Early Embryo Loss Research Articles

Vitamin D3 pretreatment protects against lipopolysaccharide-induced early embryo loss through its anti-inflammatory effects.

Increasing evidence demonstrates that inflammatory cytokines are involved in LPS-induced adverse pregnant outcomes including early embryo loss. Vitamin D3 (VitD3) has anti-inflammatory activity. We aimed to investigate the effects of vitamin D3 (VitD3) on LPS-induced early embryo loss in mice. All pregnant mice except controls were intraperitoneally (ip) injected with LPS on GD7. In VitD3 alone and LPS+VitD3 groups, pregnant mice were pretreated with VitD3 by gavage daily from GD5 to GD7. LPS caused 62.5% pregnant mice with early embryo loss. Interestingly, the rate of abortion dropped to 14.3% when pregnant mice were pretreated with VitD3. Additional experiment showed that VitD3 significantly attenuated LPS-evoked elevation on TNF-α, IFN-γ, MIP-2, and nitrate plus nitrite in maternal serum. In addition, VitD3 alleviated LPS-induced COX-2 expression in the decidua and attenuated the elevation of PGF2α in maternal serum. Although VitD3 had no effect on IL-10 in maternal serum, it induced further elevation of serum IL-10 level in LPS-treated mice. Further analysis showed that VitD3 activated VDR signaling, simultaneously inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the decidua. VitD3 protects mice from LPS-induced early embryo loss at least partially through its anti-inflammatory effects.

Early embryo mortality in natural human reproduction: What the data say.

It is generally accepted that natural human embryo mortality during pregnancy is high - losses of 70% and higher from fertilisation to birth are frequently claimed. The first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period. Establishing the fate of embryos before this is challenging, and hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are cited to justify quantitative claims regarding embryo loss: (i) a hypothesis published by Roberts & Lowe in TheLancet is widely cited but has no quantitative value; (ii) life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii) studies that measure human chorionic gonadotrophin (hCG) reveal losses in the second week of development and beyond, but not before; and (iv) the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig's data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data suggests that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, it is clear that some published estimates of natural embryo mortality are exaggerated. Although available data do not provide a precise estimate, natural human embryo mortality is lower than is often claimed.

Early embryo loss, morphology, and effect of previous immunization against androstenedione in the ewe

In a naturally mated cycle, ova and viable embryo number as well as embryo size were assessed on Day 4, 10, 14, 18, and 30 of gestation in Romney ewes (n = 38–44 per gestational group). For Days 4–18 of gestation, embryos were recovered by flushing the reproductive tract after slaughtering of the ewe. Ovulation rate was determined by counting the number of corpora lutea present on both ovaries. For the Day 30 group, number of ovulations was measured by laparoscopic examination of the ovaries at Day 9–12 of the cycle, and number of embryos present was determined by ultrasound examination at approximately Day 30 of pregnancy. Most of embryo loss occurred before Day 14 of gestation with 6% loss before Day 4, and 12% loss between Day 4 and 14 of gestation. A similar proportion of viable embryos per number of ova ovulated were recovered on Day 14 and 18 (82%) and Day 30 (81%) of gestation. Fertilization failure was estimated at 1%. Conceptus and embryo size was most variable on Day 14, representing a period of rapid growth (conceptus length ± standard deviation); Day 4 (169 ± 8 μm), Day 10 (379 ± 93 μm), Day 14 (23 ± 32 mm), Day 18 (embryo length ± standard deviation; 5.0 ± 0.7 mm). Vaccination with commercially available fertility vaccines targeting androstenedione (Androvax and Ovastim) in previous seasons resulted in reduced conceptus size compared with controls. However, no difference in the proportion of viable embryos was observed between treatments, signifying maternal tolerance for considerable variation at this stage of development. Furthermore, the finding that most of loss occurs within the first 14 days of gestation highlights the importance of both oocyte quality and the uterine environment for the embryo to successfully overcome the challenges leading up to and including pregnancy recognition in the ewe.

A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus 'Nicoletti assay'.

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value.

