Definition by FSH, AMH and embryo numbers of good-, intermediate- and poor-prognosis patients suggests previously unknown IVF outcome-determining factor associated with AMH.

BACKGROUND: Though outcome models have been proposed previously, it is unknown whether cutoffs in clinical pregnancy and live birth rates at all ages are able to classify in vitro fertilization (IVF) patients into good-, intermediate- and poor prognosis. METHODS: We here in 3 infertile patient cohorts, involving 1247, 1514 and 632 women, built logistic regression models based on 3 functional ovarian reserve (FOR) parameters, including (1) number of good quality embryos, (2) follicle stimulating hormone (FSH, mIU/mL) and (3) anti-Müllerian hormone (AMH, ng/mL), determining whether clinical pregnancy and live birth rates can discriminate between good, intermediate and poor prognosis patients. RESULTS: All models, indeed, allowed at all ages for separation by prognosis, though cut offs changed with age. In the embryo model, increasing embryo production resulted in linear improvement of IVF outcomes despite transfer of similar embryo numbers; in the FSH model outcomes and FSH levels related inversely, while the association of AMH followed a bell-shaped polynomial pattern, demonstrating "best" outcomes at mid-ranges. All 3 models demonstrated increasingly poor outcomes with advancing ages, though "best" AMH even above age 43 was still associated with unexpectedly good pregnancy and delivery outcomes. Excessively high AMH, in contrast, was at all ages associated with spiking miscarriage rates. CONCLUSIONS: At varying peripheral serum concentrations, AMH, thus, demonstrates hithero unknown and contradictory effects on IVF outcomes, deserving at different concentrations investigation as a potential therapeutic agent, with pregnancy-supporting and pregnancy-interrupting properties.

Different effectiveness of closed embryo culture system with time-lapse imaging (EmbryoScope(TM)) in comparison to standard manual embryology in good and poor prognosis patients: a prospectively randomized pilot study.

BACKGROUND: Previously manual human embryology in many in vitro fertilization (IVF) centers is rapidly being replaced by closed embryo incubation systems with time-lapse imaging. Whether such systems perform comparably to manual embryology in different IVF patient populations has, however, never before been investigated. We, therefore, prospectively compared embryo quality following closed system culture with time-lapse photography (EmbryoScope™) and standard embryology. We performed a two-part prospectively randomized study in IVF (clinical trial # NCT92256309). Part A involved 31 infertile poor prognosis patients prospectively randomized to EmbryoScope™ and standard embryology. Part B involved embryos from 17 egg donor-recipient cycles resulting in large egg/embryo numbers, thus permitting prospectively alternative embryo assignments to EmbryoScope™ and standard embryology. We then compared pregnancy rates and embryo quality on day-3 after fertilization and embryologist time utilized per processed embryo. RESULTS: Part A revealed in poor prognosis patients no differences in day-3 embryo scores, implantation and clinical pregnancy rates between EmbryoScope™ and standard embryology. The EmbryoScope™, however, more than doubled embryology staff time (P < 0.0001). In Part B, embryos grown in the EmbyoScope™ demonstrated significantly poorer day-3 quality (depending on embryo parameter between P = 0.005 and P = 0.01). Suspicion that conical culture dishes of the EmbryoScope™ (EmbryoSlide™) may be the cause was disproven when standard culture dishes demonstrated no outcome difference in standard incubation. CONCLUSIONS: Though due to small patient numbers preliminary, this study raises concerns about the mostly uncontrolled introduction of closed incubation systems with time lapse imaging into routine clinical embryology. Appropriately designed and powered prospectively randomized studies appear urgently needed in well-defined patient populations before the uncontrolled utilization of these instruments further expands. TRIAL REGISTRATION: NCT02246309 Registered September 18, 2014.

IVF outcomes in average- and poor-prognosis infertile women according to the number of embryos transferred.

