Preimplantation Embryo Loss Research Articles

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization invitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Role of the hypothalamus in ghrelin effects on reproduction: sperm function and sexual behavior in male mice.

Ghrelin signals to the hypothalamus inhibit reproduction during times of food scarcity. In this study, we demonstrate that ghrelin impairs sperm quality in male mice. Ghrelin (GHRL) is an orexigenic peptide that has been investigated as one of the signals responsible for the reproductive performance of mammals under fluctuating metabolic conditions. Central GHRL administration impairs spermatogenesis in mice by regulating the hypothalamic-pituitary-gonadal axis function. In the present study, the hypothalamus role as a mediator of GHRL effects on sperm fertilizing capacity and male sexual behavior was evaluated. After 42 days of hypothalamic GHRL infusion or artificial cerebrospinal fluid, in vitro and in vivo sperm fertilizing capacity, testicular α-tubulin, speriolin gene expression and spermatic α-tubulin protein were evaluated. Hypothalamic expression of genes Kiss1, Gpr54 and Gnrh was also studied. The second group of animals was infused with one time only GHRL or artificial cerebrospinal fluid into the hypothalamus to evaluate the effects on sexual behavior. Results demonstrated that chronic GHRL administration to male mice significantly increased the percentages of pre-implantation embryo loss and the number of post-implantation embryo loss. In relation to the gene expression, our results show a relative decrease of Kiss1, Gpr54 and Spatc1. Although no significant differences were observed in the quantitative expression of α-tubulin protein, qualitative changes in its expression pattern were observed. In addition, a dual effect on sexual behavior was observed: 40% of the treated animals showed a significant reduction in the number of mounts and intromissions, while a 60% showed a significant decrease in ejaculation latency vs control animals. In conclusion, our results provide evidence that central GHRL administration possibly induces failure in embryo development and/or implantation in the females mated with treated males, possibly because of a negative effect in the α-tubulin pattern.

O-289 Effects of Taxol on the developmental potency of human and mouse GV oocytes

Abstract Study question Is it possible to improve the quality of oocytes with delayed maturation or aging? Summary answer Yes, modification of oocyte microtubules at the germinal vesicle (GV) stage inhibits abnormal chromosome separation in in-vitro maturation and enhances early cleavage. What is known already Oocyte aging is characterized by an increase of aneuploidy and a decrease of the developmental potency with maternal ages, which will results in low frequencies of the blastocyte formation and implantation. In addition to the 1st meiotic division, it has become clear that the 2nd meiotic division also significantly contributes to aneuploidy production which is followed by pre-implantation embryo loss. However, no method for overcoming the oocyte aging has been established. Study design, size, duration Human GV oocytes with delayed maturation obtained from consented female patients were used. Mouse GV oocytes collected from 9-15 month old ICR mice were also used. After exposure to Taxol for 1hr, the oocytes were matured in vitro and examined cytogenetically and cytologically at the GV stage, the MII stage and pronuclear stage. Participants/materials, setting, methods RNA transcripts of the GV stage oocytes were compared before and after Taxol exposure with microarray in both species. Chromosome aberrations at the MII stage and blastocyst formation rate were examined in human IVM oocytes. Mouse IVM oocytes were evaluated on 2nd polar body (PB) extrusion and O2 consumption (CRAS3.0, Crino Co Inc.) at the pronuclear stage after parthenogenetic activation with electro-stimulation. Main results and the role of chance After Taxol treatment, premature chromosome separation was significantly reduced from 96% to 7% and the blastocyst formation rate increased from 3% to 16% in human. In mice, normal PB extrusion rate increased from 29% to 92%, and oxygen consumption was higher in some pronuclear oocytes. In human GV oocytes, taxol treatment altered gene expression only in a few factors related to chromosome attachment and segregation. On the other hand, in mouse GV oocytes, gene expression was significantly altered by oocyte aging and Taxol treatment. Limitations, reasons for caution The present results were obtained in human GV oocytes with delayed maturation which were collected after PMS and HCG administrations. It is not clear whether Taxol is effective on the GV oocytes collected before HCG administration. Wider implications of the findings Our results indicate that the combination of biochemical modification and IVM in the GV stage may be able to restore the aging of human oocytes. Trial registration number not applicable

Early pregnancy loss: A critical review of the evidence for embryo “wastage”

