Sperm-carried IGF2: towards the discovery of a spark contributing to embryo growth and development.

Infertility is defined as the inability to achieve conception after one year of unprotected intercourse. It is a serious public problem causing significant psychological, economic, and social challenges for couples wanting children. The most recent report of the World Health Organization (WHO) of 277 health surveys concluded that 48 million couples were infertile in 2010 ( Mascarenhas et al. , 2012 ), while the current prevalence of infertility is likely to be even higher. Despite decades of research, the etiology of infertility remains poorly understood and no actual cause has been identified for approximately 15–30% of cases ( Gelbaya et al. , 2014 ). Therefore, further research is urgently needed to identify new causes and targeted therapeutics for infertile couples, including those with male infertility. In the last 40 years, ART, mainly including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and intracytoplasmic morphologically selected sperm injection (IMSI), have increasingly gained ground in reproductive medicine, as they are believed to resolve infertility issues for many couples. The first child born after IVF was Louise Joy Brown, born on 25 July 1978 ( Yovich, 2020 ). Since then, ART has been increasingly offered to infertile couples and its use amongst those with male infertility has increased from 36.4% in 1996 to 76.2% in 2012, in the absence of a real increase in cases ( Boulet et al. , 2015 ). Despite these advances, failure of embryo development remains a challenge in some cases. On this basis, several studies have been conducted on spermatozoa to understand the multifactorial etiology of male infertility and to evaluate whether embryo development may be influenced by sperm biology. This has led to the discovery that a high percentage of fragmented DNA in sperm can interfere with embryo growth and cause pregnancy loss ( McQueen et al. , 2019 ; Tan et al. , 2019 ). The term imprinting refers to a genetic process that regulates the monoallelic expression of genes in a parent-of-origin specific manner. A gene is defined maternally imprinted when it is silenced through hypermethylation in the maternal chromosome and expressed by the paternal one, from which the term ‘paternally expressed imprinted gene’ derives. Conversely, a paternally imprinted gene is hypermethylated, therefore silenced, in the paternal chromosome, and is expressed by the maternal one (maternally expressed imprinted gene). Imprinted genes control a wide range of physiological functions, including growth and development ( Morison et al. , 2005 ). Many paternally expressed imprinted genes have been demonstrated to influence trophoblast proliferation and invasiveness, thereby modulating placental proliferation ( Rachmilewitz et al. , 1992 ; Goshen et al. , 1994 ). Interestingly, infertile male patients are more likely to have abnormal methylation pattern of imprinted genes in their spermatozoa ( Cannarella et al. , 2023 ). More recently, the development of next-generation sequencing (NGS) technology has enabled the identification and characterization of thousands of RNAs in spermatozoa. This complex RNA population appears to arise from spermatogenesis, although it also shares an epididymal origin. Being packed with histones, it has even been hypothesized that a minority of sperm genes are capable of transcription (for review, see Santiago et al. , 2021 ). Sperm RNAs appear to play a role in the regulation of spermatogenesis, capacitation ( Ali et al. , 2023 ), fertilization ( Wang et al. , 2021 ), pre-implantation, and post-implantation embryo development ( Guo et al. , 2017 ; Conine et al. , 2018 ; for review: Yang et al. , 2023 ) and, fascinatingly, intergenerational inheritance, by modulating the phenotype of offspring ( Hammer et al. , 2021 ; Ren et al. , 2022 ; da Cruz et al. , 2023 ; Ribas-Aulinas et al. , 2023 ). In this context, a potential key regulator is insulin-like growth factor 2 ( IGF2 ), a highly conserved gene involved in growth and proliferation ( Gao et al. , 2012 ; Keniry et al. , 2012 ). This is a paternally expressed imprinted gene and its expression is controlled by the methylation rate of H19 ( Srivastava et al. , 2000 ). H19 / IGF2 represents the first historically characterized set of imprinted genes ( De Chiara et al. , 1991 ). Both H19 and IGF2 map to chromosome 11p15.5. H19 encodes a long noncoding RNA (lncRNA), which negatively modulates human placental trophoblast cell proliferation ( Gao et al. , 2012 ). In particular, lncRNA H19 targets miR-675, which in turn represses insulin-like growth factor 1 receptor ( IGF1R ) gene transcription ( Keniry et al. , 2012 ). IGF2 encodes a growth factor that, by triggering IGF1R, promotes fetal and placental growth ( Baker et al. , 1993 ; Constância et al. , 2002 ; Fowden, 2003 ; Qiu et al. , 2005 ). In contrast to IGF1 , which is preferentially expressed after birth, IGF2 is mainly expressed during the early stages of embryo development. A lower methylation of the H19 gene has been found in infertile patients ( Cannarella et al. , 2023 ) and this leads to a decreased sperm expression of IGF2 ( Thorvaldsen et al. , 1998 ). One of the mechanisms by which low H19 methylation in the paternal allele may affect fertility is the induction of reduced IGF2 expression in the embryo, as the methylation pattern of imprinted genes is transferred to the offspring ( Matsuzaki et al. , 2015 ). However, to date, it is not known whether, in addition to this mechanism, a reduction in sperm levels of IGF2 conveyed to the oocyte could contribute to the pathogenesis of infertility. We have recently demonstrated that IGF2 is expressed in human spermatozoa ( Cannarella et al. , 2020 , 2022 ), but its role in human fertility remains to be elucidated. In particular, it is not known whether, being a growth factor, sperm IGF2 mRNA could potentiate zygote mitosis and influence embryo morphokinetics. The discovery of this aspect could potentially lead to the identification of a sperm marker to be used clinically for both the diagnosis and possibly treatment of infertility. We hypothesized that sperm IGF2 gene expression and transmission at fertilization is required to support early embryo development. To test this hypothesis, we analyzed sperm IGF2 mRNA levels in the same semen aliquot used for homologous ART in infertile couples, whose embryo morphokinetics was evaluated using time-lapse technology. We then analyzed the relationship between sperm IGF2 mRNA levels and embryo morphokinetics, after correcting for confounding factors. In the attempt to find a mechanistic explanation for the observed results, we analyzed the transcriptome of blastocysts obtained after injection of Igf2 mRNA in mouse parthenotes.

