A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9±0.1 h (range 0.70-10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p<0.05) with shrinkage ceasing after 2h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4±0.2 vs. 2.8±0.1, p<0.01) or 35-38 years (3.4±0.2 vs. 2.8±0.1, p<0.01), but timing was not related to the Day 3 morphological grades (1-4) of subsequent embryos (2.9±0.1, 2.9±0.1, 2.8±0.2 and 3.0±0.1; p>0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC=0.596 or 0.601, p=0.006 or 0.004) and usable embryos (AUC=0.638 or 0.632, p=0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.
RU486 is a partial progesterone and estrogen receptor antagonist, functioning to actively silence progesterone receptor gene-associated transcription. For this reason, it has been used as both a contraceptive and an abortive agent. In the present study, cellular and gene specific effects of RU486 were investigated in a rat model of early pregnancy, including key phases of the window of receptivity and early implantation. As these stages are hormonally regulated by progesterone and estrogens, the focus here was to elucidate the mechanism of action of a single dose of RU486, used as a postcoital contraceptive, to successfully prevent implantation of a viable blastocyst. Immunofluorescent techniques were used to examine the change in protein levels of PR in RU486-treated endometria at days 4.5, 5.5 and 6.5 of pregnancy. Changes in the Pgr gene expression level as a consequence of RU486 administration was evaluated using quantitative real-time reverse transcription polymerase chain reaction. The progesterone receptor gene and protein expression was ubiquitously decreased throughout pregnancy as a direct consequence of RU486 administration. The overall effects of postcoital RU486 administration during early pregnancy indicate highly effective inhibition of progesterone and estrogen effects on the endometrium, mediated by their receptors. More specifically, the expression and localization of the progesterone receptor mirrors that described in ovariectomized animal models, suggesting a hormonally under-stimulated endometrium. Clearly from the present study, the precise priming of the endometrium by progesterone, in preparation for blastocyst implantation, is severely impaired by RU486, thus predisposing the uterus to pregnancy failure.
The aim of the present study was to identify differentially expressed genes and their related biological pathways in the secretory phase endometrium from patients with recurrent miscarriage (RM) and fertile subjects. Endometrial samples from RM and fertile patients were analyzed using the Affymetrix GeneChip® ST Array. The bioinformatic analysis using the Partek Genomic Suite revealed 346 genes (175 up-regulated and 171 down-regulated) that were differentially expressed in the endometrium of RM patients compared to the fertile subjects (fold change ≥1.5, p<0.005). Validation step using quantitative real-time polymerase chain reaction (qPCR) confirmed a similar expression pattern of four exemplary genes: one up-regulated gene (fibroblast growth factor 9, FGF9) and three down-regulated genes: integrin β3 (ITGB3), colony stimulating factor 1 (CSF1) and matrix-metalloproteinases 19 (MMP19). The Gene Set Enrichment Analysis (GSEA) and the Pathway Studio software have found 101 signaling pathways (p<0.05) associated with the affected genes including the FGFR3 /signal transducer and activator of transcription (STAT) pathway and the CSF1R/STAT pathway. Cell adhesion, cell differentiation and angiogenesis were among biological processes indicated by this system. In conclusion, microarray technique is a useful tool to study gene expression in the secretory phase-endometrium of RM patients. The differences in endometrial gene expressions between healthy and RM subjects contribute to an increase in our knowledge on molecular mechanisms of RM development and may improve the outcome of pregnancies in high-risk women with RM.
The possible role of insulin-like growth factors (IGFs) and their receptors (IGFRs) in the pathogenesis of diabetic embryopathy was investigated. Sexually mature female ICR mice of 6-8 weeks old were made diabetic by a single intraperitoneal injection with 200 mg/kg streptozotocin ten days prior to mating. Fallopian tubes and uterine tissues were obtained from the superovulated diabetic and normal mice 48, 72 and 96 hours following human chorionic gonadotropin (hCG) injection. The mRNA expression of IGF-1 and IGF-2 as well as their receptors was determined in the tissues using Real-time Polymerase Chain Reaction (Real-time PCR). The mRNA expression of IGF-1 in the fallopian tube and uterus of the diabetic mice was significantly lower 72 and 96 hours after hCG treatment, respectively, as compared to the controls. The mRNA expression of IGF-1R at 96 hours post-hCG treatment was significantly higher in the fallopian tube and lower in the uterus of the diabetic mice as compared to the controls. The mRNA expression IGF-2 in the fallopian tube was significantly higher 48 and 96 hours after hCG treatment, but was lower in the uterus of diabetic mice 96 hours after hCG treatment as compared to controls. The mRNA expression of IGF-2R in the diabetic mice was significantly higher 48 and 96 hours (the fallopian tube) and 48 hours (uterus) after hCG treatments as compared to the controls. In conclusion, an alteration in mRNA expression of IGFs and their receptors in the diabetic mice as observed in this study could possibly result in diabetic embryopathy.