Outcome measures of IVF success, which account for effectiveness of IVF and perinatal outcome risks, have recently been described. The association between number of embryos transferred in average and poor-prognosis IVF patients, and the chances of having good or poor IVF and perinatal outcomes, was investigated. Good IVF and perinatal outcome was defined as the birth of a live, term, normal-weight infant (≥2500 g). Poor IVF and perinatal outcome was defined as no live birth or birth of a very low weight neonate (<1500 g) or severe prematurity (birth at <32 weeks gestation). Each neonate was analysed as a separate outcome. A total of 713 IVF cycles in 504 average and poor-prognosis patients from January 2010 to December 2013 were identified. The odds of having good IVF and perinatal outcomes increased by 28% for each additional embryo transferred. The odds of poor IVF and perinatal outcome decreased by 32% with an additional embryo transferred. The likelihood of live birth with good perinatal outcome in average- and poor-prognosis patients after IVF increases with additional embryos being transferred. These data add to recently reported evidence in favour of multiple embryo transfer in older women and those with average or poor IVF prognosis.

Some aspects of interactivity between endocrine and immune systems required for successful reproduction.

BACKGROUND: In successful reproduction, endocrine and immune systems closely interact. We here attempt to further elucidate the relationship between androgen levels, systemic activation of the immune system and reproductive success in infertile women, utilizing 2 distinct infertile patient cohorts. METHODS: In Group 1, we investigated 322 women (ages 38.6+/-5.4 years) at initial presentation; in Group 2 125 women undergoing in vitro fertilization (169 IVF cycles, ages 38.9+/-5.5 years). In Group 1, we assessed androgens and an immune panel, previously demonstrated to discriminate between activated quiescent immune systems; in Group 2, utilizing the same immune panel, we investigated whether immune system activation relates to embryo quality in IVF cycles. RESULTS: No individual immune test within the immune panel was associated with androgen levels. The total/free testosterone ratio (TT/FT) was, however, significantly associated with presence of gammopathies (in IgG, IgM, IgA, IgE; P=0.026). Surprisingly, immune system activation was associated with significantly improved embryo quality (P=0.008), a finding persistent after adjustment for age and repeat IVF cycles (P=0.006). CONCLUSIONS: Association of immune system activation with improved embryo quality concurs with previously reported immune activation in association with normal functional ovarian reserve (FOR) and normal androgen levels, while, counter intuitively, hypoandrogenism and low FOR are associated with lack of immune system activation. Mild immune system activation, therefore, likely appears essential for establishment of pregnancy, and may be regulated by androgens.

Glucose metabolism in mouse cumulus cells prevents oocyte aging by maintaining both energy supply and the intracellular redox potential.

Inhibiting oocyte postovulatory aging is important both for healthy reproduction and for assisted reproduction techniques. Some studies suggest that glucose promotes oocyte meiotic resumption through glycolysis, but others indicate that it does so by means of the pentose phosphate pathway (PPP). Furthermore, although pyruvate was found to prevent oocyte aging, the mechanism is unclear. The present study addressed these issues by using the postovulatory aging oocyte model. The results showed that whereas the oocyte itself could utilize pyruvate or lactate to prevent aging, it could not use glucose unless in the presence of cumulus cells. Glucose metabolism in cumulus cells prevented oocyte aging by producing pyruvate and NADPH through glycolysis and PPP. Whereas PPP was still functioning after inhibition of glycolysis, the glycolysis was completely inactivated after inhibition of PPP. Addition of fructose-6-phosphate, an intermediate product from PPP, alleviated oocyte aging significantly when the PPP was totally inhibited. Lactate prevented oocyte aging through its lactate dehydrogenase-catalyzed oxidation to pyruvate, but pyruvate inhibited oocyte aging by its intramitochondrial metabolism. However, both lactate and pyruvate required mitochondrial electron transport to prevent oocyte aging. The inhibition of oocyte aging by both PPP and pyruvate involved regulation of the intracellular redox status. Together, the results suggest that glucose metabolism in cumulus cells prevented oocyte postovulatory aging by maintaining both energy supply and the intracellular redox potential and that) glycolysis in cumulus cells might be defective, with pyruvate production depending upon the PPP for intermediate products.

Mouse cumulus-denuded oocytes restore developmental capacity completely when matured with optimal supplementation of cysteamine, cystine, and cumulus cells.

Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

Pyruvate prevents aging of mouse oocytes.

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus-oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.

Demecolcine-assisted enucleation of goat oocytes: protocol optimization, mechanism investigation, and application to improve the developmental potential of cloned embryos.