This prospective, uncontrolled, observational study was conducted on infertile couples referred to an ART center. The following parameters were collected from female partners: etiology of infertility, age, body mass index (BMI), luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17β-estradiol (E 2 ), progesterone, anti-Müllerian hormone (AMH), and the type of stimulation protocol adopted for ART. Male partners’ age, BMI, sperm parameters, and, if available, LH, FSH, total testosterone, karyotype, and microdeletions of the long arm of the Y chromosome were collected. The following information were prospectively collected: sperm concentration in the semen sample and after gradient centrifugation of the same specimens used for ART, type of ART employed (ICSI, IMSI, or IVF plus ICSI), type of protocol used for ovarian stimulation, oocyte morphology and quality, embryo kinetics, embryo morphology, day of embryo transfer (ET), serum levels of β-human chorionic gonadotropin (βhCG) after ET, type of pregnancy, when achieved (biochemical, extrauterine, or clinical), miscarriage, and live birth. After clinical use, an aliquot of the sperm sample used for ART was employed to evaluate IGF2 mRNA levels. Embryo morphokinetic parameters were monitored using time-lapse technology. The levels of IGF2 mRNA were correlated to embryo kinetics and morphology after correcting for confounding factors. The human study was conducted at the Division of Endocrinology, Metabolic Diseases and Nutrition of the University-Teaching Hospital Policlinico ‘G. Rodolico-San Marco’, University of Catania (Catania, Italy), and the ART center ‘HERA—Unità di Medicina della Riproduzione’ (Catania, Italy). The protocol was approved by the Ethic Committee ‘Catania 1’ of the University-Teaching Hospital Policlinico ‘G. Rodolico-San Marco’ (n° 18189, approved on 27 March 2023). Informed consent was obtained from the patients after a full explanation of the purpose and nature of any procedures used. The study was conducted according to the principles expressed in the Declaration of Helsinki. Couples who underwent homologous ART were considered eligible if their infertility lasted ≥12 months and in the presence of male factor or idiopathic infertility. Female factor of infertility consisting of impaired oocyte reserve (as in patients with severe endometriosis or premature ovarian failure) was considered grounds for exclusion. Instead, couples with female infertility due to tubal factors or anovulation were included. Couples with repeated implantation failure or recurrent miscarriage and couples undergoing heterologous ART were excluded. In detail, couples where each partner met the criteria specified below, were considered eligible. For the female partners, these included age ≥18 years, no history of or ongoing major comorbidities/organ failure (heart failure, kidney failure, liver failure, diabetes, tumors, thyroid disease, hyperprolactinemia), absence of anamnestic or current alcohol abuse, drug use or cigarette smoking, regular menses (25–35 days), and AMH >0.83 ng/ml ( Cai et al. , 2022 ). The exclusion criteria included the collection of fewer than five mature follicles on the day of oocyte retrieval. For the male partners, inclusion criteria were age ≥18 years, no history of major comorbidities/current organ failure (heart failure, kidney failure, liver failure, diabetes, or tumors), and no history or current alcohol abuse, drug use, or cigarette smoking. Patients with cryptozoospermia or azoospermia were excluded, as their semen samples could not be used to study sperm IGF2 expression. Long or short agonist protocols were used for ovarian stimulation. In detail, luteal gonadotrophin-releasing hormone analog (GnRHa) (Suprefact ® , Hoechst Marion Roussel Deutschland GmbH, Frankfurt, Germany), followed by recombinant FSH (Gonal-F ® , Merck-Serono, London, UK, or Puregon ® , MSD, Franklin Lakes, USA), were administered starting on Day 3 of the cycle. At 35 h after the administration of 10 000 IU hCG (Gonasi ® , IBSA, Lodi, Italy), transvaginal ultrasound-guided aspiration of oocyte–cumulus complexes was carried out. After removing the cumulus, oocyte morphology was classified into four categories, according to the status of the first polar body, size of the perivitelline space, and cytoplasmic inclusions, as described elsewhere ( Xia, 1997 ). Based on their morphology, oocytes were scored as grade 1–2 (poor) or grade 3–4 (good). Semen samples were collected by masturbation in a sterile container after 3–4 days of sexual abstinence. Conventional sperm parameters were evaluated according to the World Health Organization (WHO) criteria ( WHO, 2010 ). After liquefaction, spermatozoa were separated by density gradient centrifugation following the manufacturer’s instructions (SpermGrad™, Vitrolife, Englewood, CO, USA) ( Ali et al. , 2022 ). Motile spermatozoa were then selected for ART. The sample aliquot collected following density gradient centrifugation that remained after being used for ART was stored at −80°C until RNA extraction. Total RNA was extracted with Trizol ® Reagent (Life Technologies, Waltham, MA, USA) according to the manufacturer’s instructions. RNA concentration and purity were determined using an Eppendorf Biophotometer (Hamburg, Germany). Reverse transcription (RT) of RNA was performed for each sample using a cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Specifically, RT of 2.5 µg of total RNA was performed to obtain a final volume of 20 µl of cDNA. qPCR was carried out using 50 ng of cDNA and SYBR Green Master Mix (Stratagene, Amsterdam, The Netherlands). For this purpose, an Mx3000P thermal cycler (Stratagene) using FAM for detection and ROX as a reference dye was used. The mRNA level of each sample was normalized against β-actin mRNA and expressed as fold change. The following primers were used for RT-PCR analysis, as reported previously ( Cannarella et al. , 2022 ): IGF2 forward primer 5′-CCCGTGGGCAAGTTCTTCC-3′, reverse primer 5′-CGCTGGGTGGACTGCTTC-3′, β-actin forward primer 5′-ACCTTCTACAATGAGCTGCG—3′, reverse primer 5′- TCCATCACGATGCCAGTGGTA-3′. Data were expressed using an index, given by (IGF2 mRNA/β-actin mRNA) × 100. The moment in which a spermatozoon was injected into the oocyte was considered time zero ( t 0). The time of appearance of both pronuclei (PN) was tPN, the time of the last observation of the two PN was t PN fading , time 2, 3, 4, …, n ( t 2, t 3, t 4, …, t n ) corresponded to the time of the embryo at stage 2, 3, 4, …, n cell. The time when the embryo was in the morula stage was defined as t M and the frame when a crescent-shaped area began to emerge from the morula was defined as t B. The time when an increase in volume and expansion of the blastocoel cavity was visible was defined as the time of the expanded blastocyst ( t EB). All times were expressed in hours and fractions of an hour ( Ciray et al. , 2014 ). Embryo morphology was graded on Days 2–4 and at the blastocyst stage (Day 5). Early embryos were graded using a score