Approximately 10% of clinically recognized pregnancies end in miscarriage. Many authors suggest that this number greatly underestimates the death of early human embryos, commonly asserting that in addition to clinical pregnancy loss, as many as 60% of all conceptions are lost before a recognized pregnancy is established. However, a critical review of the literature indicates that previous analyses of early embryo loss have relied on inadequate data, unwarranted speculation and insufficient clarity about what constitutes a human embryo. Anovulation, fertilization failure and non-development (none of which entail embryo demise) make significant contributions to observed fecundity, yet have often been ignored. Although the actual percentage of early human embryo loss is not known with precision, based on multiple lines of evidence, preimplantation embryo loss is likely to be negligible (if it occurs at all). When all early pregnancy loss is considered, embryo wastage does not exceed 35%, and is likely to be lower than this maximum figure, possibly as low as 7%. In light of this conclusion, the relevance of embryo wastage to an assessment of the moral worth of the human embryo is briefly considered.

Congenital Malformations Attributed to Prenatal Exposure to Cyclophosphamide.

Cyclophosphamide (CPA) remains one of the most widely prescribed anticancer drugs. It is also used in the treatment of rheumatoid arthritis, childhood nephrotic syndrome and systemic lupus erythematosus. It is a potent immunosuppressive agent. It is commonly used in blood and bone marrow transplantation. With the growing trend among women postponing childbearing, the number of women who are diagnosed with breast cancer is also increasing thus escalating the chances of exposure of the unborn child to antineoplastic drugs. A review of the literature provides strong evidence for the teratogenic effects on infants prenatally exposed to CPA. Both sporadic case reports and larger case series have demonstrated that babies with cyclophosphamide embryopathy are afflicted with intrauterine growth restriction, small for gestational age, and craniofacial malformations including eye anomalies, cleft/arched palate, hydrocephaly, micrognathia, low set microtia, hearing defects, craniosynostosis, and facial asymmetry. Also observed in these cases are limb defects such as radial, ulnar and tibial hypoplasia, club foot, digital defects of the hand and feet as well as vertebral fusion, brevicolis, and occasional Sprengel's deformity. These anomalies vary in consistency of occurrence and severity of the phenotype across cases and lack the specificity of thalidomide embryopathy or rubella embryopathy. However, they do occur is no longer in doubt. First trimester of pregnancy seems to be particularly susceptible to fetal malformations, although CPA effects on fetuses of later stages of gestation (hearing defects, growth restriction for example) are also reported occasionally. One of the major concerns from a mechanistic point of view is our inability to dissect the teratogenic effects of CPA from those of other drugs administered together with CPA as combination therapy. Animal experiments have been of particular value in that they are able to circumvent the numerous extraneous variables inherent to human case reports. They have also revealed the detrimental effects of CPA on gametes, preimplantation embryos, organogenesis as well as their potential teratogenic mechanisms. Of particular importance are the role of genetic polymorphisms, male mediated teratogenesis, ovarian failure, preimplantation embryo loss, epigenetic modifications, proxidant-antioxidant imbalance, autophagy, apoptosis, microRNAs and postclosure neural tube defects induced by CPA -all of which are areas for further research in CPA teratogenesis.

Effects of oocytes exposure to bovine diarrhea viruses BVDV-1, BVDV-2 and Hobi-like virus on in vitro-produced bovine embryo development and viral infection

As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 × 102 - 2.50 × 103 TCID50/100 μL), BVDV-2 (6.25 × 101 - 5.20 × 102 TCID50/100 μL) or Hobi-like virus (1.90 × 102 - 1.58 × 103 TCID50/100 μL) for 22–24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50–100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2–64.1% vs 49.8%; P = 0.003–0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20–50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3–38.0% vs 49.8% for negative controls; P ≤ 0.001–0.014). Blastocyst rates were only reduced in the 7.9 × 102 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P = 0.009), when calculated by oocyte number. 50–80% of degenerated embryos tested positive for Hobi-like virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and -2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.

Fertility defects in Surfactant associated protein D knockout female mice: altered ovarian hormone profile.