Although demecolcine-assisted enucleation has been performed successfully in porcine and cattle, the mechanism and protocol optimization of chemically assisted enucleation need further investigation. The present study optimized the protocol for goat oocyte enucleation and demonstrated that a 30-min treatment with 0.8 ng/mL demecolcine-induced cytoplasmic protrusions in over 90% of the oocytes. Rates of enucleation, cell fusion, and blastocyst formation were significantly higher after demecolcine-assisted than after blind aspiration enucleation, although differences in rates of live births remain to be unequivocally determined between the two treatments. The ability to form protrusions decreased significantly as spindles became less organized in aged oocytes and the oocytes with a poor cumulus expansion. More than 93% of the demecolcine-induced protrusions persisted for 2 h in the absence of cytochalasin B (CB) but most disappeared within 30 min of CB treatment. The spindle disintegrated, an actin-rich ring formed around the chromosome mass and the MAP kinase activity increased significantly after demecolcine treatment. When oocytes with induced protrusions were treated with CB, however, the contractile ring disappeared, the spindle reintegrated, and both MPF and MAP kinase activities decreased significantly. It is concluded that (1) cytoplasmic protrusions can be induced in goat oocytes with a very low concentration of demecolcine; (2) oocyte selection and enucleation can be achieved simultaneously with demecolcine treatment; and (3) an interactive effect between the MAP kinase, MPF, microfilaments and microtubules might be implicated in the control of cytoplasmic protrusion formation after demecolcine treatment.

The interactions between cysteamine, cystine and cumulus cells increase the intracellular glutathione level and developmental capacity of goat cumulus-denuded oocytes.

To improve in vitro maturation (IVM) of denuded oocytes (DOs), we observed the interactive effects of cysteamine, cystine and cumulus cells on the glutathione (L-gamma-glutamyl-L-cysteinyl-glycine; GSH) level and developmental capacity of goat IVM oocytes. Cysteamine supplementation increased the GSH level and blastocyst rates of both cumulus-oocyte complexes (COCs) and DOs, while the addition of cystine increased the GSH level and blastulation only in the presence of cumulus cells (COCs or DOs co-cultured on a cumulus cell monolayer). Simultaneous supplementation of cysteamine and cystine increased the GSH content and blastulation of co-cultured DOs to a level similar to that of COCs matured without thiol supplementation. Co-culture without thiol supplementation improved DOs' GSH synthesis but not blastulation. The results suggest that DOs cannot utilize cystine for GSH synthesis unless exogenous cysteamine is supplied by either cumulus cells or supplementation. Thus, while the addition of cystine alone is enough to improve IVM of COCs, improvement of DOs requires supplementation of both cystine and cysteamine. Synergic actions between cysteamine, cystine and cumulus cells restore the GSH level and developmental capacity of goat DOs.

Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes.

The objective of this article was to study the effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes. Goat oocytes were cultured at different temperatures in medium containing different concentrations of ROS, and at the end of culture, oocytes were either matured or processed for light/confocal microscopy. The matured oocytes were activated chemically or fertilized in vitro for embryo development. Meiotic arrest was successfully maintained for 24 hr with 0, 50, and 200 microM ROS at 5, 20, and 38.5 degrees C, respectively. Following chemical activation, morulae/blastocysts (M/B) rates similar to untreated oocytes were obtained in oocytes that had been inhibited for 24 hr at 5 degrees C without ROS (Protocol 5C) or at 20 degrees C with 50 microM ROS (Protocol 20C) or for 8 hr at 38.5 degrees C with 200 microM ROS (Protocol 8 hr), but no blastulation was observed after oocytes were inhibited at 38.5 degrees C with 200 microM ROS for 24 hr. Following fertilization, however, while M/B rates similar to controls were achieved in oocytes treated with protocols 5C and 20C, few oocytes inhibited with Protocol 8 hr developed into morulae, due to a high incidence of polyspermy. Changes in GV chromatin configuration were not observed after inhibition with Protocol 5C, but were apparent after inhibition with protocols 20C and 8 hr, leading to a precocious germinal vesicle breakdown (GVBD) during subsequent maturation. Cortical granule (CG) migration and the formation of microtubule organizing centers occurred during inhibition and were more obvious in the absence of ROS. Significantly more oocytes inhibited by protocols 5C and 20C than by Protocol 8 hr completed CG migration after maturation. In conclusion, goat oocytes were tolerant to chilling and culture at lower temperatures with less ROS was better than culture at higher temperatures with more ROS for oocyte GVBD inhibition.