ranging from 1 to 4 (1–2 indicated a good-quality embryo and 3–4 a poor-quality embryo), following previously established criteria ( Gardner and Balaban, 2016 ). The blastocysts were evaluated for the degree of expansion (scoring from 1 to 6, where 1 indicated a degree of blastocoel expansion that is less than half of the embryo and 6 indicated a hatched blastocyst, where the blastocyst had completely escaped from the zona pellucida) ( Gardner and Schoolcraft, 1999 ), organization of the trophoblast, and inner cell mass (ICM). The trophoblast and ICM scores have been adapted from Gardner’s’ original score ( Gardner and Schoolcraft, 1999 ). Specifically, for blastocysts graded as 3–6 (i.e. full blastocysts onward), ICM development was assessed as follows: grade A, tightly packed, many cells; grade B, loosely grouped, several cells; grade C, very few cells. Trophectoderm was classified as follows: grade A, many cells forming a cohesive epithelium; grade B, few cells forming a loose epithelium; grade C, very few large cells. Embryos were selected for transfer based on a previously published algorithm grounded on embryo morphokinetic parameters ( Chamayou et al. , 2013 ). Data are shown as mean ± standard deviation (SD) for non-skewed variables, while non-normally distributed continuous variables are shown as the median and interquartile range (IQR), across the manuscript. Data distribution was evaluated with the Shapiro - Wilks test. Evaluation of the relationship between the amount of IGF2 mRNA measured in the same post-gradient semen sample used for ART and embryo morphokinetics was initially performed using unadjusted linear regression. We then built two stepwise multi-regression analyses models, in which specific variables were added as confounding factors. In particular, the female partner’s age, BMI and serum AMH levels were considered in Model 1. The confounding factors of model 2 were the same of those of the model 1 plus oocyte quality. Differences in the frequency of sperm IGF2 mRNA quartile distribution between the morphologically good and poor embryo groups were assessed using the Chi-squared test. IGF2 mRNA quartiles were calculated using the IGF2 expression values of patients enrolled in the present study. The distribution of absolute sperm IGF2 mRNA levels between the groups of morphologically good and poor embryos was analyzed using one-way analysis of variance (ANOVA) or Kruskal–Wallis test, for skewed and non-skewed variables, respectively. To understand whether sperm IGF2 mRNA could predict the ability of the embryo to reach the blastocyst stage, a receiver operating characteristic (ROC) curve was built, using sperm IGF2 mRNA levels as the dependent variable, and the achievement of the blastocyst stage on Day 5 as the positive outcome. Finally, differences in sperm IGF2 mRNA values according to the ART outcome were assessed using the Student t -test for independent samples or Mann–Whitney U test for skewed and non-skewed variables respectively, as appropriate. Since the primary endpoint of this study was to evaluate the correlation between sperm IGF2 mRNA levels and embryo morphokinetics, a power analysis using a correlation coefficient was employed to calculate the sample size. The sample size was estimated at 46 blastocysts, assuming a hypothesized absolute correlation coefficient of 0.4, a type I error (alpha) of 0.05, a type II error (beta, 1-Power) of 0.20, and a null hypothesis value of 0.0. Considering a blastocyst formation rate of approximately 50% ( Thomas et al. , 2010 ), no fewer than 92 zygotes were considered in the present study. Statistical analysis was performed using MedCalc Software Ltd (Version 19.6–64 bit) (MedCalc Software Ltd, Ostend, Belgium). A P -value less than 0.05 was accepted as statistically significant. Igf2 mRNA was synthesized from a plasmid carrying the mouse Igf2 gene and injected together with Gfp mRNA into 45 oocytes, immediately after their retrieval and incubation in parthenogenic medium, with a 1:1 ratio. Another set of 45 parthenotes was injected with the same amount of Gfp mRNA only. At 96 h after injection, embryos reaching the blastocyst stage were harvested and subjected to transcriptome analysis ( Fig. 1 ). Animal experimental protocol . One Shot™ TOP10 Chemically Competent E. coli colonies were transfected with a plasmid carrying the Igf2 gene, which served as gene reservoir. Plasmids were used for gene transcription and Igf2 mRNA synthesis. Oocytes were retrieved from 8 to 12-week-old superovulated FVB/NJ mice. They were separated from the cumulus and incubated in a parthenogenic medium. Three hours after incubation, parthenotes were injected with a fluorescent Gfp mRNA (control group, n = 45) or with Igf2 plus Gfp mRNAs (experimental group, n = 45). After reaching the blastocyst stage, 96 h after injection, embryos were harvested and sequenced. Gfp, Green fluorescent protein; Igf2, insulin-like growth factor 2. The studies were conducted on FVB/NJ mice purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The animals were used as oocyte and spermatozoon donors. All animal care and use procedures were conducted in accordance with guidelines of the University of Massachusetts Medical School Institutional Animal Care and Use Committee (Protocol # 201200029). Mouse Igf2 gene was purchased from Twist Bioscience HQ (South San Francisco, CA, USA) and used as a PCR template. The primers were designed to include the restriction enzymes present in the pBluescriptRN3P plasmid (CRC Institute, Cambridge, MA, USA), i.e. Eco RI and Not I. PCR was performed according to the instruction provided in the Invitrogen Platinum SuperFi II PCR master mix (Thermo Fisher, Waltham, MA, USA) kit. The product was then analyzed on 1% agarose gel. After gel excision and DNA purification with PureLink Quick Gel Extraction & PCR purification (Invitrogen, Waltham, MA, USA), the purified DNA was quantified with Nanodrop and digested with Eco RI and Not I using Anza™ 11 Eco RI and Anza™ 1 Not I (Thermo Fisher) for insertion of the gene into the plasmid. The digested DNA product was purified with a PureLink Quick Gel Extraction & PCR purification kit (Invitrogen). One Shot™ TOP10 Chemically Competent E. coli (Thermo Fisher) were transformed with 1 ng pBluescriptRN3P (CRC Institute) and plated on cells cultured in an LB agarose plate with ampicillin. The day before harvesting some colonies, the cells were cultured overnight in LB broth containing ampicillin. Plasmid DNA was isolated with the Pure Link Quick Plasmid Miniprep kit (Invitrogen) and quantified. It was then digested with 1 µl (20 units) Eco RI and 1 µl (20 units) Not I using Anza™ (Thermo Fisher). The linearized plasmid was visualized on an agarose gel and the band was excised. Plasmid DNA was purified with a PureLink Quick Gel Extraction & PCR purification kit (Invitrogen). The linearized DNA was dephosphorylated with Anza™ Alkaline Phosphatase, using a 20-µl reaction volume as per the protocol. Ligation was then performed between the