Global gene expression in endometrium of high and low fertility heifers during the mid-luteal phase of the estrous cycle

BackgroundIn both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. During this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. The objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high (HF) or low fertility (LF) (following four rounds of artificial insemination (AI)) using the Affymetrix® 23 K Bovine Gene Chip.ResultsConception rates for each of the four rounds of AI were within a normal range: 70–73.3%. Microarray analysis of endometrial tissue collected on day 7 of the estrous cycle detected 419 differentially expressed genes (DEG) between HF (n = 6) and LF (n = 6) animals. The main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. DEG included, FST, SLC45A2, MMP19, FADS1 and GALNT6.ConclusionsThis study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. Differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-234) contains supplementary material, which is available to authorized users.

DNA methylation analysis at imprinted locus and global methylation in spermatozoa of normozoospermic individuals of idiopathic Recurrent Spontaneous Miscarriage

Background Gametic imprint marks play an essential role in dictating the expression profile to the developing embryo as they are faithfully inherited in the somatic tissues. Insulin-like growth factor(IGF2) is a mitogen and IGF2-H19 locus, a paternally imprinted locus, has been found to be associated with embryo loss in numerous publications. Aberrations in its methylation levels in tamoxifen treated rats evidenced despite any change in sperm parameters [1]. DLK1(Drosophila-like homologue1) is also involved in embryogenesis and the IGDMR(a paternally methylated locus) methylation aberrations has been found to be associated in oligozoospermic patients [2]. Recurrent Spontaneous Miscarriage (RSM) is the spontaneous consecutive loss of >3 pregnancies in the first trimester. Although many factors have been attributed to its cause many cases remain idiopathic and undiagnosed. Based on evidences supporting the prevalence of paternal epigenetic marks associating with embryo loss we hypothesized the involvement of aberrant methylation of imprinted genes IGF2H19 imprint control region, DLK1-GTL2 IGDMR and global methylation in spermatozoa of idiopathic Recurrent Spontaneous Miscarriage (RSM) case participants having normozoospermic profile in order to understand its involvement in causing early embryo loss in these cases.

Methylation status of imprinted genes DLK1-GTL2, MEST (PEG1), ZAC (PLAGL1), and LINE-1 elements in spermatozoa of normozoospermic men, unlike H19 imprinting control regions, is not associated with idiopathic recurrent spontaneous miscarriages

To study methylation aberrations in spermatozoa at developmentally important imprinted regions to ascertain their role in early embryo loss in idiopathic recurrent spontaneous miscarriages (RSM). Case-control study. Academic research setting at National Institute for Research in Reproductive Health, Parel, Mumbai. Male partners of couples with a history of RSM and male partners of couples with proven fertility (control group). None. DNA methylation levels at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) by Epityper Massarray and global methylation levels as measured by LINE-1 methylation and anti-5-methyl cytosine antibody in spermatozoa of 23 men in control group and 23 men in RSM group. We did not observe any aberration in the total methylation levels in any of the imprinted genes or global methylation analyzed. Our results indicate that paternal methylation aberrations at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) and global methylation levels are not associated with idiopathic RSM and may not be good epigenetic markers (unlike the H-19 imprinting control region) for diagnosis of idiopathic RSM.

Reproductive response of fat‐tailed Barbarine ewes subjected to short‐term nutritional treatments including spineless cactus (Opuntia ficus‐indica f. inermis) cladodes

Reproductive outputs in fat-tailed Barbarine sheep in central Tunisia are often low because of feed shortage and the low nutritive value of diets. Supplementation with conventional concentrates is economically unsuitable in central Tunisia, so more cost-effective and sustainable alternative feeding strategies need to be developed. We tested effects of short-term nutritional treatments including cactus cladodes during the induction of 'male effect' on fertility and prolificacy parameters (follicular growth, ovulatory response and early embryo losses). One hundred and twenty ewes were distributed in 4 equal groups balanced for live weight grazed natural pastures and were supplemented for 21days, starting day 10 after introduction of rams, with cactus cladodes (CA), cactus cladodes and soybean meal (CAS), concentrate (CC) or only soybean meal (S). Nutritional treatment did not affect live weight in this experiment. Ewes receiving cactus had higher number of large pre-ovulatory follicles (≥6mm; 1.08±0.05), between days 14 and 19 after introduction of rams, than females in the CC and S ewes (0.64±0.06; p<0.05). However, there were no differences in the onset of oestrous behaviour in response to 'male effect' or in the number of corpora lutea. Average ovulation rates were 1.42±0.16 for CC, 1.47±0.13 for CAS, 1.31±0.15 for CA and 1.31±0.13 for S groups respectively. Finally, reproductive wastages at day 35 after mating were not different between groups being 0.33±0.19 for CC, 0.60±0.17 for CAS, 0.43±0.16 for CA and 0.31±0.15 for S groups respectively. It is concluded that Barbarine ewes fed nutritional treatments including cactus performed similarly to those receiving diets including conventional concentrate feeds.

