Altered helper T cell-mediated immune responses in male mice conceived through in vitro fertilization

Reproductive Toxicology 69 (2017) 196–203

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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox

Altered helper T cell-mediated immune responses in male mice conceived through in vitro fertilization Hiwa Karimi a,b , Pooya Mahdavi a,b , Shohreh Fakhari a,b , Mohammad Reza Faryabi b , Parisa Esmaeili a,b , Omid Banafshi b , Ebrahim Mohammadi d , Fardin Fathi b,∗∗ , Aram Mokarizadeh b,c,e,∗ a

Student Research Committee, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran c Department of Immunology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran d Environmental Health Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran e Department of Research and Development, Asian Jivan Teb Science-based Company, Sanandaj, Iran b

a r t i c l e

i n f o

Article history: Received 5 October 2016 Received in revised form 4 March 2017 Accepted 7 March 2017 Available online 9 March 2017 Keywords: Immune response IVF-conceived mouse Naturally-conceived mouse

a b s t r a c t A study using a mouse IVF model was conducted to examine the hypothesis that in vitro fertilization (IVF) treatment may lead to immune system alteration in the offspring. Phagocytic activity and lymphocyte proliferative responses to mitogen, alloantigen, and purified protein derivative (PPD) of Mycobacterium bovis were investigated in the splenocytes of BCG-treated male mice conceived by IVF or natural conception. Intracellular expression of T-bet and GATA3 in helper Tcell population were examined in both groups. Moreover, the serum levels of IFN-␥ and IL-4 along with BCG-specific levels of IgG1 and IgG2a were assessed by ELISA. In comparison with naturally-conceived mice, PPD-specific proliferative response and T-bet/GATA3 ratio were significantly decreased in IVF-conceived mice. Moreover, IVF-conceived mice exhibited marked decreases in IFN-␥/IL-4 and IgG2a/IgG1 ratios. Results indicate that in comparison with male mice conceived by natural conception, IVF counterparts exhibit less efficient immune responses against BCG through further promotion of Th2 responses. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Recently, the role of assisted reproductive technologies (ART) in the treatment of infertility has been greatly accentuated [1]. In vitro fertilization (IVF) can be considered as one of the main ART techniques, in which an ovum is fertilized by sperm in vitro. The IVF process involves hormonally stimulating a woman’s ovulatory process, removing ova from the ovaries, and letting the sperm fertilize them in a culture medium. Subsequently, the fertilized zygote is cultured for a few days (2–6 days) in a growth medium and is then implanted in the same or another woman’s uterus [2]. Since the birth of the first IVF-conceived child in 1978, IVF has contributed to the birth of more than 4 million babies worldwide [2]. Although the great majority of children conceived through ART

∗ Corresponding author at: Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran. ∗∗ Corresponding author. E-mail addresses: [email protected] (F. Fathi), [email protected], [email protected], [email protected] (A. Mokarizadeh). http://dx.doi.org/10.1016/j.reprotox.2017.03.005 0890-6238/© 2017 Elsevier Inc. All rights reserved.

seem to be healthy with normal development, literature review of epidemiological studies reveals an increased prevalence of birth defects, adverse pregnancy outcomes (such as low birth weight, and shorter gestational age) [3,4], cardiovascular diseases [5], high blood pressure,and high fasting glucose levels [6,7] among these offspring. As the possible etiologic factors, procedures used in IVF process including hormonal stimulation, oocyte retrieval, in vitro fertilization, exposure to the culture medium, and in vitro oocyte maturation have been suggested to be responsible for the reported adverse outcomes [8–10]. However, as many possible confounding factors could influence the results of epidemiological studies, the reported adverse outcomes have not been definitely attributed to IVF treatment. For example, even without IVF, infertile women who become pregnant by natural means have an increased risk of an offspring with birth defects (Odds ratio, 1.29; 95% CI, 0.99–1.68) [3]. Nonetheless, some health risks associated with IVF treatment (e.g. altered glucose metabolism, reprogramming metabolic hemostasis, placental morphological abnormalities, and changes in methylation status of genes) have been clearly demonstrated in animal models and humans [11–16]. In addition to these findings, impressive