insert (6 µl) and the plasmid (2 µl) using 1 µl of Anza™ T4 DNA Ligase, 2 µl of Ligase and water up to a total reaction volume of 10 µl. Ligation to transform One Shot™ TOP10 Chemically Competent E. coli (Thermo Fisher) was performed. Igf2 colonies were cultured overnight in LB broth containing ampicillin. Plasmid DNA was isolated with the Pure Link Quick Plasmid Miniprep kit (Invitrogen). The presence of the insert within the plasmid was verified by sequencing. Then, it was digested and analyzed on an agarose gel. Two bands were visualized: one corresponding to the plasmid and one corresponding to the insert (Igf2). The band corresponding to the mouse Igf2 gene was excised and purified. The product was used for the preparation of RNA (mMESSAGE mMACHINE™ T3 Transcription Kit (25 reactions) (Thermo Fisher)). The Igf2 mRNA thus obtained was stored at −80°C at a concentration of 3000 ng/µl. Quartile distribution of sperm IGF2 mRNA levels according to blastocyst morphology. IGF2, insulin-like growth factor 2. Statistical analysis was performed using the Chi-squared test. Significance was considered for P  <   0.05. Superovulation was induced in females by an intraperitoneal (i.p.) injection of pregnant mare’s serum gonadotropin (PMSG; 5 IU) followed 48 h later by an intraperitoneal injection of human chorionic gonadotropin (hCG; 5 IU). Oocytes were then collected from the female oviducts after placing the dissected ampulla of the oviduct into KSOM containing 0.1% hyaluronidase to digest cumulus cells away from the oocytes. After 5 min in hyaluronidase, the oocytes were washed 4–5 times in KSOM and incubated in an activation medium for 60 min in a CO 2 incubator at 37°C, 5% CO 2 , 5% O 2 , and 95% relative humidity. The activation medium contained cytochalasin B at a concentration of 5 µg/ml, 4 mM EGTA and 10 mM SrCl 2 . Subsequently, the oocytes were finally placed in KSOM in an incubator at 37°C until injection/incubation. There were 45 parthenotes injected with Igf2 mRNA (3 ng/µl) plus Gfp mRNA (3 ng/µl) (experimental group) and 45 parthenotes injected with Gfp mRNA alone (3 ng/µl) (control group). Injection and successful transcription were confirmed by fluorescence detected on Day 2 ( Supplementary Fig. S1 ). Blastocysts were harvested 96 h after incubation or injection, placed in TCL buffer with 1% β-mercaptoethanol, and then stored at −80°C for processing into individual embryo RNA sequencing libraries. Three pools of 15 embryos each per experimental condition were analyzed. The length of the reverse transcribed cDNA fragments from the samples and the quality of the libraries were verified by measuring the fragment lengths. The good-quality samples were then sequenced ( Supplementary Fig. S2 ). RNA-seq libraries were generated according to the Smart-seq3 protocol with several modifications ( Hagemann-Jensen et al. , 2020 ). Briefly, individual two-cell embryos were collected in a 1× TCL buffer (Qiagen, cat #1070498, Germantown, MD, USA) containing 1% β-mercaptoethanol (Sigma, cat #M6250, St Louis, MO, USA). Total RNA was purified using 2.2× SPRI beads (Beckman, B23318 , Jersey City, NJ, USA) and the beads were re-suspended in 6 μl of lysis buffer (0.5 U/μl) of recombinant RNase inhibitor (RRI) (Takara, 2313B, Kusatsu, Shiga, Japan), 0.15% Triton X-100, 0.5 mM dNTP (Thermo Fisher Scientific, R0181), 1 μM Smart-seq3 oligo-dT primer (5 ′ -biotin-ACGAGCATCAGCAGCATACGA T30VN-3 ′ ; IDT), and 5% PEG 8000 (Sigma, P2139). Samples were incubated at 72°C for 10 min and on ice afterwards. Next, 2 μl of reverse transcription mix (25 mM Tris-HCl, pH 8.3) (Sigma, T6791), 30 mM NaCl (Ambion, AM9759, Austin, TX, USA), 1 mM GTP (Thermo Fisher Scientific, R0461), 2.5 mM MgCl 2 (Ambion, AM9530G), 8 mM DTT (Thermo Fisher Scientific, R0861, Waltham, MA, USA), 0.5 U/μl RRI (Takara, 2313B, Kusatsu, Shiga, Japan), 2 μM of different Smart-seq3 TSOs (5 ′ -biotin AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG-3 ′ ; IDT), and 2 U/μl of Maxima H-minus reverse transcriptase enzyme (Thermo Fisher Scientific, EP0751) were added to each sample. Reverse transcription and template switching were carried out at 42°C for 90 min, then 10 cycles of 50°C for 2 min, 42°C for 2 min, and 85°C for 5 min. PCR preamplification was performed by adding 12 μl of PCR mix (1× KAPA HiFi master mix, 0.1 μM Smartseq3 forward PCR primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG-3′; IDT) and 0.1 μM Smartseq3 reverse PCR primer (5′-ACGAGCATCAGCAGCA TACGA-3 ′ ; IDT)). PCR was conducted as follows: 3 min at 98°C, 19 cycles of 20 s at 98°C, 30 s at 65°C, 6 min at 72°C, and 5 min at 72°C. The PCR product was purified using 0.8x SPRI beads and eluted in nuclease-free water (Ambion, AM9932). Purified cDNA was quality checked by a bioanalyzer and quantified using Qubit 1× dsDNA HS Assay Kit (Thermo Fisher Scientific, Q33231 ). Sequence libraries were prepared using the Illumina Nextera XT DNA Library Prep kit (Illumina, FC- 131-1096, San Diego, CA, USA). Libraries were sequenced at paired ends (45 bp for read 1 and 30 bp for read 2) on an Illumina NextSeq500 instrument. Raw fastq files were separated into 5 ′ end UMI-containing reads and internal reads based on the 11-bp tag sequence (ATTGCGCAATG). For the 5 ′ end reads, UMI sequence was extracted by UMI tools ( Smith et al. , 2017 ), followed by transcriptome alignment using STAR ( Dobin et al. , 2013 ). Only unique mapping reads were kept, and PCR duplicates were removed by UMI tools. For the internal reads, read alignment was performed using STAR, and deduplication was carried out by Picard ( Liao et al. , 2014 ). The resulting bam files from the 5 ′ end and internal reads were merged. Quantification of read counts was performed by using FeatureCounts ( Liao et al. , 2014 ), and batch-effect correction was performed using Combat-seq ( Zhang et al. , 2020 ). Differentially expressed genes were identified using DESeq2 ( Love et al. , 2014 ), using a multiple hypothesis-adjusted P- value of 0.05 and fold change greater than 2. Two different pathway enrichment analysis (PEA) tools were employed in our functional enrichment analysis: g:Profiler g:GOSt ( Reimand et al. , 2007 , 2011 , 2016 ), and EnrichR ( Chen et al. , 2013 ; Kuleshov et al. , 2016 ), using modified Fisher’s exact test, and the Fisher’s exact test variant, respectively. Specifically, g: Profiler g: GOSt (version e109_eg56_p17_1d3191d) was applied to the gene list, with the g: SCS multiple testing correction method used and an adjusted P -value <0.05. For EnrichR, an unadjusted P -value <0.005 was accepted as significant. The results were validated through a literature review ( Chicco and Agapito, 2022 ). In detail, animal experiments have been carried out to find molecular evidence for the ability of sperm IGF2 mRNA to influence early embryo growth, its ability to reach the blastocyst stage, and, to some extent, its early embryo morphology. Therefore, GO terms were validated by studying research literature for connections between GO terms and early embryo growth.