Cumulus cell and oocyte mRNA expression of hormone and growth factor receptors in mares

determined in old mares, but could represent a cause of age-associated early embryo loss. We hypothesized that alterations in the meiotic spindle and chromosomal alignment occur more frequently in the oocyte of old versus young mares. Oocytes were collected from the dominant follicles of young (4-11 yr, n 1⁄4 7) and old ( 20 yr, n 1⁄4 8) estrous mares and fixed at 44 h after administration of a GnRH analog to the donor. Oocytes were stained for DNA (Hoechst 33258) and microtubules (mouse anti a and b tubulin and goat anti-mouse-alexa488). Confocal images of the oocytes' meiotic spindles were analyzed to determine spindle integrity and alignment of chromosomes. Spindle conformation was considered normal when the following criteria were observed: bipolar organization, microtubules converging at both poles, and chromosomes evenly aligned at the equatorial plate. Atypical meiotic spindle morphology was observed in more oocytes from old versus youngmares (7/8 and 0/7, respectively; P< 0.05, Fisher's Exact Test), with oocytes from old mares having normal chromosomal alignment (n 1⁄4 1), nonaligned chromosomes (n 1⁄4 5), or a disarrangement of the spindle (n 1⁄4 2). Results of our study support that advanced mare age is associated alterations in morphology of the meiotic spindle and an increase in misalignment of chromosomes, which could affect embryo viability and developmental competence.

A comparison of the bovine uterine and plasma proteome using iTRAQ proteomics

Early embryo loss is a key factor affecting fertility in dairy and beef herds. Prior to implantation, the bovine embryo spends around 16 days free-floating in the uterine environment and is dependent on the composition of uterine fluid for normal growth and development. However, there is a lack of information regarding the protein composition of the bovine uterus and how it relates to plasma. In this study, uterine flushings (UF) (n = 6) and blood plasma (n = 4) were collected from beef heifers on day 7 of the oestrous cycle, albumin depleted and compared using iTRAQ proteomics. A total of 35 proteins were higher and 18 were lower in UF including metabolic enzymes, proteins with anti-oxidant activity and those involved in modulation of the immune response. This study confirms the dynamic nature of the bovine uterine proteome and that it differs from plasma. Factors affecting the uterine proteome and how it impacts on embryo survival warrant further study.

Inherited Thrombophilia and IVF Failure: The Impact of Coagulation Disorders On Implantation Process

In connection with the embryo acceptance process after IVF procedure, endometrial cells surface receptors, extracellular matrix (ECM) molecules, endothelium and blood circulation factors were involved in remodelling of endometrium. Plasminogen activator inhibitor type 1 plays a significant role during the early phases of placental vascular remodelling and regulates the trophoblast invasion through controlling plasmin activity. Endometrial cell surface protein integrin alphaV/beta3, responsible for the adhesion of the embryo, has had also the same subunit beta3, which is component of integrin alphaIIb/beta3 connected with platelet aggregability. Prothrombin, furthermore, has had a debatable effect upon endothelial and mesenchymal cells and possible contribution on embryo vascular development. Confoundable data have been present about the role of coagulation factor V and its role for implantation. These and other coagulation factors have relatively common gene polymorphisms that enhanced their activity. This review discusses the effect of increased coagulation activity on implantation process, which is not yet fully determined. The establishment of the positive or negative impact of mother hypercoagulability on the success of embryo implantation after assisted reproduction technology could determine the timing of preventing anticoagulant therapy in women with history of early embryo loss.