Surfactant associated protein D (SFTPD, also known as SP-D), a pattern recognition molecule, is an integral component of the mucosal immune system of female reproductive tract (FRT). In addition to host defense functions in the FRT, recent evidences indicate immunomodulatory role of SFTPD in parturition and pre-term labor. Regulation of SFTPD expression by ovarian hormones in the mouse uterus implicates SFTPD of FRT in pregnancy establishment and maintenance. In the current study, we attempted to decipher the functional relevance of SFTPD in FRT by characterizing the fertility parameters of surfactant associated protein D knockout (Sftpd(tm1Jhf)/Sftpd(tm1Jhf)) female mice. Knockout female mice exhibited extended estrous cycle with altered serum profile of ovarian hormones. We also demonstrate altered expression of ovarian hormone receptors and hormone responsive genes ITGB1, LIF and HOXA10 in uteri of these mice. Knockout females mated with wild type males had significantly smaller litter size due to increased pre-implantation embryo loss. We also observed an altered immune profile in knockout mice uteri with elevated levels of inflammatory cytokines, increased numbers of pro-inflammatory monocytes/macrophages and lower FOXP3 levels during the pre-implantation period. LPS administration to pregnant knockout mice did not result in any increase in embryo implantation loss and was associated with a blunted uterine pro-inflammatory response, plausibly due to higher levels of serum progesterone. Taken together, our results demonstrate that SFTPD deficiency affects female fertility, highlighting roles for SFTPD in ovarian and uterine physiology.

Reproductive effects of the tumor necrosis factor (TNF) deficiency in mice

TNF is a multifunctional cytokine that, at physiological concentrations, maintains the balance between apoptosis and survival of male germ cells and, at higher concentrations, has adverse effects on various stages of the reproductive process. Although ant-cytokine therapies have been used in millions of patients, the consequences of cytokine deficiency for reproductive functions are poorly understood and need attention. In this work, we have studied behavioral interactions between males and females, spermatogenesis, male fertility, and embryonic developmental characteristics of the progeny in TNFα knockout mice (TNF-/-). We have demonstrated that TNF is involved in the regulation of sexual behavior, spermatogenesis, pre- and postimplantation development. Complete TNF deficiency led to decreased reproductive efficiency: a lower number of viable embryos were observed in TNF-/- mice than in wild-type mice. The decrease in fertility was caused by preimplantation embryo loss in TNF-/- mice. Preimplantation loss in females might be caused by asospermia in TNF-/- males. Additionally, the sensitivity of reproductive functions to female stimuli was different between TNF-/- mice and wild-type mice, while interactions with females increased the concentrations of sper­matozoids in both TNF-/- and wild-type mice. Still higher levels were observed in knockout animals, which led to increase in the number of immature spermatozoids in epididymides.

Neonatal Phytoestrogen Exposure Alters Oviduct Mucosal Immune Response to Pregnancy and Affects Preimplantation Embryo Development in the Mouse1

Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are "posteriorized" as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype.

Permanent Oviduct Posteriorization after Neonatal Exposure to the Phytoestrogen Genistein

Background: Preimplantation embryo loss during oviduct transit has been observed in adult mice after a 5-day neonatal exposure to the phytoestrogen genistein (Gen; 50 mg/kg/day).Objective: We investigated the mechanisms underlying the contribution of the oviduct to infertility.Methods: Female mice were treated on postnatal days 1–5 with corn oil or Gen (50 mg/kg/day). We compared morphology, gene expression, and protein expression in different regions of the reproductive tracts of Gen-treated mice with those of control littermates at several time points.Results: Neonatal Gen treatment resulted in substantial changes in expression of genes that modulate neonatal oviduct morphogenesis, including Hoxa (homeobox A cluster), Wnt (wingless-related MMTV integration site), and hedgehog signaling genes. An estrogen receptor antagonist blocked these effects, indicating that they were induced by the estrogenic activity of Gen. Oviducts of adults treated neonatally with Gen had abnormal morphology and were stably “posteriorized,” as indicated by altered Hoxa gene patterning during the time of treatment and dramatic, permanent up-regulation of homeobox genes (e.g., Pitx1, Six1) normally expressed only in the cervix and vagina.Conclusions: Neonatal exposure to estrogenic environmental chemicals permanently disrupts oviduct morphogenesis and adult gene expression patterns, and these changes likely contribute to the infertility phenotype.