Fabrication of Porous Polyvinylidene Fluoride/Multi-Walled Carbon Nanotube Nanocomposites and Their Enhanced Thermoelectric Performance.

In this paper, a solvent vapor-induced phase separation (SVIPS) technique was used to create a porous structure in polyvinylidene fluoride/Multi-walled carbon nanotube (PVDF/MWNTs) composites with the aim of increasing the electrical conductivity through the incorporation of MWNTs while retaining a low thermal conductivity. By using the dimethylformamide/acetone mixture, porous networks could be generated in the PVDF/MWNTs composites upon the rapid volatilization of acetone. The electrical conductivity was gradually enhanced by the addition of MWNTs. At the same time, the thermal conductivity of the PVDF film could be retained at 0.1546 W·m-1·K-1 due to the porous structure being even by loaded with a high content of MWNTs (i.e., 15 wt.%). Thus, the Seebeck coefficient, power factor and figure of merit (ZT) were subsequently improved with maximum values of 324.45 µV/K, 1.679 µW·m-1·K-2, and 3.3 × 10-3, respectively. The microstructures, thermal properties, and thermoelectric properties of the porous PVDF/MWNTs composites were studied. It was found that the enhancement of thermoelectric properties would be attributed to the oxidation of MWNTs and the porous structure of the composites. The decrease of thermal conductivity and the increase of Seebeck coefficient were induced by the phonon scattering and energy-filtering effect. The proposed method was found to be facile and effective in creating a positive effect on the thermoelectric properties of composites.

Reduced RNA expression of the FMR1 gene in women with low (CGGn<26) repeats.

Low FMR1 variants (CGGn<26) have been associated with premature ovarian aging, female infertility and poor IVF treatment success. Until now, there is little published information concerning possible molecular mechanisms for this effect. We wished to examine whether relative expression of RNA and the FMR1 gene's fragile X mental retardation protein (FMRP) RNA isoforms differ in women with various FMR1 sub-genotypes (normal, low CGGn<26 and/or high CGGn≥34). This prospective cohort study was conducted between 2014 and 2017 in a clinical research unit of the Center for Human Reproduction in New York City. The study involved a total of 98 study subjects, including 18 young oocyte donors and 80 older infertility patients undergoing routine in vitro fertilization (IVF) cycles. The main outcome measure was RNA expression in human luteinized granulosa cells of 5 groups of FMRP isoforms. The relative expression of FMR1 RNA in human luteinized granulosa cells was measured by real-time PCR and a possible association with CGGn was explored. All 5 groups of FMRP RNA isoforms examined were found to be differentially expressed in human luteinized granulosa cells. The relative expression of four FMR1 RNA isoforms showed significant differences among 6 FMR1 sub-genotypes. Women with at least one low allele expressed significantly lower levels of all 5 sets of FRMP isoforms in comparison to the non-low group. While it would be of interest to see whether FMRP is also decreased in the low-group we recognize that in recent years it has been increasingly documented that information flow of genetics may be regulated by non-coding RNA, that is, without translation to a protein product. We, thus, conclude that various CGG expansions of FMR1 allele may lead to changes of RNA levels and ratios of distinct FMRP RNA isoforms, which could regulate the translation and/or cellular localization of FMRP, affect the expression of steroidogenic enzymes and hormonal receptors, or act in some other epigenetic process and therefore result in the ovarian dysfunction in infertility.

With low ovarian reserve, Highly Individualized Egg Retrieval (HIER) improves IVF results by avoiding premature luteinization.

