less than 10% in all cases (Supplementary table 1). The representative images of gating strategy are presented in Supplementary Fig. 1.

3.3 Relative levels of modified histones in sperm

The levels of H4Ac were estimated in control and case groups by measuring the mean MFI and no significant difference could be observed (Fig. 2A). Levels of the modified histones H3K4me3, H3K27me3 and H3K9me3 in sperm were assessed in the study groups. H3K4me3 MFI levels were found to be significantly higher in the iRPL group as compared to the control group (Fig. 2B). ~20% of the iRPL study population’s sperm had H3K4me3 levels higher than that of the highest in control group. No significant differences were observed in the levels of H3K27me3 and H3K9me3 in the sperm of iRPL group as compared to control (Fig. 2C and 2D). The CV% for H4Ac, H3K4me3, H3K27me3 and H3K9me3 estimation were calculated and averaged for intra and inter day reproducibility and was found to be less than 10% in all cases (Supplementary table 3). The gating representative images are presented in Supplementary Fig. 1.

3.4 Correlation analysis of sperm H4 levels with sperm DFI% and 5-mC%

The sperm H4 MFI values were correlated with their respective DFI% and 5-mC% levels for both groups. This was done to check if higher overall histone content in sperm makes the genome susceptible to fragmentation and if their enrichment depends on the genomic methylation. Upon correlation analysis of H4 with DFI%, significant positive correlations were observed in both groups (Fig. 3A). However, no correlation was observed between the sperm H4 levels and 5-mC content in both groups (Fig. 3B).

Over the last decade, many studies have shed light on the involvement of the sperm epigenome in maintenance of a pregnancy, and not just till fertilization [12]. Even though epigenetic reprogramming events occur after fertilization, certain loci escape this phenomenon. One of the well know regions to escape epigenetic reprogramming are imprinted genes [20]. While differentially methylated imprinted genes within human sperm have been implicated in iRPL [6], our previous study also reported differentially methylated CpGs within genes involved in embryo and placenta development specific only to the iRPL study population [5]. This strengthened the implication of an altered DNA methylome in the condition of RPL. Another aspect of the sperm epigenome is the retained histones. In humans, dramatic chromatin remodeling events occur during spermatogenesis process wherein testis-specific histone variants and other histone modifications are incorporated throughout meiosis and the vast majority of canonical nucleosomes are evicted and replaced by protamines [21, 22], resulting in the packaging of the paternal chromatin into three major structural domains: one attached to the nuclear Matrix Attachment Regions [23], second one containing the majority of sperm DNA, coiled into toroids by protamines [24]; and, a third one corresponding to DNA bound to approximately 5–15% of the preserved histones [7, 9, 25]. It has been hypothesized that histone retention is non-random phenomenon which regulates gene expression, totipotency, normal development and inheritance of DNA methylation in the early embryo [26, 27, 28, 29, 30]. In case of RPL, the protamine and nucleosome levels have been previously measured by indirect methods such as Chromomycin A3 (CMA3) staining and mRNA quantification. One study reports significantly higher sperm protamine deficiency in case of unexplained recurrent abortions by CMA3 staining [31]; whereas a study from our lab has shown no such difference in the iRPL population using the same technique [32]. Rogenhofer et al., 2017 have reported lower Protamine-1 and Protamine-2 mRNA levels indirectly by their Ct values in cases of iRPL [33]. In this study, we aimed to show a more sensitive approach of intracellular protein estimation of protamine and histone. With no noticeable differences observed in the fertile and iRPL study populations, we checked whether differences in their levels are inter-dependent. A stark significant negative correlation between the Histone H4 levels (representative of the nucleosomal octamer) and Protamine-2 (Protamines 1 and 2 are known to be present in the ratio 1:1 within human sperm [34] was noted. This indicated that with each individual, the levels of histones and protamines vary with negative correlation which, additionally, also credited our method of quantification.