MRNA transcription of prostaglandin synthases and their products in the equine endometrium in the course of fibrosis

Accurate regulation of the reproductive cycle and successful implantation depend on proper functioning of the endometrium. The aim of this study was to determine whether mRNA transcription of specific enzymes responsible for prostaglandin (PG) synthesis (prostaglandin-endoperoxide synthase, PTGS-2; prostaglandin F2α synthase, PGFS; and prostaglandin E2 synthases, PGES) and PG concentrations in endometrial extracts would change in moderate (Kenney's Category II) and severe phases of fibrosis (Kenney's Category III; endometrosis), compared with healthy endometrium (Kenney's Category I), during the estrous cycle. Endometrial tissues samples were obtained from mares at the early (n = 12), mid (n = 12) and late (n = 12) luteal phases and the follicular phase (n = 12) of the estrous cycle. Additionally, all endometria were classified microscopically as belonging to Categories I and II or III according to the Kenney classification, resulting in allocation of 4 samples for each subcategory, e.g., mid luteal I, II and III. Relative mRNA transcription was quantified using Real-time PCR. Concentrations of PGE2 and PGF2α in the endometrial extracts were determined using enzyme-linked immunosorbent assay (EIA). In Category I, PTGS-2 mRNA transcription was upregulated at the mid (P < 0.05) and late luteal phases (P < 0.001) and at the follicular phase (P < 0.05) compared to the early luteal phase. PGFS mRNA transcription as well as PGF2α concentrations increased at the mid (P < 0.01) and late (P < 0.05) luteal phases compared to the early luteal phase in Category I. PGES mRNA transcription was higher at the mid (P < 0.01) and late luteal phases (P < 0.05) compared to the early luteal and follicular phases in Category I. Prostaglandin E2 concentration in Category I was higher at the mid luteal phase (P < 0.01) compared to all other phases of the estrous cycle. During incipient endometrosis (Category II) and under full endometrosis (Category III), PTGS-2, PGFS and PGES mRNA transcription and PG concentration were altered compared to the respective estrous phases in healthy endometria (P < 0.05). It may be concluded that serious changes in mRNA transcription of PG synthases and PG production that occur in the equine endometrium during the course of fibrosis in the estrous cycle could be responsible for disturbances leading to disorders of the estrous cycle and early embryo losses.

The interaction between ovulation rate and embryo survival in determining prolificacy of different strains of obese swine with gene polymorphisms for leptin receptors

The Mediterranean Iberian pigs show a clear predisposition to obesity, due to gene polymorphisms for leptin receptors, and a lower prolificacy when compared with lean breeds. Previous studies indicate a bimodal effect of obese genotypes on prolificacy; either by lowering ovulation rate or by increasing embryo losses when compared with lean genotypes. The present study aimed to discriminate the relative influence of both parameters by comparing strains of Iberian females with different prolificacy (14 original purebred Retinto, group R, and 20 Retinto × Torbiscal females selected for prolificacy, group RT). Examination of ovaries around Day 40 of pregnancy showed a higher ovulation rate in the RT than in the R sows (21.5 ± 1.4 versus 12.3 ± 1.0 corpora lutea, respectively; P &lt; 0.0005). However, there was a higher incidence of implantation failures and early embryo losses in the RT females (42.9 versus 14% for the group R; P &lt; 0.005). Thus, the number of viable embryos was higher in the RT line (10.8 ± 0.5 versus 7.6 ± 0.7 in the R sows, P &lt; 0.0005), but also limited when compared with ovulation rate. Conversely, this effect was not found in the R sows; however, prolificacy of these females was limited by higher embryo mortality (24.1 versus 4.6% for RT females). In conclusion, the present study evidences dissimilar reproductive behaviour among strains of the Iberian pig differing in prolificacy and confirms previous evidences suggesting the existence of intrinsic factors avoiding excessively large litter sizes in Iberian females.