The effects of estrus synchronization with prostaglandin F(2α) (PGF(2α)) and Controlled Internal Drug Release Device (CIDR) on ensuing antral follicular development were documented and compared to natural estrous cycles of non-seasonal tropical goats. Two to six follicular waves were observed, with the three-follicular wave pattern being most frequently observed (58%), followed by four follicular waves (31.6%) per estrous cycle. There were no significant differences (p>0.05) between the PGF(2α)- or CIDR-synchronized and natural estrous cycles nor between the synchronized and subsequent non-synchronized cycles in terms of the time of ovulation, the duration of inter-ovulatory intervals, daily numbers of antral follicles ≥3mm in diameter, and the number of follicular waves per cycle in the goats of the present study.
Advanced age is an increasing trend for both males and females seeking in vitro fertilization (IVF). This retrospective cohort study investigated the outcomes of 1280 IVF-related treatment cycles, selecting the first treatment for couples utilizing autologous gametes and who underwent single fresh embryo transfer. Males aged 40-49 years had a 52% reduction in normal sperm motility, while it was markedly reduced by 79% at 50 years or older. However, neither semen parameters nor male age were predictive of clinical pregnancy or live birth chance. In a combination of age groups, cases with Younger Females had the greatest chance of successful outcomes and this was independent of having a younger or older male partner. Specifically, Young Female-Young Male combinations (≤ 35 years) were the most likely to succeed in achieving a clinical pregnancy or live birth (OR 2.84, p 35 years, respectively) had a similar increased chance (OR 2.07, p
Levels of leptin and marinobufagenin (MBG), a cardiotonic steroid, are elevated in the serum of women with pre-eclampsia. Besides this, leptin administration to pregnant rats increases systolic blood pressure (SBP), urinary protein excretion and serum markers of endothelial activation. The link between leptin and MBG is unknown and it is also unclear if leptin-induced increases in blood pressure and proteinuria in the pregnant rat could be prevented by an MBG antagonist. To ascertain this link, this study investigated the effect of resibufogenin (RBG), a marinobufagenin antagonist, on leptin-induced increases in blood pressure and proteinuria during pregnancy in rats. Four groups of Sprague-Dawley rats, aged 12 weeks, were given either normal saline (CONTROL) or 120 μg/kg/day of leptin (LEP), or 120 μg/kg/day of leptin+30 μg/kg/day of resibufogenin (L + RBG) or 30 μg/kg/day of resibufogenin (RBG) from Day 1-20 of pregnancy. Systolic blood pressure and urinary protein excretion (UPE) were measured during the study period. Animals were euthanized on day 21 of pregnancy and vascular cell adhesion molecule 1, (VCAM-1), soluble intracellular cell adhesion molecule 1 (sICAM-1), E-selectin and endothelin-1 (ET-1) were estimated in the serum. SBP, UPE, VCAM-1, sICAM-1 and ET-1 were significantly higher only in the LEP group when compared with those in CONT and in L + RBG and RBG groups. The prevention by RBG of leptin-induced increases in SBP, proteinuria, and endothelial activation during pregnancy seem to suggest a potential role for MBG in leptin-induced adverse effects on blood pressure, urinary protein excretion and endothelial activity during pregnancy in the rat.