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evidence exists in support of belief that IVF treatments can induce immune system alteration in the offspring. For example, altered expression of genes involved in immune response such as ERAP2 and STAT4 has been discerned in placentae from patients undergone ART treatments [17]. Moreover, available reports regarding health risks associated with in vitro fertilization (IVF) have mainly been focused on asthma and cancer in which dysregulation of immune system may be a causal factor [18,19]. These findings may accentuate the possible alteration of immune system in individuals conceived through IVF. However, since this possibility has not been verified by any experimental study, an animal study was conducted to examine the hypothesis that IVF treatment may lead to immune alteration in the offspring. Accordingly, a mouse IVF model using inbred C57BL/6J mice was developed to remove the effects of potential confounding variables (e.g. genetic background, maternal environment, and litter size). Subsequently, phagocytic activity, lymphocyte proliferative response, and helper T-cell mediated immune responses including serum levels of IL-4, IFN-␥, IgG1, and IgG2a were assessed in BCG- treated male mice conceived by IVF or natural conception. Changes in these immunological parameters not only influence the efficacy of generated immune response against BCG but also may predispose the offspring to different types of infections, cancer, allergies or auto immunities. 2. Materials and methods 2.1. Animals In bred male and female C57BL/6J mice originally obtained from Pasteur Institute (Tehran, Iran) were used in this study. Mice were kept in pathogen free condition with ad libitum access to food and water, photoperiod of 14 h of light and 10 h of dark, temperature 22 ± 1 ◦ C, and 50 ± 10% humidity. All procedures were performed according to the regulations defined by the National Institute of Health Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23. Revised) and approved by the Ethics Committee of Kurdistan University of Medical Sciences. 2.2. Off spring production by in vitro fertilization and natural mating Male and female mice at the age of 8 weeks were randomly and equally subjected to conception either by IVF treatment or natural mating. The selected parents were matched based on age, weight, and health status. In IVF group, female C57BL/6J mice were used to prepare the oocytes. Sperm were obtained from 8-week-old male C57BL/6 j mice. Surrogate females were C57BL/6J ones mated with vasectomized males. For production of naturally-conceived mice, one male mouse was placed with one female mouse (breeding monogamous) for 5 days. Afterwards, the female mouse was placed in separate cage. IVF and natural mating procedures were performed serially with the identical protocol to prepare demographically matched offspring (Table 1). 2.3. Oocyte collection In order to minimize the possible effects of maternal stress and general anesthetics on oocyte quality, oocyte retrieval was performed following cervical dislocation [20,21]. Accordingly, immediate after cervical dislocation, mature oocytes were collected from the oviducts of 8 week-old female mice. The mice were superovulated with injections of 7.5 IU of equine chorionic gonadotropin (eCG; Sigma-Aldrich, Cat:G4527) and 7.5 IU of human chorionic gonadotropin (hCG; Sigma-Aldrich, Cat:C8554) administered at 48 h interval. Mature oocytes were collected from the oviducts 14–15 h after hCG injection. Subsequently, oocytes were

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placed in M2 medium (EmbryoMax M2 Medium (1x), liquid, with Phenol Red, Merck Millipore; Cat:MR-015-D) supplemented with 300 ␮g/mlhyaluronidase (Sigma-Aldrich, Cat: H4272) until the cumulus cells dispersed. 2.4. In vitro fertilization (IVF) and embryo development Immediate after cervical dislocation, sperm samples were collected from the cauda epididymis of mature male C57BL/6J mice. The collected sperm were capacitated for 1.5 h at 37 ◦ C, and diluted in human tubal fluid (HTF) medium (Sigma-Aldrich, Cat: MR070-D) to obtain a final concentration of 0.7–1.3 × 106 sperm/ml. The obtained oocytes were incubated with spermatozoa for 5 h at 37 ◦ C under 5% CO2 , 5% O2 balanced in 90% N2 . Subsequently, the oocytes were washed to eliminate the excess of spermatozoa and then cultured overnight under same conditions in a drop of potassium simplex optimization medium (KSOM-aa, Sigma-Aldrich, Cat: MR-106-D). After further incubation, when embryos reached the blastocyst stage they were transferred into the uterus. 2.5. Embryo transfer Embryos were transferred to the uterus of pseudo-pregnant females by a non-surgical embryo transfer (NSET) technique. [22]. This procedure avoids anesthesia, stress-related physiological disturbance and other disadvantages of surgery [22]. On day 19.5 of pregnancy, recipients had natural delivery, and pups were born (Table 1). 2.6. Experimental groups In each IVF experiment, one offspring was selected to be matched with one of the naturally conceived off springs. Naturallyconceived and IVF-conceived mice matched based on age, male gender, female litter size, and offspring sex ratio were selected for comparisons in the study (Table 1). Mice in both groups were weaned at 4 weeks after birth. Afterwards, mice were kept in separate polyethylene cage with equal housing condition. At 8 week old, male mice matched based on weight and health status were subjected to subcutaneous immunization with 2 × 105 colony-forming units (CFU) of Mycobacterium bovis-Bacille Calmette-Guérin (BCG; Pasture Institute, Iran) (n = 10/group). Furthermore, a booster dose containing the same amount of BCG vaccine was injected on day 7. All mice were sacrificed by cervical dislocation 14 days after the last injection. Then, phagocytic activity, lymphocyte proliferative response and helper T cell-mediated immune responses were investigated in IVF mice and mice conceived naturally. 2.7. Flow cytometric analysis of phagocytic activity The phagocytic ability of CD45+ splenocytes was assessed through yellow-green (YG) bead uptake measurement with the application of two-color flow cytometry [13]. Briefly, the spleens of naturally-and IVF-conceived mice were harvested immediately after sacrificing. Then, the spleens were passed into a RPMI-1640 medium containing plate by the utilization of sterile piston syringe. The resultant splenocytes were passed through a sterile mesh to remove debris. Subsequently, splenocytes were suspended in ACK lysing buffer (Lonza Walkersville, Inc, USA) to remove red blood cells. When the cells were twice washed in the phosphate-buffered saline (PBS; Sigma-Aldrich, St Louis, MO), they were suspended in serum-free Na+ medium (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.1% BSA, and 5 mM d-glucose, pH:7.5 at 37 ◦ C) with 0.1 mM Ca2+ .