English

Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects of MTHFR polymorphism on cow abortion were investigated. The PCR-SSCP and DNA sequencing were applied to detect the polymorphisms in exon 4 and 7 of MTHFR gene in a total of 569 Chinese Holstein cattles. The homocysteine levels during various periods of pregnancy were detected by HPLC. The results show that 8137C/T SNP was identified in exon 7 of MTHFR, but no polymorphism was detected in exon 4. Association analysis revealed that the 8137C/T mutation was significantly associated with cow abortion (P = 0.0002). Regarding relative risk of incidence, cows with CT genotype showed greater relative risk than those with CC (OR = 2.05, 95% CI = 1.40 to 3.00). The blood homocysteine levels of individuals with CT genotype were significantly higher than those with CC genotype during the first 6 months of pregnancy and in non-pregnant cows (P &lt; 0.05). In conclusion, MTHFR may be a beneficial candidate gene to control cow abortion. &nbsp; Key words:&nbsp;Methylenetetrahydrofolate reductase (MTHFR), abortion, Chinese Holstein cattle, SNP.

Bone morphogenetic protein 4 accelerates the establishment of bovine trophoblastic cell lines

Trophoblastic cells play a crucial role in implantation and placentogenesis. A large proportion of the failures of conception in cows occur in the peri-implantation period, which are known as early embryo losses. In exploring this critical phenomenon, trophoblastic cell lines can provide substantial information. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stock in the long term. In this study, 12 new cell lines were established using bone morphogenetic protein 4 (BMP4). BMP4 stimulated embryonic cells to enter the trophoblastic cell lineage but there were no significant differences between intact and BMP4-treated groups. Only one out of 49 embryos developed trophoblastic cells in the intact group. Finally, 12 cell lines were maintained for around 30 passages, and they retained trophoblastic characteristics and expressed bovine trophoblastic genes: placental lactogen, interferon-τ, pregnancy-associated glycoprotein 1, and prolactin-related protein 1. Although the gene expression patterns were different among cell lines and depended on the cells, there was no significant relationship between the expression intensities of genes and the treatment dose of BMP4. All of them expressed bovine POU domain class 5 transcription factor 1 and caudal-type homeobox 2. The expression of these genes was confirmed by quantitative RT-PCR and immunohistochemical detection. These results suggest that BMP4 is involved in the raising of trophoblast cell lines from early embryonic cells and the newly developed cell lines can provide different types of bovine trophoblastic cells with different cell lineages. This may constitute a significant new tool for the examination of trophoblastic differentiation.

English

The objective of this work was to investigate the significance of macrophages and IL-10 in uterus in early embryo loss (or resorption), and to elucidate the anti-abortive effect and the immunological modulation of maternal-fetal interface with quercetin and bornyl Acetate. Lipopolysaccharide (LPS) (0.10 &mu;g/mouse) was injected via the tail vein in order to induce abortion in 7-day-gestation mice which received quercetin and bornyl acetate at days 4 to 7 of gestation.&nbsp;Levels of IL-10 in uterus supernatant were measured using enzyme-linked immuno-absorbent assay (ELISA), and uterine macrophages of each group (n=10) were detected by immunohistochemistry. The levels of IL-10 declined significantly in uterus with LPS treatment. The amount of macrophages in the uterus of LPS-induced abortion mice was much higher than that of the control mice. When quercetin and bornyl acetate were used to prevent LPS-induced abortion, the effect of quercetin combined with bornyl acetate on anti-LPS-induced abortion was more significant, and the IL-10 content was close to normal and the amount of macrophages was decreased to 16.199 &plusmn; 0.802, which was significantly different from that of LPS-induced abortion group. The decrease of IL-10 and the increase of macrophage number in the LPS-treated mice uterus were associated with the embryo loss, and quercetin and bornyl acetate has the anti-abortive effect through modulation of maternal-fetal interface immunity balance. &nbsp; Key words:&nbsp;Embryo resorption, lipopolysaccharide, macrophages, IL-10, mice.