There is a paucity of studies on effect of iron in embryo culture procedures. This study aims to ascertain the optimal, tolerance and toxic levels of iron in a protein-free embryo culture medium (PFM) to determine the effect of iron on embryonic development. The application of PFM in assisted reproductive technologies (ART) is intended to eliminate disease transmission and improve ART treatment outcome. The optimal, tolerance and toxic levels of iron on human spermatozoa and mouse embryos were determined by challenging them with different levels of iron (ferric iron; Fe(+3)). Human normozoospermic semen samples (n=24) and days 1-4 Quakenbush Special (Qs) mouse embryos (n=1160) were incubated in PFM supplemented with different concentrations of Fe(+3) over different periods of time. 2.0μg/mL (35.8μM) of Fe(+3) was the optimal level of Fe(+3) for human spermatozoa with a tolerance range of 0.5-2μg/mL; whereas a level of 0.11μg/mL (2μM) of Fe(+3) was the optimum for day 2 embryos. Levels of ferric iron at 0.11 to 2.8μg/mL appear to enhance spermatozoa motility, preserve its DNA integrity and possibly increase percentage of blastocysts developed but levels of ferric iron >16μg/mL is hazardous for both spermatozoa and embryos. In spite of beneficial effects of iron it is premature to recommend its supplementation in embryo culture media because of the known deleterious nature of iron and the paucity of toxicological data. Toxicological studies must be performed following which it can be decided whether it is safe to consider iron as a supplement in human embryo and spermatozoa culture media.
The objectives of this study is to evaluate the efficacy of protein-free media in the preparation, holding and crypreservation of spermatazoa for use in ART. Normozoospermic semen samples (N=71) were used to compare the effects of media on the survival and quality of spermatozoa when washed and cultured with different media with and without added proteins at 4°C, 15°C, 22°C and 37°C for 0, 4-7 and 24h. Survival and quality of spermatozoa were assessed after freeze-thaw with synthetic cryoprotectant with and without proteins. Ethics/IRB approval was obtained (Ref. 1073.52). Spermatozoa parameters were similar in all media after washing and culture for 24h. Post-thaw survival and quality of spermatozoa was not significantly different 24h after thawing of samples frozen in all cryoprotectant medium. In conclusion synthetic protein-free culture and cryoprotectant media are equal in efficacy to protein-containing media in culture and cryopreservation of spermatozoa . Use of these synthetic media are anticipated to significantly reduce the risk, potentially associated with conventional protein-containing media, of transmission of disease and possibly harmful undeclared proteins to the patient, baby and the healtcare worker. Synthetic media also ensure consistency of quality between batches of media.
Amniotic fluid (AF) is now known to harbor highly potent stem cells, making it an excellent source for cell therapy. However, most of the stem cells isolated are from AF of mid-term pregnancies in which the collection procedure involves an invasive technique termed amniocentesis. This has limited the access in getting the fluid as the technique imposes certain level of risks to the mother as well as to the fetus. Alternatively, getting AF from full-term pregnancies or during deliveries would be a better resolution. Unfortunately, very few studies have isolated stem cells from AF at this stage of gestation, the fluid that is merely discarded. The question remains whether full-term AF harbors stem cells of similar potency as of the stem cells of mid-term AF. Here, we aim to review the prospect of having this type of stem cells by first looking at the origin and contents of AF particularly during different gestation period. We will then discuss the possibility that the AF, at full term, contains a population of highly potent stem cells. These stem cells are distinct from, and probably more potent than the AF mesenchymal stem cells (AF-MSCs) isolated from full-term AF. By comparing the studies on stem cells isolated from mid-term versus full-term AF from various species, we intend to address the prospect of having highly potent amniotic fluid stem cells from AF of full-term pregnancies in human and animals.
Low sperm concentration, increased fraction of morphologically abnormal sperm, and raised levels of markers of oxidative stress are often reported in the seminal plasma of infertile obese males. The precise reason for changes remains unknown. This short review summarises evidence from human and animal studies linking leptin to the reproductive dysfunction reported in obese males and presents a possible mechanism for this based on the available data in the literature. Serum leptin concentrations correlate positively with body fat mass but its precise link to semen abnormalities reported in obese males has yet to be conclusively established. Decreased sperm concentration, increased fraction of morphologically abnormal sperm and increased markers of oxidative stress have been reported following six weeks of daily leptin treatment to normal weight rats. In addition, decreased expression of endogenous antioxidant enzymes and increased expression of respiratory chain enzymes noted in the testes of leptin treated rats increases the propensity to oxidative stress. Besides that, leptin's interference with histone to protamine transition in the DNA of sperm increases the susceptibility of sperm to free radical attack and may explain the often reported higher DNA fragmentation index in sperm of obese males. Concurrent supplementation of melatonin, a natural anti-oxidant, to these rats prevents the effects of leptin. The role of leptin in obesity-related reproductive dysfunction has to be considered seriously and these effects of leptin might involve increased oxidative stress.