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)

The unique transcriptome through day 3 of human preimplantation development

Successful human development is dependent upon a cascade of events following fertilization. Unfortunately, knowledge of these critical events in humans is remarkably incomplete. Although hundreds of thousands of human embryos are cultured yearly at infertility centers worldwide, the vast majority fail to develop in culture or following transfer to the uterus. In this study, we sought to characterize global patterns of gene expression in individual, normal embryos during the first three days of embryonic life using microarrays; we then compared gene expression between normally growing and growth-arrested embryos using quantitative PCR. Our results documented several novel findings. First, we found that a complex pattern of gene expression exists; most genes that are transcriptionally modulated during the first three days following fertilization are not upregulated, as was previously thought, but are downregulated. Second, we observed that the majority of genes exhibiting differential expression during preimplantation development are of unknown identity and/or function. Third, we show that embryonic transcriptional programs are clearly established by day 3 following fertilization, even in embryos that arrested prematurely with 2-, 3- or 4-cells. This indicates that failure to activate transcription is not associated with the majority of human preimplantation embryo loss. Finally, taken together, these results provide the first global analysis of the human preimplantation embryo transcriptome, and demonstrate that RNA can be amplified from single oocytes and embryos for analysis by cDNA microarray technology, thus lending credence to additional studies of genetic regulation in these cell types, as well as in other small biological samples.

Mastitis and fertility in cattle - possible involvement of inflammation or immune activation in embryonic mortality.

Causes for pre-implantation embryo loss, which can be as high as 50% or more of fertilized embryos, are multifactorial and largely undescribed. Studies in cattle using mastitis as a model indicate that one cause of early embryonic loss is infectious disease or activation of immune responses at sites outside the reproductive tract. Infection of the mammary gland in dairy cattle is associated with a reduction in pregnancy rate (proportion of inseminated cows that become pregnant) and an increase in the number of inseminations required to establish pregnancy. Also, intravenous challenge with bacterial peptidoglycan and polysaccharide at approximately days 3-5 after breeding reduced subsequent pregnancy rate in sheep that had been previously immunized against the same material. The mechanism by which extrauterine activation of immune and inflammatory responses leads to embryonic loss is not clear although cytokines probably play a crucial role. Effects could be exerted at the level of the hypothalamic-pituitary axis, ovary, reproductive tract or embryo. Interferon (IFN)-alpha, for example, which can reduce pregnancy rate in cattle when injected around 13-19 days after breeding, increases body temperature, inhibits secretion of luteinizing hormone, and reduces circulating concentrations of progesterone. Other cytokines or products of cytokine activation could cause embryonic loss by causing hyperthermia (as elevated temperature blocks oocyte function and embryonic development), exerting toxic effects on the corpus luteum [for example, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) and prostaglandin F(2alpha)], stimulating endometrial prostaglandin synthesis [TNF-alpha and interleukin(IL)-1beta], reducing endometrial cell proliferation (IL-1beta), and interfering with oocyte maturation and embryonic development (TNF-alpha, nitric oxide, and prostaglandin F(2alpha)). Although largely neglected by reproductive immunologists, study of the involvement of the immune system in pre-implantation embryonic loss is likely to lead to new methods for enhancing fertility.

Effect of tamoxifen on sperm fertilising ability and preimplantation embryo development

Recent evidences point to a role of estrogens in males. We have earlier reported that tamoxifen, a synthetic non-steroidal antiestrogen, when administered to adult male rats, in the dose range of 0.04–0.4 mg/kg per day, reduced fertility. The reduced fertility was measured in terms of fertility index (a measure of the efficiency of the ovulated ovas to fertilise and implant), fecundity (siring ability) and litter size. The present study was done to investigate whether the reduction in fertility index was due to reduction in fertilising ability or increase in pre-implantation embryo loss. Also a dose related effect of tamoxifen from 0.02 mg to 2 mg/kg per day on the fertility of the male rats was studied. To study the fertilising ability, control and tamoxifen (0.4 mg/kg per day, the most effective dose) treated adult male rats were mated with normal cycling females and the females sacrificed at day 0–4 of gestation. Eggs fertilised/unfertilised were flushed from the oviduct/uterus and the number and types of eggs were noted. The index of fertilisation, a measure of the fertilising ability was determined. The studies demonstrate that the reduction in fertility is not due to decreased fertilising ability but because of the increased pre-implantation embryo loss as evident from an increase in number of abnormal eggs in the treated group with no change in index of fertilisation. A dose related decrease in fertility was observed. The present study suggests that tamoxifen at 0.02–2-mg dose is predominantly estrogenic in males and paternal factor/s sensitive to tamoxifen is involved in embryogenesis.