BACKGROUND: Highly Individualized Egg Retrieval (HIER), defined as age-specific early oocyte retrieval (ER), has been demonstrated to avoid premature luteinization in women ≥43. We here investigated whether HIER also applies to younger women with premature ovarian aging (POA), and what best lead follicle size should be for administration of ovulation-triggers. METHODS: Fifty-six women ≥43, and 37 POA patients underwent IVF cycles. Granulosa cells (GCs) were isolated, cultures were established, RNA was extracted and real-time PCR analyses performed, with gene expressions at mRNA level investigated for FSH receptor (FSHR), luteinizing hormone receptor (LHCPR), P450 aromatase (CYP19a1) and progesterone receptor (PGR). POA was defined by age < 40, FSH above 95%CI and/or AMH below 95%CI for age. Women ≥43 years were divided into very early retrieval (VER), with human chorionic gonadotropin (hCG) trigger at 13.5-15.5 mm, ER at 16.0-18.0 mm or standard retrievel (SR) at 18.5-20.5 mm; POA patients were divided into ER and SR. Pregnancy rates and and molecular markers of premature luteinization (PL) were main outcome measures. RESULTS: ER resulted in a significantly higher clinical pregnancy rate (16.7%) than VER (5.9%) or SR (6.7%; both P < 0.05). Molecular markers of PL were highest with SR and lowest with VER. In POA, ER improved pregnancy chances even more than in women ≥43 (7.7% with SR vs. 41.7% with ER), while also reducing molecular markers of PL. With low ovarian reserve (LOR), by avoiding PL, ER with hCG trigger at 16.0-18.0 mm, thus, improves clinical pregnancy rates at all ages. As VER demonstrated lowest molecular PL marker but equally poor pregnancy rates as SR, too early ovulation triggers, likely, result in cytoplasmatic immaturity. CONCLUSIONS: HIER is even more effective in POA patients than women above age 43, demonstrating that HIER should be further investigated going into even more advanced ages.

PEDOT:PSS/graphene quantum dots films with enhanced thermoelectric properties via strong interfacial interaction and phase separation.

The typical conductive polymer of PEDOT:PSS has recently attracted intensive attention in thermoelectric conversion because of its low cost and low thermal conductivity as well as high electrical conductivity. However, compared to inorganic counterparts, the relatively poor thermoelectric performance of PEDOT:PSS has greatly limited its development and high-tech applications. Here, we report a dramatic enhancement in the thermoelectric performance of PEDOT:PSS by constructing unique composite films with graphene quantum dots (GQDs). At room temperature, the electrical conductivity and Seebeck coefficient of PEDOT:PSS/GQDs reached to 7172 S/m and 14.6 µV/K, respectively, which are 30.99% and 113.2% higher than those of pristine PEDOT:PSS. As a result, the power factor of the optimized PEDOT:PSS/GQDs composite is 550% higher than that of pristine PEDOT:PSS. These significant improvements are attributed to the ordered alignment of PEDOT chains on the surface of GQDs, originated from the strong interfacial interaction between PEDOT:PSS and GQDs and the separation of PEDOT and PSS phases. This study evidently provides a promising route for PEDOT:PSS applied in high-efficiency thermoelectric conversion.

The effects of delayed activation and MG132 treatment on nuclear remodeling and preimplantation development of embryos cloned by electrofusion are correlated with the age of recipient cytoplasts.

The electrofusion method, used extensively in livestock cloning, cannot be used in mice, because it is believed that the mouse oocytes are more susceptible to detrimental effects of electrical stimulus than oocytes from other species. Reports on whether a delayed activation after electrofusion and a premature chromosome condensation (PCC) is essential for efficient cloning are inconclusive. To address these issues, effects of pulsing on activation and MPF activity of nonenucleated oocytes and effects of delayed activation and MG132 treatment on donor nuclear PCC and preimplantation development of embryos cloned by electrofusion or nuclear injection were compared between different cytoplast ages in mice and goats. The results indicated that the use of oocytes collected early after donor stimulation would make it possible to conduct somatic cell nuclear transfer in mice by electrofusion. Whether a delayed activation is essential was dependent upon the age, or rather, the level, of MPF activity of the cytoplasts at the time of electrofusion, as was the requirement for MG132 treatment. The competence for blastocyst formation of cloned embryos was highly correlated with the level of donor nuclear PCC in recipient cytoplasts. The nuclear injection technique was more adaptable to older cytoplast ages, and hence less dependent on drugs for inhibition of MPF inactivation, compared to electrofusion.

Rescue in vitro maturation (IVM) of immature oocytes in stimulated cycles in women with low functional ovarian reserve (LFOR).