There were 16 couples who met the inclusion criteria and were enrolled in the study. Six were referred to the ART Center for unexplained infertility, eight for a male factor infertility, one for tubal factor infertility, and one for anovulation. Specifically, the mean sperm concentration of male partners was 32.84 mil/ml, with an oligozoospermic condition (<15 mil/ml) present in 5/16 patients (31.3%). The mean age of female partners was 37.0 ± 4.7 years and 4/16 (25.0%) women were over 40 years old. Ovarian stimulation for the female partner was performed using a long protocol with GnRHa in seven couples and a short protocol in nine couples. Eleven couples underwent ICSI, three underwent IMSI, and two underwent IVF+ICSI. The clinical characteristics and hormonal values of the couples enrolled in the study are summarized in Supplementary Table S1 . ART yielded 106 embryos, the kinetics of which were observed with time-lapse technology ( Supplementary Table S2 ). A total of 8.1 ± 3.0 embryos per couple was included in the analysis, resulting in the transfer of two blastocysts in all couples. After ET, which was performed in all patients, implantation occurred in 7 (43.8%) of them, all of whom showed positivity for βhCG. In six women (37.5%), a clinical pregnancy was noted through observation of the embryo cavity and listening to the fetal heartbeat, while in one (6.3%), a miscarriage occurred. All partners who achieved a clinical pregnancy delivered live children, and two were twins. Sperm IGF2 mRNA was normally distributed ( P  = 0.99), with a mean value of 6.26 ± 2.55 [range (min–max): 1.30–11.4]. In the unadjusted analysis, sperm IGF2 mRNA negatively correlated with t PN fading , t 2, t 4, and t 6 ( Fig. 2 ). The adjusted analysis showed a negative correlation with t 2, t 3, t 4, and t 5 (Models 1 and 2), an unexpected positive correlation with t M (Model 2) and t B (Models 1 and 2). The negative correlation between sperm IGF2 mRNA and the time to stages of very early embryo development was consistent in the unadjusted and adjusted models ( Table 1 ). This indicates that the higher the sperm levels of IGF2 mRNA, the more rapid the growth of the early embryo, especially during the first cell divisions. Unadjusted correlation analysis between IGF2 mRNA and tPN fading (n = 106 embryos), t 2 (n = 106 embryos), t 4 (n = 96 embryos), and t 6 (n = 77 embryos) . Only the times with significant results are shown. Sperm IGF2 expression (calculated as the IGF2 mRNA/β-actin mRNA ratio) negatively correlated with embryo development times: tPN fading , t 2, t 4, and t 6. t , time; PN, pronuclei. Regression analysis between sperm IGF2 mRNA levels and embryo morphokinetics. t , time; PN, pronuclei; t M, time of morula; t B, when the frame showed a crescent-shaped area began to emerge from the morula; t EB, time when an increase in volume and expansion of the blastocoel cavity was visible. Unadjusted statistical analysis was performed using linear regression. Adjusted stepwise multi-regression analyses models include the following confounding factors: female partner’s age, body mass index (BMI), and serum anti-Müllerian hormone (AMH) levels in Model 1; female partner’s age, BMI, serum AMH levels and oocyte quality in Model 2. Significance was considered for P  4.9 predicted the capability of the embryo to reach the blastocyst stage on Day 5, with a sensitivity of 100% and a specificity of 71.6% (AUC 0.845; P  < 0.001) ( Fig. 3 ). Thus, these findings suggest that sperm IGF2 mRNA index levels greater than 4.9 could predict complete development of the early embryo. Sperm IGF2 expression: predictive analysis for embryo capability to reach the blastocyst stage on Day 5 . An IGF2 mRNA index of >4.9 predicted the capability of the embryo to reach the blastocyst stage on Day 5, with a sensitivity of 100% and a specificity of 71.6% (AUC 0.845; p < 0.001) The sample size of the analysis was 131 embryos. Sperm IGF2 expression was calculated as the IGF2 mRNA/β-actin mRNA ratio. Early embryo morphology (Days 2–4) was evaluated in 58 embryos and was scored to be good in 36 (62.1%) and poor in 22 (37.9%). The distribution frequency of sperm IGF2 mRNA values in quartiles I to IV was calculated in morphologically good and poor embryos, resulting in a significantly different distribution ( Fig. 4 , panel A). Among the embryos falling into quartile I (n = 21), 8 (38.1%) had good morphology, while 13 (61.9%) were of poor morphology. Meanwhile, embryos from quartiles II (n = 11) and III (n = 12) were more frequently of good morphology (n = 9, 81.8% and n = 11, 91.7%, respectively) than those with poor morphology (n = 2, 18.2% and n = 1, 8.3%). Finally, the 14 embryos falling in quartile IV were similarly distributed between good morphology (n = 8, 57.1%) and poor morphology (n = 6, 42.9%) ( Supplementary Table S3 ). Distribution of sperm IGF2 mRNA according to embryo morphology from Day 2 to Day 4 . Panel A shows the quartile distribution of sperm IGF2 mRNA in the two groups of morphologically good (n = 36) and poor (n = 22) embryos. Significant differences in quartile distribution were found between the two groups, with a higher prevalence of low IGF2 levels (I quartile) and a lower prevalence of IGF2 levels falling into the II and III quartiles in morphologically poor compared to morphologically good embryos. * P  < 0.05, Chi-squared test. Panel B shows the violin plots of the distribution of sperm IGF2 mRNA values in the groups of morphologically good (n = 36) and poor (n = 22) embryos. The distributions of the two groups were not significantly different. Sperm IGF2 expression was calculated as the IGF2 mRNA/β-actin mRNA ratio. Of note, the distribution of good and poor-quality oocytes did not differ between the two embryo morphology groups ( Table 2 ), likely indicating no influence of oocyte quality on the results. Oocyte quality in early embryos of good or poor morphology. Statistical analysis was performed using the Chi-squared test. Significance was considered for P  <   0.05. The difference in the absolute levels of IGF2 mRNA was also analyzed showing higher levels in morphologically good embryos compared to morphologically poor ones. However, the difference did not reach statistical significance [0.068 (0.049–0.082) vs. 0.046 (0.043–0.083), P  = 0.12] ( Fig. 4 , panel B). A total of 48 blastocysts were evaluated for the degree of expansion and organization of the trophoblast and ICM. No significant differences were found in the distribution frequency of sperm IGF2 mRNA levels in quartiles I to IV between the blastocysts groups ( Table 3 ). Similarly, absolute IGF2 mRNA levels were similar across groups of blastocysts based on degree of expansion, trophoblast and ICM (data not shown). Patients were grouped according to ART outcome. The mean IGF2 mRNA fold-change was not significantly different in the group that achieved clinical pregnancy