Another layer to epigenetic regulation is by enrichment of modified histones and gene regulatory regions. These modifications include methylation at lysine residues, acetylation, ubiqutinylation and phosphorylation. The various modifications have different roles in gene regulation. In our study we estimated levels of the well-studied modified histones H4 Acetylation (H4Ac), H3 lysine 4 trimethylation (H3K4me3), H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 9 trimethylation (H3K9me3). H4Ac is a modified histone with acetyl groups at the lysine residues of H4 histone tail. It is known to be an activating mark for transcription and leads to open chromatin structure especially at Transcription Start Sites of developmental genes [17]. H3K4me3 is a modified histone with methyl groups at the 4th lysine residue of H3 histone tail. It is known to be an activating mark for transcription and large blocks of H3K4me3 localize to a subset of developmental promoters, regions in HOX gene clusters and selective non-coding RNAs. Also, in general, these blocks are enriched on paternally-expressed imprinted loci rather than paternally-repressed loci [9]. H3K27me3 is a modified histone with methyl groups at the 27th lysine residue of H3 histone tail. It is significantly enriched at developmental promoters that are repressed in early embryos [9]. And lastly, H3K9me3 is a modified histone with methyl groups at the 9th lysine residue of H3 histone tail; and is a mark of heterochromatin and is found at pericentric regions of the chromosomes [9]. We report no significant alterations in the levels of the modified histones H4Ac (activating mark), H3K27me3 and H3K9me3 (repressive marks) in sperm of the iRPL study population. Interestingly, a subset of the iRPL study population were found to have higher H3K4me3 levels compared to the fertile population.

Many recent studies have shown that histone H3K4me3 in sperm may be instructive in terms of early gene expression in the developing embryo. Oikawa et al., 2020 showed that a fraction of the genome of the human and xenopus sperm population has loci specific high methylation density of H3K4 and H3K27 which is homogenously present in the sperm population [14]. These marks were found to be enriched especially on broad domains around transcription start sites on limited genes of developmental importance. Interestingly, majority of these marks, especially H3K4me3 sites, were also observed in Embryonic Stem Cells (ESCs) and 8-celled embryo. This indicated that a fraction of the paternal H3K4me3 marks escape epigenetic reprogramming. Another study by Lismer et al., 2021 affirms this by showing that post-natal folate deficiency alters the H3K4me3 enrichment patterns in sperm which do not get erased after fertilization, but persist throughout preimplantation development leading to congenital anomalies [35]. Therefore, one could assume that even during adulthood, a man’s diet and environment could affect his gametic epigenome such as the histone profile, which could cause aberrant fetal development. This further helps us to assume that the differential H3K4me3 enrichment in the sperm genome could reflect in differential gene expression during early embryo development leading to an aberrant transcriptome. Such an event could manifest into fetal malformations and defective placentation leading to the condition of miscarriage. And a persistent aberrant sperm H3K4me3 profile in the cases of male partners of iRPL couples could explain the repetitive miscarriages in the first trimester. However, one limitation of this study is that the higher H3K4me3 levels noted may not be homogenously present in the subset of the iRPL population.

Studies have reported the inter-dependence of DNA methylation and histone enrichment which regulate gene expression [20, 36]. But understanding of this phenomenon is still obscure. We wanted to check if the total 5’ methyl cytosine content would correlate to the H4 histone levels in sperm of both groups. But, overall, the sperm nucleosome levels did not seem to be strongly influenced by the 5-mC DNA content. During spermatogenesis, the protamines are cross-linked by disulphide bonds which densely compact the sperm chromatin. This gives protection against exogenous assault to the sperm DNA [37]. Despite this protection, basal levels of sperm DNA damage occurs even in fertile men [38]. We checked weather higher histone levels i.e. lower protamine levels lead to higher levels of DNA fragmentation in the fertile and iRPL groups. As anticipated, a positive correlation trend was noted in this study. A recent study by our lab also reported that sperm DFI and 5-mC levels are significantly inversely correlated in the fertile population but such was not the case in the iRPL study population (Irani et al., 2024). Therefore, we can infer that higher histone retention in the human sperm makes the sperm more susceptible to DNA fragmentation.

It is a distressing situation for couples undergoing repetitive testing to find an underlying cause for experiencing iRPL. This study unraveled that higher H3K4me3 levels in the male gamete, an epigenetic mark which can escape reprogramming in the embryo, could contribute to RPL. This could be considered as a potential biomarker to account for male factors associated with 1st trimester pregnancy loss. Additionally, this study affirmed that higher nucleosome content bound to sperm DNA leads to a higher incidence of DNA fragmentation.

iRPL

idiopathic Recurrent Pregnancy Loss

CASA

Computer-assisted semen analysis

PBS

Phosphate Buffer Saline

5mC

5’ methyl cytosine

DFI

DNA Fragmentation Index

Standard Deviation

Co-efficient of variation

Ethics approval and consent to participants

Ethical approval from two centres were obtained from the respective institutional ethics committees of ICMR-National Institute for Research in Reproductive and Child Health, Parel, Mumbai (Ethics Approval Ref: D/ICEC/Sci-60/64/2018); and Nowrosjee Wadia Maternity Hospital, Parel, Mumbai (Ethics Approval Ref: IEC/NWMH/01/2019). All study participants were enrolled after screening and obtaining the signed informed consent documents.