Rescue in vitro maturation (IVM) is currently not a routine procedure in association with in vitro fertilization (IVF). We compared in a prospectively cohort study of 10 patients with normal functional ovarian reserve (NFOR) and of 25 with low functional ovarian reserve (LFOR), defined by abnormally high FSH and/or abnormally low AMH levels), IVM dynamics of immature oocytes. Following controlled ovarian hyperstimulation in IVF cycles, only immature oocytes underwent rescue IVM (for up to 48 h). Oocyte maturation dynamics, fertilization rates, embryo development, and pregnancy rates were then compared between NFOR and LFOR patients. Though proportion of MI and GV oocytes reaching MII stages within 48 h and rate of maturation of MI oocytes did not differ, in women with LFOR significantly more GV oocytes reached MII stage within 24 h (30.4 vs. 66.9 %; P = 0.013), while fertilization rates and embryo generation numbers were similar between both groups. Rescue IVM, thus, produced 1.5 additional embryos for transfer in women with LFOR and 1.6 in patients with NFOR, a highly significant difference in relative improvement in available embryo numbers for LFOR (+60.0 %) and NFOR women (+16.5 %). Rescue IVM, thus, not only demonstrates different time dynamics between women with LFOR and NFOR but also disproportionate efficacy in improving available embryo numbers for transfer in favor of LFOR patients. 1/7 patients, who reached embryo transfer with only embryos produced via rescue IVF conceived and delivered, proving that rescue IVF in women with LFOR also improves pregnancy and delivery chances. Because of the small number of embryos LFOR patients produce, every additional embryo is of considerable potential clinical significance for them, suggesting that rescue IVM in women with LFOR should become routine practice.

Associations between peripheral androgens and cortisol in infertile women.

Testosterone has in recent years been proven essential for normal growth and maturation of small growing follicles. Concomitantly, low functional ovarian reserve (LFOR), characterized by a small growing follicle pool, has been associated with low testosterone levels, which can be of ovarian and/or adrenal origin. In this study we, therefore, investigated whether peripheral sex steroid precursors and testosterone levels potentially reflect on adrenal function. In a retrospective cohort study of 355 consecutive infertile women, who presented to an academically affiliated fertility center in New York City, we investigated in a series of statistical models whether low peripheral sex steroid precursors and testosterone are associated with peripheral cortisol (C) levels, reflecting adrenal function. To determine potential correlations, we investigated the dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), androstenedione (AD), total testosterone (TT), free testosterone (FT); sex hormone binding globulin (SHBG), anti-Müllerian hormone (AMH), thyroid stimulating hormone (TSH) and C in a series of multivariate and logistic regression analyses, utilizing C either as a continuous variable or with cut off <5.0µg/dL, and TT only as a continuous variable. Practically all models demonstrated significant predictability of peripheral sex hormone precursors for C levels, with DHEA demonstrating the strongest and most consistent predictability as an individual parameter and as part of the DHEAS/DHEA ratio. We conclude that in infertile women peripheral sex hormone precursors, especially DHEA, reflect C levels and, therefore, adrenal function. In infertile women, at all ages low levels of sex hormone precursors, therefore, should be considered indications for further adrenal assessments.

Aging-related premature luteinization of granulosa cells is avoided by early oocyte retrieval.

Why IVF pregnancy rates decline sharply after age 43 is unknown. In this study, we compared granulosa cell (GC) function in young oocyte donors (n=31, ages 21-29), middle-aged (n=64, ages 30-37) and older infertile patients (n=41, ages 43-47). Gene expressions related to gonadotropin activity, steroidogenesis, apoptosis and luteinization were examined by real-time PCR and western blot in GCs collected from follicular fluid. FSH receptor (FSHR), aromatase (CYP19A1) and 17ß-hydroxysteroid dehydrogenase (HSD17B) expression were found down regulated with advancing age, while LH receptor (LHCGR), P450scc (CYP11A1) and progesterone receptor (PGR) were up regulated. Upon in vitro culture, GCs were found to exhibit lower proliferation and increased apoptosis with aging. While FSH supplementation stimulated GCs growth and prevented luteinization in vitro. These observations demonstrate age-related functional declines in GCs, consistent with premature luteinization. To avoid premature luteinization in women above age 43, we advanced oocyte retrieval by administering human chorionic gonadotropin at maximal leading follicle size of 16  mm (routine 19-21  mm). Compared to normal cycles in women of similar age, earlier retrieved patients demonstrated only a marginal increase in oocyte prematurity, yet exhibited improved embryo numbers as well as quality and respectable clinical pregnancy rates. Premature follicular luteinization appears to contribute to rapidly declining IVF pregnancy chances after age 43, and can be avoided by earlier oocyte retrieval.