compared with the group with no clinical pregnancy (5.59 ± 3.28 vs. 6.78 ± 1.86; P  = 0.140) nor in the group that delivered a live baby compared with those who did not (6.30 ± 2.94 vs. 6.23 ± 2.46; P  = 0.609). The only miscarriage observed occurred in a couple whose male partner had the lowest sperm IGF2 mRNA level of the entire cohort (1.30). A total of 65 genes were found to be up-regulated and 36 down-regulated in parthenotes injected with Igf2 mRNA, compared with controls ( Fig. 5 , Supplementary Fig. S3 and Table S4 ). Heatmap showing differently expressed genes in parthenotes injected with Igf2 mRNA (n = 45) compared to controls (n = 45) . An adjusted P -value 2 indicate a statistically significant change in differential gene expression. A total of 65 genes were found to be up-regulated and 36 were down-regulated. Four replicates for un-injected (controls) and for Igf2 mRNA injected samples were used for the analysis, with several animals used for each replicate. Using g: Profiler g: GOSt, seven GO biological processes, two GO molecular function terms, and one GO cellular component were retrieved. No KEGG terms or Wiki Pathways were found. The most associated term was ‘regulation of biological quality’ by GO, with P  = 4.132 × 10 −5 ( Fig. 6 ), and was enriched by the following genes: B2m , Ctsh, Fkbp1b, Nr1d1 , Rab38, Rnd1 , Scnn1a, Tspan7 , and Zdhhc2 from the list of down-regulated genes, and Abca7, Abcg1, Atp1a3 , Crhr1 , Cyp11a1 , Ehd3 , Eif4a3l1 , Flrt3 , Gabbr1 , Gata1, Ikbke, Inhbb , Nectin1 , Palm , Pim2 , Pin1rt1 , Plaur, Ptgs1 , Sh3kbp1 , Slc4a5 , Sncg , Syt2 , and Zdhhc15 , from the up-regulated genes ( Supplementary Fig. S4 ). This term is defined as ‘any process that modulates a qualitative or quantitative trait of a biological quality. A biological quality is a measurable attribute of an organism or part of an organism, such as size, mass, shape, colour, etc.’ (see GO:0065008, definition). Thus, injection of Igf2 mRNA may have triggered a pathway that regulates embryo size and mass, and, thus, its growth. Overview of enrichment analysis results obtained with g:Profiler . Upper panel. The Manhattan interactive plot illustrates the results of the enrichment analysis. The x -axis represents functional terms grouped and color-coded according to data sources [Molecular Function from GO is red; Biological Process from GO is orange, Cellular Component from GO is green, KEGG from biological pathways is fucsia, Reactome from biological pathways is electric blue, Wiki Pathways from biological pathways is sky blue, TRANSFAC from regulatory motifs in DNA is blue, mirTarBase from regulatory motifs in DNA is water green, CORUM from protein databases is green, HP from Human phenotype ontology is violet]. The y -axis shows the P -values of enrichment adjusted to the negative log10 scale. Light circles represent not significant terms (if present). Bottom panel. Source, Term ID, Term Name, and adjusted P -values of significant terms are shown. The query can be found at: https://biit.cs.ut.ee/gplink/l/9vD3nuvtTh . Abbreviations. BP, biological process; CC, cellular component; GO, gene ontology; HP, human phenotype; MIRNA, miRTarBase; MF, molecular function; REAC, Reactome; TF, TRANSFAC; WP, WikiPathways. ‘Regulation of Toll-like receptor 4 signaling pathway’ resulted in the top associated term from GO ( P  = 2.06 × 10 −4 ) with EnrichR ( Supplementary Fig. S5 ). The close GO term ‘positive regulation of Toll-like receptor 4 signaling pathway’ was also found with EnrichR ( P  = 1.11 × 10 −3 ) ( Supplementary Fig. S5 ). Toll-like receptor 4 (Tlr4) is involved in the controlled inflammatory response, which is elicited by the male partner’s seminal fluid. Tlr4 null mutation causes regulatory T (Treg) cells in the uterus to fail to activate, interfering with embryo implantation in mice. Tlr4 −/− mice have a lower pregnancy rate, increased fetal loss and fetal growth restriction ( Chan et al. , 2021 ). Gene up-regulation in the embryo enhances the attachment and migration of trophoblast cells to endometrial cells, thus promoting implantation ( Hosseini et al. , 2020 ). The role of TLR4 in mediating trophoblast cell migration has been documented in human trophoblast cell lines ( Leon-Martinez et al. , 2018 ). TLR4/NF-kB signaling also modulates cell proliferation/apoptosis in embryos in response to pathological stimuli in rats with maternal diabetes ( Chen et al. , 2017 ). These findings support the link between these GO terms and pre-implantation embryo development. The GO term ‘Cellular response to thyroid hormone stimulus’ was found in both g: Profiler g: GOSt and EnrichR PEA. ‘Response to thyroid hormone’ was also listed among the biological processes found by EnrichR ( P  = 1.61 × 10 −3 ) ( Supplementary Fig. S5 ). Thyroid hormone receptor (THR) transcripts and proteins have been documented in the preimplantation developing embryo, up to the blastocyst stage, with levels increasing after fertilization. Their inhibition has been reported to reduce the developmental rate and number of cells ( Rho et al. , 2018 ). Thyroid hormones support early embryo development ( Ashkar et al. , 2010 ), with in vitro embryos supplemented with thyroid hormones showing significant increases in blastocyst formation and hatching rates ( Ashkar et al. , 2010 ). Collectively, these findings indicate a connection between these GO terms and early embryo growth. ‘ Cell differentiation ’ (GO:0030154) [definition: ‘The cellular developmental process in which a relatively unspecialized cell, e.g. embryonic or regenerative cell, acquires specialized structural and/or functional features that characterize a specific cell. Differentiation includes the processes involved in commitment of a cell to a specific fate and its subsequent development to the mature state’] and ‘multicellular organism development’ (GO:0007275) [definition: ‘The biological process whose specific outcome is the progression of a multicellular organism over time from an initial condition (e.g. a zygote or a young adult) to a later condition (e.g. a multicellular animal or an aged adult)’] are GO biological processes found using g: Profiler g: GOSt, with a P  = 7.834 × 10 −3 , and a P  = 4.511 × 10 −2 , respectively, both intrinsically connected with early embryo development ( Fig. 6 ). The GO term ‘Positive regulation of EGFR signaling pathway’ (GO:0045742) was found using EnrichR ( P  = 6.11 × 10 −4 ) ( Supplementary Fig. S5 ). The epidermal growth factor receptor (EGFR) is known to trigger cell survival, proliferation and differentiation ( Dellaqua et al. , 2023 ), and influence trophoblast function in various species ( Cheng et al. , 2022 ). More recently, EGF has been demonstrated to modulate mice trophoblast differentiation through EGFR ( Nishitani et al. , 2023 ). This evidence supports the existence of a connection between this GO term and trophoblast development.