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Table 1 The number of ovulated oocytes, transferred embryos, and off spring as well as fertilization and birth rate of IVF experiments. In each IVF experiment, one offspring was selected to be matched with one mouse of control group. The matching technique applied in this study was based on male gender, age, weight, litter size, off spring sex ratio, and health status. Weight (gr) IVF mouse Control mouse

No. of IVF offspring matched with control offspring

Birth rate (%)

No. of off spring

No. of recipients

No. of transferred embryos

Fertilization No. of Embryos rate (%)

No. of Ovulated Sperm oocytes donor

No. of Oocyte donor

22.5 20.4 21.8 18.9 19.8 23.2 20.1 22.4 23.1 20.6 18.6 19.4 18.5 21.1 19.8

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

31.8 25 22.2 31.2 35.7 33.3 25 28.5 27.7 31.2 25 41.6 33.3 25 35.7

7 5 4 5 5 6 4 4 5 5 5 5 4 4 5

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

22 20 18 16 14 18 16 14 18 16 20 12 12 16 14

66.6 76.9 58 78.2 56.1 86.3 53.1 76.9 65.5 76.2 88.8 72.2 66.6 72.7 62.5

33 26 31 23 41 22 32 26 29 21 27 18 18 22 24

2 2 2 3 3 2 3 2 2 2 2 2 2 3 2

22.3 20.6 21.7 19.1 19.8 23.5 20.5 22.5 23.1 20.3 18.3 19.1 18.3 20.8 20

The obtained cells were stained with APC-conjugated anti-CD45 antibody (eBioscience; Cat:17-045-83) in Na+ medium for 30 min at 18 ◦ C. APC-conjugated rat IgG2b k (eBioscience; Cat: 17-403182) was used as isotype control. After being washed in Na+ medium with 0.1 mM Ca2+ , the cells (2 × 106 cells) were again suspended in a pre-heated (37 ◦ C) Na+ medium supplemented by FBS-pretreated 2 ␮m YG latex beads (5 ␮l) (Sigma-Aldrich, Cat: L4530). After 5 min, the cells were washed three times. Then, by the application of a FACS Calibur flow cytometer (Beckman Dickinson, San Jose, CA), the percentage of cells ingested beads was measured within the CD45+ population in the specified regions of monocytes/macrophages and granulocytes. Gating of these cells within the CD45+ population was performed based on SSC-H parameter according to the previously published protocol [14]. Moreover, the number of ingested beads within each population was determined by measuring mean fluorescence intensity (MFI). At least 20000 events for each sample were acquired. Version 7.6 of Flow Jo software was used to perform data analysis.