Competing interests

The authors declare no competing interests.

Author details

¹Dept. of Neuroendocrinology, ICMR-National Institute for Research in Reproductive and Child Health, Mumbai, India

²Dept. of Clinical Research, ICMR-National Institute for Research in Reproductive and Child Health, Mumbai, India

³Department of Gynaecology and Obstetrics, Nowrosjee Wadia Maternity Hospital

Funding

This work is supported by the ICMR-NIRRCH core grant (Serial No. RA/1653/04-2024).

Author Contribution

DI and DS designed the study. DT, VB and AP referred participants towards the study. DI carried out experimental work along with data analysis and DS provided all materials and lab facilities for it. DI wrote the manuscript and DS carried out manuscript revision. DI and DS are the guarantors of this work and take responsibility for the integrity of the data analysis.

Acknowledgement

We are grateful to the study participants for their enrollment in this study. We thank Deepshikha Arya, Sunita Kharat, Sharmila Kamat, Suryakant Mandavkar, Tejashree Sontakke, Reshma Goankar, Shobha Sonawane, Deepak Shelar for their assistance in participant recruitment. We thank Dr. Sharvari Deshpande for her guidance during experiments and Dr. Nafisa Balasinor for her support. We are grateful to Dr. Srabani Mukherji, Sushma Khavale, Gayatri Shinde for flow cytometry facility usage. Delna Irani thanks Lady Tata Memorial Trust for her research fellowship.

Data Availability

All data would be made available upon reasonable request.