Despite its prevalence, the etiology of infertility remains elusive in some cases ( Punab et al. , 2017 ). The diagnostic workup of the male partner has demonstrated its efficacy in improving the outcome of ART ( Cannarella et al. , 2023 ). Furthermore, the advancements in the field of molecular biology have made it possible to highlight the complexity of spermatozoa, as capable of influencing the growth and development of the embryo through various mechanisms (e.g. DNA fragmentation, centrioles, methylation, transcriptome, proteome) ( Vallet-Buisan et al. , 2023 ). In this context, we focused our interest on sperm-carried IGF2 mRNA as recently reported ( Cannarella et al. , 2020 , 2022 ), which, after fertilization, is transferred within the oocyte and here translated into protein. We hypothesized that transcript levels may be able to influence the early mitosis rate, based on the different methylation pattern of the IGF2 gene in germ cells (hypermethylated in spermatozoa and hypomethylated in oocytes) ( Mendonça et al. , 2015 ), which argues for a different pattern of expression between spermatozoa and oocytes. We, therefore, investigated the influence of sperm IGF2 mRNA on embryokinetics and morphology in 16 infertile couples undergoing ART, whose embryo morphokinetics was monitored using time-lapse technology before being the embryos transferred into the uterus. The results showed a negative correlation between sperm IGF2 mRNA levels and t 2, t 3, t 4, t 5, and t EB, independent of maternal age, BMI, AMH, and oocyte quality. Furthermore, interestingly, a mRNA expression index greater than 4.9 predicted the ability of the embryo to reach the blastocyst stage on Day 5. We also found an influence of the sperm-delivered IGF2 mRNA levels on early embryo morphology, as they occurred more frequently in morphologically poor embryos obtained from spermatozoa with lower IGF2 mRNA levels (falling into the first quartile). Experiments were then performed in mice to obtain information that could help clarify the results seen in humans. After incubation in a parthenogenic medium, 45 mouse oocytes were injected with Igf2 and Gfp mRNAs. Another 45 oocytes were injected with Gfp mRNA only. All underwent transcriptome analysis, performed at the blastocyst stage. The results revealed the differential expression of 65 and 36 genes that were up- or down-regulated, respectively, in the experimental group compared to the control group. PEA has shown enrichments of pathways regulating early embryo development, thus providing evidence to understand the outcomes obtained in humans. Overall, we provide evidence for the role of sperm-carried IGF2 mRNA in early embryo growth and development. Being a paternally expressed imprinted gene, Igf2 can also be expressed by the embryo. To prevent any influence of the zygote genome expression in the results of the transcriptome, we used parthenotes as an experimental model. These are embryos with only the maternal genome, obtained from the activation of oocytes. Parthenotes can develop up to a certain stage, but never to full term because of the lack of the paternal genome ( Brevini and Gandolfi, 2008 ). Therefore, this prevented any bias due to Igf2 expression by the zygote. This allowed us to control the source of Igf2 mRNA, which was represented exclusively by the amount injected into the oocytes. One might then ask whether the results found in the human experimental protocol might be due to IGF2 expression by the zygote, rather than sperm-derived IGF2 expression. Indeed, the human findings are based on diploid embryos, which also have the paternal genome. Although established during gametogenesis, genomic imprinting is maintained throughout embryogenesis. Indeed, methylation imprints in differently methylated regions (DMRs) of the germline escape the global epigenetic reprogramming that occurs in the pre-implantation embryo ( Li and Sasaki, 2011 ). This implies that, when the zygote genome is ready to begin its transcriptional activity, the imprinted genes will maintain their monoallelic expression, which will occur in a parent-of-origin specific manner. Zygote genome activation (ZGA) does not begin immediately after fertilization, as the two DNA strands (maternal and paternal) must recombine and organize themselves. The processes regulating ZGA are complex and differ slightly in mice and humans. In mice, the ZGA can be divided into two waves: the minor ZGA and major ZGA, which occur at the intermediate 1-cell (1C) stage and the late 2-cell (2C) stage, respectively ( Tadros and Lipshitz, 2009 ). However, in humans, it occurs at the 8-cell stage and is preceded by maternal RNA degradation (MRD) ( Lee et al. , 2014 ; Schulz and Harrison, 2019 ). Recent findings have shown that both processes (ZGA and MRD) are initiated by the paternal genome and rely on paternally specific expressions of ZNF675 and LSM1 in human embryos ( Yuan et al. , 2023 ). Overall, the current knowledge indicates that, in humans, ZGA begins at the 8-cell stage physiologically at Day 3. Our data show an influence of sperm-carried IGF2 mRNA levels on early stages of human embryo development; particularly from t 2 to t 5, consistently across the models corrected for confounding factors ( Table 1 ). The influence of IGF2 mRNA at times from t 2 to t 5 cannot be due to IGF2 expression by the zygote, since gene expression is still silent until t 8 (corresponding to Day 3). Indeed, paternally specific gene expressions have been reported only for ZNF675 and LSM1 in human embryos before ZGA, while it has never been demonstrated for IGF2 ( Yuan et al. , 2023 ). Given the above, even in the human experimental model, our results can hardly be ascribed to zygote IGF2 expression, but rather to sperm-carried IGF2 mRNA levels. One interesting finding that corroborates our results is that the number of 8-cell embryos, which is not related with the ZGA, appears to influence the blastocyst formation rate at Day 5 ( Carrillo et al. , 1998 ; Jones et al. , 1998 ; Racowsky et al. , 2000 ). Therefore, by consistently influencing the timing until the 8-cell stage (from t 2 to t 5), IGF2 mRNA levels in spermatozoa may indirectly impact on blastocyst formation. This explains the results of our ROC curves. The introduction of time-lapse technology in ART has allowed observing a strong correlation between embryokinetics and embryo viability ( Pribenszky et al. , 2010 ; Wong et al. , 2013 ; Cruz et al. , 2011 ; Meseguer et al. , 2011 ; Azzarello et al. , 2012 ; Chamayou et al. , 2013 ; Herrero et al. , 2013 ; Campbell et al. , 2013 ; Aguilar et al. , 2014 ; Guo et al. , 2021 ). Embryos that reach a good quality blastocyst stage on Day 5 are associated with higher implantation and live birth rates than those that reach a good quality blastocyst stage on Day 6 ( Irani et al. , 2018 ), supporting the importance of timing of blastocyst development for ART outcome. A recent retrospective clinical study of over 17 000 embryos confirmed the association between blastocyst formation times and ART outcomes. Specifically, the authors reported that the later the blastocyst formation, the lower the likelihood of a successful ART outcome in terms of implantation, ongoing pregnancy and live birth rates, even after correcting for maternal age and other confounding factors (e.g. paternal age, embryo morphology, and number of previous transfers). Strikingly, a worse ART outcome was associated with delayed maturation already during the post-fertilization phase and, in particular, with tPN fading and during the first or second/third division cycle. Longer times to reach post-fertilization stages or first divisions have also been associated with longer times to blastocyst development ( Coticchio et al. , 2023 ). This evidence indicates that starting from fertilization and the very first division of the embryo, rapid kinetics leads to non-delayed blastocyst development and a viable embryo, which is able to implant and develop. In this context, the ability of