2.8. Cell proliferation assay In order to assess the proliferative responses of lymphocytes to mitogen, alloantigen, and purified protein derivative (PPD), we performed a cell proliferation assay using a colorimetric cell viability kit (WST-8) (Promokine, Heidelberg, Germany, Cat:PKCA705-CK04). In brief, splenic mononuclear cells (MNCs) were suspended in RPMI-1640 medium (Invitrogen, Carlsbad, CA) and supplemented by 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). Then, the cells were seeded at 2 × 105 cells/well without (control) or with I: 2% (v/v) phytohemagglutinin (PHA; Gibco-BRL), II: mitomycin-C-pretreated MNCs (MMC, 50 ␮g/ml; Sigma-Aldrich) from NMRI mice (2 × 105 cells/100 ␮l), and III: 10 ␮g/ml Purified Protein Derivative (PPD) of Mycobacterium bovis (National Institute for Biological Standards and Control, Potters Bar, Hertfordshire). Subsequently, the cells were incubated at 37 ◦ C in a 5% CO2 incubator for 72 h (mitogenic and antigenic stimuli) or 96 h (alloantigenic stimuli). After incubation, the wells were treated with 10 ␮l CCVK solution and incubated for another 4 h. The wells containing culture medium with blocked cells or without cells were set as blank ones. Subsequently, the plate was gently shaked, and the absorbance (OD) of the samples against blanks was measured with ELISA-reader at 490 nm [23]. The stimulation index was calculated using the following formula:

22 20 18 18 23 19 17 20 19 16 24 13 12 16 15

C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J

Stimulation index = [OD490(MNCs + PHA or PPD or alloantigen) − OD490(blank)]/[OD490(MNCs without antigenic or mitogenic stimuli) − OD490(blank)]

2.9. Flow cytometric analysis for intracellular expression of T-bet and GATA3 in CD4+ T cells Unimmunized and immunized mice born by IVF or natural conception were sacrificed at 8 and 11 weeks, respectively. Subsequently splenic MNCs were separated by density gradient centrifugation over ficoll-hypaque (Sigma-Aldrich, St. Louis, MO). After magnetic separation of TCD4 cells by negative selection (Affymetrix eBioscience, Cat: 8804-6821-74), staining of TCD4 cells was performed using the fluorescent-conjugated monoclonal antibodies (mAbs) against transcription factors. Concisely, after being washed with PBS, the cells (106 cells) were permeablized with methanol (MeOH; 1 min −20 ◦ C). Then, intracellular staining was performed using anti-T-bet (eBioscience; Cat: 12-5825) or antiGATA3-PE (eBioscience; Cat: 12-9966-42) conjugated mAbs. Rat IgG2b k PE (eBioscience; Cat: 12-4031) was used as isotype control. After washing the cells, the proportions of T-bet+ cells and GATA3+ cells within the TCD4+ population were measured using a FACS Calibur flow cytometer (Beckman Dickinson, San Jose, CA). At least 20000 events for each sample were acquired. Version 7.6 of Flow Jo software was used to perform data analysis. 2.10. Cytokine assay Two weeks after the last vaccination with BCG, all mice were sacrificed, and sera were separated from the collected blood samples. Subsequently, the serum levels of IFN-␥ and IL-4 were assessed by ELISA kits (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Sensitivities of the ELISA kits for the detection of IL-4 (eBioscience, Cat: BMS613HS) and IFN-␥ (eBioscience, Cat: 88-7314-22) were 0.32 pg/ml and 15 pg/ml respectively. 2.11. Antibodyassay The levels of specific antibodies in the serum of vaccinated mice were determined by sandwich ELISA [15]. First ELISA plates were coated with 10 ␮g/ml sonicated M. bovis-BCG for 24 h. After three times of being washed with PBS containing 0.1% Tween 20,

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the plates were blocked with 10% BSA (Bovine serum albumin; Sigma-Aldrich) in PBS for 2 h at room temperature. After being washed once more, serial dilutions of sera were added into the wells and incubated at 37 ◦ C for another 2 h. Then, the plates were washed three times and incubated with goat anti-mouse IgG1 (Sigma-Aldrich, Cat: M8770) or IgG2a antibodies (Sigma-Aldrich, Cat: M4434) for 1 h. Subsequently, the plates were again washed three times, and peroxidase-conjugated rabbit anti-goat antibody (Sigma-Aldrich, Cat: A5420) was added to the wells. After 1 h of incubation at 37 ◦ C, the plates were washed and incubated with tetramethylbenzidine (TMB) substrate in dark for 30 min. The reactions were stopped by addition of 1N sulphuric acid, and the absorbance was read at 490 nm.