Bender Atik R, Christiansen OB, Elson J, Kolte AM, Lewis S, Middeldorp S, et al. ESHRE guideline: recurrent pregnancy loss. Hum Reprod Open. 2018;2018:1–12.
Ibrahim Y, Johnstone E. The male contribution to recurrent pregnancy loss. Transl Androl Urol. 2018;7:S317–27.
McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell. 1984;37:179–83.
Surani MAH, Barton SC, Norris ML. Experimental reconstruction of mouse eggs and embryos: An analysis of mammalian development. Biol Reprod. 1987;36:1–16.
Irani D, Balasinor N, Bansal V, Tandon D, Patil A, Singh D. Whole genome bisulfite sequencing of sperm reveals differentially methylated regions in male partners of idiopathic recurrent pregnancy loss cases.
Khambata K, Begum S, Raut S, Mohan S, Irani D, Singh D et al. DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss. Epigenetics. 2023;18.
Tanphaichitr N, Sobhon P, Taluppeth N, Chalermisarachai P. Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp Cell Res. 1978;117:347–56.
Balhorn R. The protamine family of sperm nuclear proteins. Genome Biol. 2007;8.
Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature. 2009;460:473–8.
Brykczynska U, Hisano M, Erkek S, Ramos L, Oakeley EJ, Roloff TC, et al. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat Struct Mol Biol. 2010;17:679–87.
Jung YH, Sauria MEG, Lyu X, Cheema MS, Ausio J, Taylor J, et al. Chromatin States in Mouse Sperm Correlate with Embryonic and Adult Regulatory Landscapes. Cell Rep. 2017;18:1366–82.
Stuppia L, Franzago M, Ballerini P, Gatta V, Antonucci I. Epigenetics and male reproduction: The consequences of paternal lifestyle on fertility, embryo development, and children lifetime health. Clin Epigenetics. 2015;7:1–15.
Wang L, Zhang J, Duan J, Gao X, Zhu W, Lu X, et al. Programming and inheritance of parental DNA methylomes in mammals. Cell. 2014;157:979–91.
Oikawa M, Simeone A, Hormanseder E, Teperek M, Gaggioli V, O’Doherty A et al. Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription. Nat Commun. 2020;11.
Lismer A, Siklenka K, Lafleur C, Dumeaux V, Kimmins S. Sperm histone H3 lysine 4 trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance. Nucleic Acids Res. 2020;48:11380–93.
Ishihara T, Griffith OW, Suzuki S, Renfree MB. Presence of H3K4me3 on Paternally Expressed Genes of the Paternal Genome From Sperm to Implantation. Front Cell Dev Biol. 2022;10:1–15.
Miller CP, Rudra S, Keating MJ, Wierda WG, Palladino M, Chandra J. Caspase-8 dependent histone acetylation by a novel proteasome inhibitor, NPI-0052: A mechanism for synergy in leukemia cells. Blood. 2009;113:4289–99.
Cao XW, Lin K, Li CY, Yuan CW. [A review of WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition)]. Zhonghua Nan Ke Xue. 2011;17:1059–63.
Deshpande SSS, Nemani H, Balasinor NH. Diet-induced- and genetic-obesity differentially alters male germline histones. Reproduction. 2021;162:411–25.
Hammoud SS, Purwar J, Pflueger C, Cairns BR, Carrell DT. Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility. Fertil Steril. 2010;94:1728–33.
Kimmins S, Sassone-Corsi P. Chromatin remodelling and epigenetic features of germ cells. Nature. 2005;434:583–9.
Castillo J, Estanyol JM, Ballescà JL, Oliva R. Human sperm chromatin epigenetic potential: Genomics, proteomics, and male infertility. Asian J Androl. 2015;17:601–9.
Martins RP, Charles Ostermeier G, Krawetz SA. Nuclear matrix interactions at the human protamine domain: A working model of potentiation. J Biol Chem. 2004;279:51862–8.
Hud NV, Downing KH, Balhorn R. A constant radius of curvature model for the organization of DNA in toroidal condensates. Proc Natl Acad Sci U S A. 1995;92:3581–5.
Gatewood JM, Cook GR, Balhorn R, Schmid CW, Bradbury EM. Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones. J Biol Chem. 1990;265:20662–6.
Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW. Sequence-specific packaging of DNA in human sperm chromatin. Science. 1987;236:962–4.
Suzuki T, Minami N, Kono T, Imai H. Comparison of the RNA polymerase I-, II- And III-dependent transcript levels between nuclear transfer and in vitro fertilized embryos at the blastocyst stage. J Reprod Dev. 2007;53:663–71.
Van De Werken C, Van Der Heijden GW, Eleveld C, Teeuwssen M, Albert M, Baarends WM, et al. Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications. Nat Commun. 2014;5:1–15.
Gannon JR, Emery BR, Jenkins TG, Carrell DT. The sperm epigenome: Implications for the embryo. Adv Exp Med Biol. 2014;791:53–66.
Nakamura T, Liu YJ, Nakashima H, Umehara H, Inoue K, Matoba S, et al. PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature. 2012;486:415–9.
Kazerooni T, Asadi N, Jadid L, Kazerooni M, Ghanadi A, Ghaffarpasand F, et al. Evaluation of sperm’s chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion. J Assist Reprod Genet. 2009;26:591–6.
Khambata K, Raut S, Deshpande S, Mohan S, Sonawane S, Gaonkar R, et al. DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss. Hum Reprod. 2021;36:48–60.
Rogenhofer N, Ott J, Pilatz A, Wolf J, Thaler CJ, Windischbauer L, et al. Unexplained recurrent miscarriages are associated with an aberrant sperm protamine mRNA content. Hum Reprod. 2017;32:1574–82.
Aoki VW, Emery BR, Liu L, Carrell DT. Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and DNA integrity. J Androl. 2006;27:890–8.
Lismer A, Dumeaux V, Lafleur C, Lambrot R, Brind’Amour J, Lorincz MC, et al. Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring. Dev Cell. 2021;56:671–86.
Arhondakis S, Varriale A. Distribution of Nucleosome-enriched Sequences of Human Sperm Chromatin Along Isochores. Explor Res Hypothesis Med. 2018;3:54–60.
Barratt CLR, Aitken RJ, Björndahl L, Carrell DT, De Boer P, Kvist U, et al. Sperm DNA: Organization, protection and vulnerability: From basic science to clinical applications-a position report. Hum Reprod. 2010;25:824–38.
Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril. 2010;93:1027–36.
Irani D, Tandon D, Bansal V, Patil A, Balasinor N, Singh D. Correlation between sperm DNA fragmentation and methylation in male partners of couples with idiopathic recurrent pregnancy loss. Sys Biol Reprod Med. 2024;70:164–73.

No competing interests reported.

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Higher sperm H3K4me3 levels are associated with idiopathic recurrent pregnancy loss

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Abstract

Background:

Results:

Conclusion:

Figures

1. Introduction

2. Methods

2.1 Study cohorts and participant enrolment

2.2 Semen collection and processing

2.3 Sperm DNA Fragmentation and 5-mC estimation

2.4 Sample preparation for flow cytometry

2.5 Gating and sample analysis by flow cytometry

2.6 Statistical analysis

3. Results

3.1 Semen analysis and sperm parameters

3.2 Relative levels of core histone H4 and Protamine-2 in sperm

3.3 Relative levels of modified histones in sperm

3.4 Correlation analysis of sperm H4 levels with sperm DFI% and 5-mC%

Discussion

Conclusion

Abbreviations

Declarations

Author details

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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