Influence of quercetin on transforming growth factor b1 in the uterus following Lps-induced abortion in pregnant mice

To investigate the significance of transforming growth factor b1(TGF-b1) in the uterus in early embryo loss (or resorption) and to elucidate the anti-abortive effect and the immunological modulation at the maternal-fetal interaction of quercetin (Que). Lipopolysaccharide (LPS) (0.10 μg/mouse) was injected via the tail vein in order to induce abortion in 7-day-gestation mice which also received quercetin at days 1 to 7 of gestation. Uterine TGF-b1 of each group (n=10) was detected by in situ hybridization. The amount of TGF-b1 in the uterus of LPS-induced abortion mice was much lower (P<0.01) than that of the control mice. When quercetin was used to prevent LPS-induced abortion, more TGF-b1mRNA was counted. The effect of high concentration quercetin on LPS-induced abortion was more significant, and the rate of TGF-b1mRNA was increased to 13.52±0.58, significantly higher than that of LPS-abortion group (P<0.01). The change of TGF-b1mRNA in the mouse’ uterus could be associated with the embryo loss, and quercetin exerts an anti-abortive effect through the modulation of the immune balance at the maternal-fetal interface. Key words: Embryo resorption, transforming growth factor-b1, lipopolysaccharide, quercetin, in situ hybridization.

The X and Y chromosome in meiosis: how and why they keep silent

The X and Y chromosome in meiosis: how and why they keep silent

Short communication: Expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies.

Prenatal Exposure to Environmental Concentrations of Organochlorines Impede Male Fertility and Subsequent Embryo Development in the Rat.

Elevated levels of organochlorines, including pesticides and industrial compounds, some of which are known endocrine disruptors, have been reported in human and wildlife populations worldwide. To test the hypothesis that prenatal exposure to organochlorines contaminating the Far North adversely affects reproductive development and function, we developed an experimental rat model. Sprague-Dawley females were gavaged thrice weekly for five weeks with environmentally-relevant concentrations (500 µg PCB/kg body weight/day) of an organochlorine mixture, resembling that found in seal blubber, or corn oil (control). Rats were then mated to untreated males and gavagedthough gestation. We obtained 31 F1 organochlorine males and 33 F1 control males; after weaning, all F1 males were fed commercial rat chow and never directly exposed to organochlorines themselves. In support of our hypothesis, 16% of the F1 organochlorine males retained residual nipples whereas none were apparent on the F1 control males (P = 0.07). Also, prenatally-exposed F1 pups underwent puberty earlier than controls (P < 0.05). To assess fertility and the ability to sire normal offspring (F2 generation), at 90 days of age, 15 F1 control and 15 F1 organochlorine males were each mated to 2 untreated females. At 19.5 days of gestation, foetuses from each female were analysed. Fourteen percent of the F2 foetuses from the prenatally-exposed F1 organochlorine fathers had gross abnormalities, including situus ambigus and cryptorchidism, whereas none of the controls were abnormal. Litter sizes from prenatally-exposed F1 organochlorine fathers were approximately 20% smaller than those from F1 controls (P < 0.05) and, surprisingly, contained fewer male pups than control litters (P = 0.06). More unfertilized oocytes and/or preimplantation embryo loss occurred in females mated to prenatally-exposed F1 males (3.8/female) than when mated to F1 controls (1.0/female) (P < 0.05). Together, these results suggest that prenatal organochlorine exposure reduces sperm function resulting in lower fertility and/or comprised embryo developmental competence. Following these initial observations, we analysed the reproductive organs and sperm quality of the F1 males at 180 days of age. Prenatally-exposed F1 males had smaller epididymides and prostates than F1 controls (P < 0.05). Although prenatally-exposed F1 males had more motile and progressively motile sperm after 2 h incubation at 37°C than F1 controls (P < 0.05), the F1 organochlorine sperm were less able to undergo capacitation in vitro (chlortetracycline fluorescent pattern B) than F1 control sperm (P < 0.05). Susceptibility of sperm DNA to denaturation, assayed by SCSA and acridine orange, were not apparent, however, the TUNEL assay revealed more single- and double-strand DNA breaks in sperm from the prenatally-exposed and control F1 males, perhaps reflecting improper chromatin packaging. In function of these results, it appears that a major impact of prenatal exposure to an environmentally-relevant concentration of organochlorines is through endocrine disruption, resulting in impaired sperm maturation and function. In addition, it is tempting to speculate that the male germ cell epigenome is modified due to prenatal organochlorine exposure, which could contribute to early embryo loss and abnormalities of foetal development. Experiments to test this new hypothesis are currently underway. Financed by FQRNT. (poster)