cells (3.434 ± 0.53 versus 3.378 ± 0.6), GATA3+ cells (1.53 ± 0.5 versus 1.5 ± 0.6), and their ratio (T-bet/GATA3 ratio: 2.481 ± 0.8 versus 2.511 ± 0.7) did not show significant differences between two groups of non-immunized mice (p > 0.05). Moreover, there were no significant changes in frequencies of T-bet+ helper T cells (25.94 ± 19.74 versus 14.96 ± 6.75) and GATA3+ helper T cells (4.56 ± 2.89 versus. 7.05 ± 3.2) between two groups of immunized mice (p = 0.07 and p = 0.08, respectively). Nonetheless after immunization in comparison with naturally-conceived mice, Tbet/GATA3 ratio was significantly decreased in IVF-conceived mice (6.198 ± 2.9 versus. 2.449 ± 1.1, p = 0.0002) (Fig. 3).

2.12. Statistical analyses

It is clear that cytokines play a key role in determining protective or noncurative immune responses. With respect to the mice immunized with BCG, any modifications in IFN-␥/IL-4 cytokine ratio may provide additional evidence regarding deviation of T cell-mediated immune responses. According to the obtained results, even though the serum levels of IL-4 did not present any significant differences between the groups (NC mice: 26.8 ± 11.17 pg/ml, IVF mice: 33.3 ± 6.49 pg/ml, p > 0.05), in comparison with naturally conceived mice, a significant decrease were observed in the serum levels of IFN-␥ (1350 ± 137 pg/ml versus 1072 ± 197 pg/ml) and IFN-␥/IL-4 ratio (61.378 ± 31.434 versus 33.412 ± 9.18) in IVF-conceived mice (p < 0.05) (Table. 2). These results indicated a shift from Th1 towards Th2 cytokine profile in BCG-treated IVF-conceived mice.

All the statistical analyses were performed using Stats Direct software. A two-tailed test with P value less than 0.05 was considered to be statistically significant. Independent t-tests were used to compare the mean differences of continuous variables. For numeric variables lacking normal distribution, Mann-Whitney U tests were used. The results are presented as mean ± standard deviation. 3. Results 3.1. No difference in phagocytic activity between naturally conceived mice and mice conceived by IVF Phagocytic activity of CD45+ splenocytes was measured either by counting the cells ingested fluorescent beads or by measuring relative mean fluorescent intensity (MFI). In dot plots, granulocytes were determined as the cells have intermediate CD45 expression and high SSC in region 1 (R1). Cells that have intermediate SSC and a slightly higher CD45 expression were identified as monocytes/macrophages in region 2 (R2). Results showed that in both regions (R1 and R2) MFI and percentage of cells ingested latex bead (fluorescence-positive cells) did not exhibit significant differences between mice conceived by natural conception and IVF-conceived mice (MFI in R1: 1523.7 ± 452 versus 1430.2 ± 306, MFI in R2: 1188.2 ± 97 versus 1238.3 ± 154, percentage of fluorescence-positive cells in R1: 16.235 ± 9.32 versus 12.961 ± 9.69, percentage of fluorescence-positive cells in R2: 15.936 ± 10.09 versus 13.675 ± 8.12, p > 0.05) (Fig.1). 3.2. Decrease of PPD-specific proliferative response in mice conceived by IVF Lymphocyte proliferative responses to PHA, MHC alloantigens, and PPD were assessed by a sensitive colorimetric cell viability assay. Fig. 2 reveals that MNCs obtained from both groups of mice, relatively equally proliferated to mitogen (proliferation index: 1.869 ± 0.369 (NC mice) versus 2.188 ± 0.459 (IVF mice) and alloantigen (proliferation index: 1.586 ± 0.432 (NC mice) versus. 1.635 ± 0.264 (IVF mice)) stimulations. Therefore, no significant differences were observed between the involved groups in this regard (p > 0.05). Nonetheless, in the case of PPD-specific proliferative response, a greater proliferative response was observed in mice born by natural conception (proliferation index: 1.831 ± 0.257 versus 1.450 ± 0.257, p = 0.014). 3.3. Decrease of T-bet/GATA3 ratio in IVF – conceived mice Two weeks after the last injection of BCG, the frequencies of Tbet- or GATA3-expressing cells were determined within the splenic TCD4+ cells by flow cytometric procedure. The results indicated that within helper T cell population the frequencies of T-bet+