sperm IGF2 to support embryo growth may be of particular clinical interest. Indeed, negative correlations with t 2, t 3, t 4, t 5, and time of t EB suggest the ability of sperm IGF2 mRNA to support early embryo divisions and the blastocyst development; on the contrary, lower expression of IGF2 is consequently associated with a delay in the kinetics of early embryo development. We were also able to define a cut-off of sperm IGF2 mRNA value that predicts the development of a blastocyst on Day 5, which could potentially open practical clinical scenarios of applying our results since spermatozoa with lower IGF2 mRNA should not be used to inject oocytes. According to the data reported here, sperm-delivered IGF2 mRNA appears to influence early embryo morphology (when evaluated from Day 2 to Day 4), while no relationship was found at a later stage. These beneficial effects are in line with the results deriving from the incubation oocytes or zygotes with IGF2-enriched cultures. An in vitro study on oocytes from obese mice reported reduced reactive oxygen species (ROS), lower spindle/chromosome defects, and improved mitochondrial function following the addition of 50 nM IGF2 to the culture. IGF2 also increased the blastocyst formation rate of zygotes incubated with this growth factor ( Wan et al. , 2022 ). Another study from the same group that incubated oocytes from aged mice with 50 nM IGF2 reported a reduction in ROS production and chromosomal abnormalities and improved mitochondrial function and oocyte maturation and morphology. Furthermore, this study also confirmed that adding IGF2 to embryo cultures increased the percentage of zygotes developing into blastocysts ( Muhammad et al. , 2020 ). This evidence seems to be confirmed in humans too. Indeed, IGF2 levels in the follicular fluid have been suggested as a clinical biomarker expressing the growth potential of human oocytes and their probability of developing ( Wang et al. , 2006 ). Furthermore, the ability of IGF2 to improve blastocyst formation rate and quality has been documented in mice and humans ( Highet et al. , 2017 ; Liu et al. , 2019 ; Muhammad et al. , 2020 ). After all, this is supported by the antioxidant activity of IGF2, which has been found to protect against ROS overproduction, to reduce lipoperoxidation, and by the activity of NADPH quinone oxidoreductase ( Martín-Montañez et al. , 2017 ) and its ability to reduce apoptotic signals ( Harris et al. , 2011 ; Sferruzzi-Perri et al. , 2017 ), to support mitochondrial function ( Zhu et al. , 2021 ) and to improve protein biosynthesis ( Matsumoto et al. , 1996 ; Muhammad et al. , 2021 ). Our findings suggest that not only the levels of IGF2 in the follicular fluid, or those used to enrich the cultures, but also those carried by spermatozoa into the oocyte can influence early embryo quality. However, it has to be emphasized that no association with blastocyst morphology was found. This could indicate a more pronounced effect of IGF2 mRNA released by spermatozoa during the first mitotic divisions, which are those influenced by the translation of the transcript into protein and, therefore, the exposure to its biological effects. Indeed, after activation of the embryo genome, which occurs at the 8-cell stage of the human embryo, ∼68 h after fertilization, around the third day ( Braude et al. , 1988 ; Tesarík et al. , 1988 ; Vassena et al. , 2011 ; Xue et al. , 2013 ; Yan et al. , 2013 ; Leng et al. , 2019 ), the embryo becomes capable of producing its own IGF2. Before this phase, the main intracellular source of the IGF2 transcript could be that transported by the spermatozoon. Notably, the evidence found in humans and discussed above aligns well with the findings of animal studies. In this regard, we decided to analyze the transcriptome of blastocysts developed from parthenotes injected with Igf2 mRNA after oocyte activation. This model was chosen to avoid interference from any other sperm-derived factor in the observed results. Furthermore, it allowed us to control the amount of Igf2 transcript introduced into the activated oocytes by injecting a standardized quantity into each cell. The differently expressed genes found in our experimental model are involved in pathways of embryo development and quality, such as pathways regulating TLR-4 and EGFR signaling, response to thyroid hormones, cell biological quality, and differentiation. TLR4 signaling enhances trophoblast proliferation ( Chen et al. , 2017 ; Leon-Martinez et al. , 2018 ; Hosseini et al. , 2020 ), thus suggesting that the influence of IGF2 on embryo development may occur by modulating this pathway. Exposure to a specific experimental condition led to the observation of differential expression of both IGF2 and of TLR-4 genes in chicken villi, thus adding evidence for the presence of a connection between these two signaling pathways ( Pham et al. , 2022 ). IGF2 and thyroid hormone sensitivity influence affect prenatal growth, and, therefore, both IGF2 defects and resistance to thyroid hormones cause growth failure ( Wit and Camacho-Hübner, 2011 ). Thyroid hormones support embryo growth ( Ashkar et al. , 2010 ; Rho et al. , 2018 ), and thus a modulation of the embryo sensitivity to these hormones may allow IGF2 to influence embryo development. Simultaneous dysregulation of Igf2 and genes involved in thyroid hormone signaling in response to harmful conditions has been reported ( Li et al. , 2010 ; Viganò et al. , 2020 ), which may further indicate a link between these pathways. IGF1R, the ligand of IGF2, activates EGFR signaling ( Desbois-Mouthon et al. , 2006 ). In turn, the latter is able to modulate trophoblast development ( Nishitani et al. , 2023 ), thus indicating an EGFR-dependent mechanism by which IGF2 could regulate embryo growth. Last but not least, the enrichment of cell biological quality and differentiation pathways also provides an explanatory link to the observed results. Our findings suggest that healthy spermatozoa provide critical support for early embryo development. The implications of these results are significant, as they may represent a new, previously unrecognized aspect of reproductive biology, provide a mechanistic explanation for a proportion of infertility cases classified as apparently idiopathic, and suggest diagnostic and therapeutic opportunities for the clinical management of infertile couples. However, the results of the present study must be taken with caution because a relatively small number of infertile couples were evaluated, although more than one hundred embryos were considered. We have included a real-world cohort here, with four of 16 couples with a female partner over 40 years, reflecting today’s epidemiological data. Although this can be seen as a limitation of the study, on the other hand, this also makes our results generalizable to patients who access ART daily and not to a specific clinical context. Furthermore, multi-regression analysis confirmed the influence of sperm-carried IGF2 mRNA on embryo morphokinetics even after adjustment for female partner age, BMI, serum AMH levels and oocyte quality. These data suggest that sperm-carried IGF2 influences embryo morphokinetics regardless of these parameters. Furthermore, the lack of a control group composed of men with recently proven fatherhood represents an additional limitation of this study. However, the findings of the present study provide the rationale for their validation in a controlled study with an adequate sample size and, possibly, a multicentric study. In conclusion, taken together, these preliminary findings raise the intriguing possibility that sperm-delivered IGF2 mRNA may play a critical role in embryogenesis by triggering the expression of genes involved in early embryo development and improving embryokinetics. This evidence may shed light on the etiology of unexplained couple infertility since low levels of IGF2 mRNA in spermatozoa may be responsible for impairments in the early steps of embryo growth and development.