This observational study examines the outcomes of pregnancies arising in women referred for infertility, where those who experienced threatened miscarriage were treated with medroxyprogesterone acetate (MPA) tablets. The 14-year study period covers comprehensive real-time data entries into the validated electronic database including details of the infertility management, pregnancy outcomes and any foetal anomalies among the infants, each being tracked and recorded. Of 4057 clinical pregnancies, 1343 received MPA for threatened miscarriage; 934 (69.6 %) of which continued to livebirths. These were compared with the remaining 2714 clinical pregnancies without threatened miscarriage or MPA and which resulted in 2075 (76.5 %) livebirths. There were 134 developmental abnormalities recorded among the 3009 livebirths of which 78 (2.6 %) were categorised appropriate for the Western Australian Developmental Abnormalities Register; WARDA. These comprised 55 in the MPA group, 36 of which were categorised as serious (being 2.7 % of clinical pregnancies and 3.9 % of births). In the group without MPA, there were 79 abnormalities, of which 42 were categorised as serious (being 1.7 % of clinical pregnancies and 2.2 % of births). Specifically, there were no cases of androgenisation noted among the female infants. The abnormality rates were low overall and well within the annual WARDA ranges. We cautiously suggest that oral MPA can be considered for studies throughout pregnancy including the early first trimester to assess a potential role in reducing miscarriage, as well as advanced pregnancies to evaluate a potential role in reducing stillbirths and preterm delivery.
The objective of this research was to study the ameliorative effects of a standardized quassinoid-rich extract (TAF 273) of Eurycoma longifolia root on some reproductive disorders in female rats. An irregular estrous cycle and ovarian cystic follicles were induced in 21-day-old females by the daily administration of testosterone (10 mg/kg, sc) for three weeks. The hormone-treated rats exhibited persistent diestrous as well as ovaries containing cystic follicles. Upon treatment with TAF 273, fewer animals showed irregular estrous cycles and there was less follicular morphological damage. The reversal effect may be derived from the anti-estrogenic properties of the plant quassinoids.
A parallel group superiority prospective randomised controlled trial was devised to compare the culture characteristics of human pre-implantation stage embryos during uninterrupted culture in a time lapse incubator (TLI) versus the conventional model of interrupted culture in a standard incubator (SI) under low oxygen tension using a single step medium. 221 patients aged 35-and-under, 124 patients aged between 36 and 39 and 86 patients aged 40-and-over years were randomised and cultured either in a SI or in a TLI. Patients in the three age groups were distributed between the TLI and SI in a 1:1 ratio. The development of embryos on days 2, 3 and 5, and the clinical pregnancy and implantation rates were recorded. The fertilisation rate, development of day 2 and clinical pregnancy rates were similar in both treatments but the 8-cell development rate in all age groups combined (p = 0.016), blastocyst development rate (p = 0.0022) and the implantation rate (p = 0.0022) was significantly higher for the uninterrupted culture. These findings demonstrated significant differences between the two incubation groups. It also indicated less efficacious embryonic development with age in both treatments which appeared more pronounced in the conventional incubator. In conclusion uninterrupted culture is superior compared to the interrupted incubation culture system.
This seven-year retrospective study analysed the live birth rate (LBR) for women undergoing IVF treatment with various antral follicle counts (AFC). The LBR decreased with lower AFC ratings, and in 290 treatment cycles for women in the poorest AFC category, ≤4 follicles (group E), the LBR was the lowest at 10.7%. The pregnancy loss rate (PLR) significantly increased with poorer AFC categories, from 21.8% in AFC group A (≥20 follicles), to 54.4% in AFC group E (p<0.0001). This trend was repeated with advancing age, from 21.6% for younger women (<35years), to 32.9, 48.5 and 100% for ages 35-39, 40-44 and ≥45 years, respectively (p<0.0001). However, LBR within the specific AFC group E cohort was also age-dependent and decreased significantly from 30.0% for <35 years old, to 13.3, 3.9 and 0% for patients aged 35-39, 40-44 and ≥45 years, respectively. Most, importantly, LBR rates within these age groups were not dependent on the number of IVF attempts (1st, 2nd, 3rd or ≥4 cycles), which indicated that cycle number should not be the primary deciding factor for cessation of IVF treatment in responding women <45years old.