3.4. Decrease of serum levels of IFN- in mice conceived by IVF

3.5. Decrease of serum levels of IgG2a in mice conceived through IVF As IgG1 and IgG2a were respectively identified as Th2- and Th1-related antibodies, specific serum levels of these antibodies were determined by ELISA.Table2 shows that specific serum levels of IgG2a were dominant in both groups of mice immunized with BCG. However, even though the serum levels of IgG1 were not significantly different between mice generated by natural conception and IVF- conceived mice (0.522 ± 0.259 versus. 0.686 ± 0.293, p > 0.05), the serum levels of IgG2a (1.386 ± 0.225 versus. 1.223 ± 0.189) and the ratio of IgG2a to IgG1 antibody (3.611 ± 2.532 versus. 2.288 ± 1.382) were shown to be significantly higher in naturally-conceived mice (p < 0.05). These results indicate that naturally-conceived mice elicit more robust Th1 responses against BCG vaccine (Table. 2). 4. Discussion Despite great advances in demonstration of health problems associated with ART, the impact of in vitro fertilization and embryogenesis on the immune system of resultant offspring has remained unknown. However, since whole DNA methylation patterns are first established after fertilization in the zygote [24], IVF treatments (including in vitro manipulation of gametes/embryos and exposure to different medium along with environmental stresses such as light, oxygen tension, and temperature) may influence the immune system development through epigenetic mechanisms such as a change in methylation status of genes involved in the immune system. Moreover, as different patterns of DNA methylation have been documented in both animals and humans conceived through IVF [15,16,25,26], the aberrant methylation of genes related to immunity is not unlikely in these cases. In the other hand, considering that available reports regarding health risks associated with IVF have mainly focused on asthma [19] and cancer [18], affection of immune system in IVF-conceived individuals is conceivable. Supporting evidence is provided by an ex vivo study indicating different expressions of genes involved in the immune responses (ERAP2 and

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Fig. 1. Phagocytic activity of CD45+ splenocytes against YG latex beads. Representative dot plots (A). Gating of monocytes/macrophages (Region 2) and granulocytes (Region 1) within the CD45+ population was performed based on SSC-H parameter. The data are summarized in dot plots representing percentage of cells ingested beads (B) and their MFI (C). According to the results obtained from flow cytometry, the percentage of cells ingested beads (B) and their related MFI value (C) did not reveal any significant differences between naturally- conceived mice (NC mice) and mice born by IVF (IVF mice) (p 䊐 0.05). Independent t-tests were used to compare the means from two different groups of data. The horizontal bars represent the mean values with standard deviations. The results were obtained from ten independent experiments.

Fig. 2. Proliferative responses of the lymphocytes obtained from naturally-and IVF-conceived mice to different stimulators. In comparison to naturally conceived mice, a decreased PPD-specific proliferative response was observed in lymphocytes obtained from mice born by IVF (p < 0.05). There were no significant differences in lymphocyte proliferative response to PHA and MHC alloantigen between naturally conceived mice (NC mice) and IVF mice. Cell proliferation was analyzed using a WST-8 Cell Counting Kit-8. The data are expressed as stimulation index. The horizontal bars represent the mean values with standard deviations. The results were obtained from ten independent experiments. * p < 0.05 (Mann-Whitney U tests) represent statistically significant differences between IVF-conceived mice and control mice.

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Fig. 3. Frequencies of T-bet+ cells and GATA3+ cells within the helper T-cell population and their ratio in mice conceived by IVF and naturally conceived mice. Representative histogram for frequencies of T-bet+ cells and GATA3+ cells within the TCD4+ population are presented (A). The data are summarized in dot plots representing percentage of T-bet+ cells and GATA3+ cells within helper T population (B) and their ratio (C). There were no significant differences in frequencies of T-bet+ cells and GATA3+ cells and their ratio between two groups of non-immunized mice (n = 5/group, p > 0.05). Also there were no significant differences in frequencies of T-bet+ helper T cells and GATA3+ helper T cells between two groups of immunized mice (n = 10/group, p = 0.07 and p = 0.08, respectively) (B). In comparison with naturally-conceived mice (NC mice), significant decreases in T-bet/GATA3 ratio were observed in IVF-conceived mice treated with BCG (n = 10/group, p < 0.05) (C). The horizontal bars represent the mean values with standard deviations. * p < 0.05 (Mann-Whitney U tests) represent statistically significant differences between IVF-conceived mice and control mice.