Fate of unfertilized ova in male rodent dominant lethal assays: Extension of the studies by Kratochvilova

Kratochvilova has described a technique whereby ova can be recovered from mated mice and their stage of division determined. This is of value to determine if reduced total implantations in a male dominant lethal (DL) germ cell mutation assay are due to pre-implantation loss of embryos, a presumed mutagenic event, or to chemically induced male infertility. Kratochvilova was not specific about the fate of unfertilized ova, but it was implied that they undergo a process of fragmentation that might be confused with the regular cleavage of fertilized ova. It became important for us to draw a firm distinction between ova fragmentation and regular ova cleavage in the rat. We therfore repeated the ova analyses of female mice mated with males exposed to iso-propyl methanesulphonate (iPMS), as described by Kratochvilova. Following that calibration study the technique was extended to the rat via ova cleavage analysis in mated female rats, coupled to a study of the normal decay of ova in virgin rats. Unfertilized ova are shown to undergo irregular fragmentations that can be clearly distinguished from normal cell division. It is concluded that the individual or combined incidences of single celled ova and fragmented ova (dependent on the cleavage stage of the concurrent control embryos) can provide a measure of male infertility as it relates to reduced implantations in DL assays. This ability to regard two morphological classifications of unfertilized ova as providing evidence for male infertility will simplify the conduct of ova analyses in both the mouse and the rat.

Mouse Model for the Treatment of Immune Pregnancy Loss

Spontaneous abortions can be associated with preimplantation embryo loss, implantation problems and a variety of postimplantation pregnancy failures. The long list of possible causes for the postimplantation pregnancy loss includes, among others, genetic abnormalities in fetus, anatomical abnormalities of the uterus, endocrinological insufficiency, and microbiological problems. However, more than 50% of recurrent miscarriages still have no recognized causes. The concept that many such abortions may be immunologically mediated has gained increasing support over the years. Moreover, immunization of such women with husband's or third party leukocytes has resulted in more than 70% of subsequent pregnancies resulting in live births. Since neither the mechanisms leading to pregnancy loss nor the success of immunotherapy are clear, the set-up of animal models for recurrent abortions would be of supreme significance. Our recent data show that immunopotentiation of maternal immune system by Complete Freund Adjuvant significantly improves pregnancy rate in CBA x DBA/2 mouse combination with high percentage of fetal resorptions. This effect is followed by decrease of IL 2 production in spleen; increase of MAC 1-positive cells at placenta; amplification of suppressive activity of local and systemic lymphocytes and by reverse of embryotoxic effect of maternal serum. Data obtained in this model seems to be valuable in substantiation of rationale for nonspecific immunotherapy of human abortions.

Role of non-luteal ovarian tissues in the maintenance of pregnancy.

ABSTRACT The maintenance of pregnancy by daily injections of progesterone from the time of nidation to term has been studied in rats following removal of the ovaries, removal of the active corpora lutea or pharmacological suppression of the corpora lutea with ergocornine. Progesterone was able to maintain normal embryogenesis following ergocornine or removal of corpora lutea but not in ovariectomized rats. This suggests that the non-luteal tissues of the ovary secrete hormones essential for the maintenance of gestation. Other observations include a high pre-implantation loss of embryos and an increase in the average conceptus weight not correlated with the litter size following removal of corpora lutea or of the ovaries. It is concluded that pregnancy is maintained by the combined activities of luteal and non-luteal ovarian tissues.

Genetic radiosensitivity of specific post-dictyate stages in mouse oöcytes

The induction of dominant lethals after X-irradiation of dictyate, later meiotic stages, and the pronuclear stage after fertilization have been compared using the technique of induced ovulation in mice. The injection of gonadotrophins ensures that the time at which the synchronously dividing oöcytes reach any particular meiotic stage is accurately known. Embryonic lethality up to 13½ days was studied after 0 r., 100 r. and 200 r. acute X-irradiation. Metaphase I, anaphase I and metaphase II were the most sensitive stages, with LD50's of about 120 r., 130 r. and 170 r. respectively. The dictyate and pronuclear stages were much less sensitive, with LD50's in the region of 500 r., and sensitivity rose steeply during prophase I. Numbers of corpora lutea decreased with irradiation, the decrease being greatest with irradiation at metaphase I and anaphase I. The ovulation of large numbers of eggs, which increased the preimplantation loss of embryos above normal, and the low luteal counts probably masked lethality to some extent. Results generally agree well with those reported in a number of plant and animal species.