Table 2 Serum levels of specific antibodies and cytokines as well as their ratio in the mice treated with M.bovis-BCG. Mouse

IgG1

IgG2a

IgG2a/IgG1 Ratio

IFN-␥ (pg/ml)

IL-4 (pg/ml)

IFN- ␥/IL-4 ratio

NCMouse IVFMouse

0.522 ± 0.259 0.686 ± 0.293

1.386 ± 0.225 1.223 ± 0.189a

3.611 ± 2.532 2.288 ± 1.382a

1350 ± 137 1072 ± 197a

26.8 ± 11.17 33.3 ± 6.49

61.378 ± 31.434 33.412 ± 9.180a

a Indicates statistical difference (p < 0.05) in IVF-conceived group vs. naturally conceived group. Results were obtained from ten independent experiments and are expressed as mean ± standard deviation. Independent t-tests were used to compare mean differences of continuous variables. For numeric variables lacking normal distribution (serum levels of IgG2a), Mann-Whitney U tests were used. The serum levels of different cytokines and antibodies were determined by ELISA. High sensitivity ELISA kits were employed for detection of serum cytokine levels.

STAT4) in the placenta of ART patients [17]. Additionally, repression of gene clusters encompassed genes of immune system (C3, C5, and ICOS) has been reported in mice born through IVF [27]. Concerning these findings, an experimental study was conducted to examine the functions of immune system in immunized mice conceived by IVF or natural conception.As a developmental

immunotoxicity study, male mice were chosen for the experiment. This choice is due the fact that estrus cycle in female mice induces a tremendous variability in the immune responses [28,29]. Furthermore, Th1- biased immunity in male mice more promote the dominant Th1 responses induced by BCG. However, given that phagocytosis against pathogens plays a crucial role in induction of

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innate and specific immune responses [30], the phagocytic potential of CD45+ splenocytes (including monocytes, macrophages, and granulocytes) was investigated in naturally-and IVF-conceived mice. According to the results obtained from flow cytometric procedure, phagocytic activity as the main effector arm of non-specific immune system did not exhibit significant differences between two groups, neither in the number of cells performing phagocytosis, nor in intensity of phagocytosis. The second issue investigated in the present study was related to the examination of proliferative responses of lymphocytes to PHA mitogen, MHC alloantigen, and PPD to provide a semi-quantitative assessment of total cell-mediated immunity. However, no significant difference was observed in lymphocyte proliferative responses to PHA and MHC alloantigen between naturally- conceived mice and mice conceived by IVF. Nonetheless, the decreased PPD-specific proliferative response in mice born by IVF may point to the decreased antigen-specific cell-mediated immune response. Considering that the differentiation of naive T helper cells into Th1 or Th2 subsets is regulated by the transcription factors (TF) Tbet and GATA3, the T-bet/GATA3 ratio reflects the Th1/Th2 status during early stages of Th1 and Th2 development [31]. Accordingly, we assessed the frequencies of helper T cells expressing T-bet or GATA3 in both groups to compare Th1/Th2 balance between IVF mice and mice conceived naturally. Since helper T cell subsets including Th1 (CD4+, Tbet+, IFN-␥+cells) and Th2 (CD4+, GATA3+, IL-4+ cells), as the effector arms of acquired immunity, have been proved to influence a wide variety of immune cells, any alterations in T cell polarization could lead to altered regulation of immune responses. Considering the critical role of helper T cell subsets in health promotion and disease prevention, it should be noted that in addition to differential roles of helper T subsets in protection against different pathogens, skewing T cell polarization towards different subsets (i.e. Th1 or Th2) can contribute to different types of immunopathological reactions [32]. Th1 subset, for example, is involved in defense mechanisms against intracellular pathogens such as BCG. Moreover, this subset can contribute to the pathogenesis of organ-specific autoimmune disorders (e.g. Multiple sclerosis, rheumatoid arthritis, and insulin-dependent diabetes mellitus). Furthermore, Th2 responses are found to be responsible for defense against helminth parasites and atopic disorders (e.g. asthma, rhinitis, and food allergies) [32,33]. Accordingly, during immune responses, proliferation and then differentiation of naive CD4T cells into appropriate subsets of T cells are indispensable to obtain optimal functioning of the acquired immunity [32–34]. However, even though immunization with high doses of BCG produces mixed Th1/Th2 responses, generation of a Th1-dominant immune response leads to more protection against Mycobacterium infection [35]. Accordingly, Th1/Th2 balance following vaccination was compared between two groups of mice to assess the efficacy of generated cell-mediated immune responses. As the results revealed, even though after immunization with BCG, helper T cells in both groups of mice were mainly expressed Th1-specific TF T-bet, a significant decrease in Th1/Th2 ratio was observed in IVF mice by presenting a decrease in T-bet/GATA3 ratio. Considering that the baseline expression of T-bet, GATA3, and its ratio within helper T cell population did not exhibit significant differences between two groups of non-immunized mice, decreased ratio of T-bet/GATA3 in immunized mice born by IVF may be occurred secondary during development of immune responses against BCG. In line with the presented findings, serum cytokine profile of IVF-conceived mice exhibited a Th2 biased immune response to BCG by presenting a decrease in IFN-␥/IL-4 cytokine ratio. Moreover, as IgG1 and IgG2a are respectively identified as Th2- and Th1-related antibodies [36], the increased IgG1/IgG2a ratio in vaccinated IVF mice supports the bias of immune responses towards Th2. Given that a Th2-biased immune response has been recognized as atypical fea-

ture of many types of allergies [37,38], male mice conceived by IVF not only may reflect more resistance to helminth infections, but also may present more potency to develop allergic immune responses. Furthermore, as these mice showed a decreased ratio of Th1/Th2, they are expected to reflect ameliorated Th1 immune responses against intracellular and tumor antigens in comparison with naturally-conceived male mice. Collectively, comparison of BCG-induced immune responses in two groups revealed that IVF-conceived male mice exhibited a significant decrease in indices related to cell-mediated immunity (i.e. significant decreases in IFN-␥/IL-4 and IgG2a/IgG1 ratios, and PPD-specific proliferative response). These results indicate that even though after immunization with BCG, the normal immune responses developed in both groups of mice, in comparison with naturally-conceived male mice, IVF counterparts presented less efficient immune responses against BCG through further promotion of Th2 immune responses. Nonetheless, in IVF mice the degree of immune perturbation may be less acute to cause clinically obvious outcomes. 5. Conclusion As far as the authors are concerned, the present study can be regarded as the first experimental study providing evidence on the possibility of immune response alternation in male offspring conceived through IVF. Although the present study has not investigated all aspects of the immune system, the results can suggest that additional immune parameters may be affected. Nonetheless, the absence of female data is a limitation of the present study. Therefore, further work is needed to determine whether immune alterations are present in female IVF offspring. Moreover, since the techniques and culture media used in animal IVF are not exactly the same as those employed in human IVF, the extent to which the results can be extrapolated from mice to the human IVF is indeterminate. Conflict of interest The authors claim no conflict of interest. Acknowledgments This work was supported by a grant from the Research Vice Chancellor of Kurdistan University of Medical Sciences, Sanandaj, Iran. The authors would like to thank Dr. Kambiz Hassanzadeh and the staff of Transgenic Mouse Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran. References [1] J.M. Goldberg, T. Falcone, M. Attaran, In vitro fertilization update, Cleve. Clin. J. Med. 74 (5) (2007) 329–338. [2] A.S.A. Desai, M. Bhanushali, V.J. Kadam, Assisted reproductive technology (ART): combating infertility, Asian J. Pharm. Clin. Res. 4 (1) (2011) 18–22. [3] M.J. Davies, V.M. Moore, K.J. Willson, P. Van Essen, K. Priest, H. Scott, E.A. Haan, A. Chan, Reproductive technologies and the risk of birth defects, N. Engl. J. Med. 366 (19) (2012) 1803–1813. [4] J. Wen, J. Jiang, C. Ding, J. Dai, Y. Liu, Y. Xia, J. Liu, Z. Hu, Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis, Fertil. Steril. 97 (6) (2012), 1331-7 e1-4. [5] M. Ceelen, M.M. van Weissenbruch, J.P. Vermeiden, F.E. van Leeuwen, H.A. Delemarre-van de Waal, Cardiometabolic differences in children born after in vitro fertilization: follow-up study, J. Clin. Endocrinol. Metab. 93 (5) (2008) 1682–1688. [6] M. Ceelen, M.M. van Weissenbruch, J.P. Vermeiden, F.E. van Leeuwen, H.A. Delemarre-van de Waal, Pubertal development in children and adolescents born after IVF and spontaneous conception, Hum. Reprod. 23 (12) (2008) 2791–2798. [7] M. Ceelen, M.M. van Weissenbruch, J. Prein, J.J. Smit, J.P. Vermeiden, M. Spreeuwenberg, F.E. van Leeuwen, H.A. Delemarre-van de Waal, Growth

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