Oocyte Scoring Enhances Embryo-Scoring in Predicting Pregnancy Chances with IVF Where It Counts Most.

CONTEXT: Our center's quality improvement optimization process on many occasions anecdotally suggested that oocyte assessments might enhance embryo assessment in predicting pregnancy chances with in vitro fertilization (IVF). OBJECTIVE: To prospectively compare a morphologic oocyte grading system to standard day-3 morphologic embryo assessment. DESIGN, SETTING, PATIENTS: We prospectively investigated in a private academically-affiliated infertility center 94 consecutive IVF cycles based on 6 criteria for oocyte quality: morphology, cytoplasm, perivitelline space (PVS), zona pellucida (ZP), polar body (PB) and oocyte size, each assigned a value of -1 (worst), 0 (average) or +1 (best), so establishing an average total oocyte score (TOS). Embryo assessment utilized grade and cell numbers of each embryo on day-3 after oocyte retrieval. Clinical pregnancy was defined by presence of at least one intrauterine gestational sac. INTERVENTIONS: Standard IVF cycles in infertile women. MAIN OUTCOME MEASURES: Predictability of pregnancy based on oocyte and embryo-grading systems. RESULTS: Average age for all patients was 36.5 ± 7.3 years; mean oocyte yield was 7.97± 5.76; Patient specific total oocyte score (PTOS) was -1.05 ± 2.24. PTOS, adjusted for patient age, was directly related to odds of increased embryo cell numbers (OR 1.12, P = 0.025), embryo grade (OR 1.19, P < 0.001) and clinical pregnancy [OR 1.58 (95%CI 1.23 to 2.02), P < 0.001]. Restricting the analysis to day three embryos of high quality (8-cell/ good grades), TOS was still predictive of clinical pregnancy (OR 2.08 (95%CI 1.26 to 3.44, P = 0.004). Among the 69 patients with embryos of Grade 4 or better available for transfer 23 achieved Clinical Pregnancy. When the analysis was restricted to the 69 transfers with good quality embryos (≥ Grade 4) the Oocyte Scoring System (TOS) (AUC±SE 0.863±0.044, oocyte score) provided significantly greater predictive value for clinical pregnancy compared to the embryo grade alone (AUC 0.646 ± 0.072, embryo grade) p = 0.015. CONCLUSIONS: Oocyte-scoring, thus, provides useful clinical information especially in good prognosis patients with large numbers of high quality embryos. This finding appears of particular importance at a time when many IVF centers are committing sizable investments to closed incubation systems with time-lapse photography, which are exclusively meant to define embryo morphology.

Early decline in functional ovarian reserve in young women with low (CGGn < 26) FMR1 gene alleles.

Mouse fmr1 models, and recent cross-sectional human data, suggest that different triple CGGn ranges of the fragile X mental retardation 1 (FMR1) gene are associated with variations in ovarian aging and infertility treatment outcomes. The FMR1 mutation affecting reproductive function most negatively in humans is the so-called low mutation, characterized by CGGn < 26. We here present a first longitudinal study of selected young women with normal functional ovarian reserve (FOR). In a prospective cohort study, we selected among 233 young oocyte donors (mean age 24.8 ± 3.3 years) as study population of 66 who had more than 1 anti-Müllerian hormone (AMH) level drawn over a 4-year period. AMH curves, as reflection of FOR, were then statistically compared between women with and without low FMR1 alleles. Biallelic low FMR1 (hom-low/low) donors already at initial presentation demonstrated significantly lower FOR than donors with biallelic normal (norm) FMR1 (CGGn = 26-34; P = 0.001). Although monoallelic low FMR1 at initial presentation was not yet associated with decreased FOR, it over 4 years did demonstrate significantly enhanced declines in FOR (P = 0.046). Including repeat measurements, low/low (P = 0.006) and high/high (CGGn > 34) alleles (P < 0.001) demonstrated lower FOR by AMH than norm donors. Even monoallelic low FMR1 alleles are, thus, already at young female ages associated with accelerated declines in FOR. Low FMR1 alleles, therefore, potentially represent a screening tool for women at genetic risk toward premature ovarian senescence, representing in all races circa 10% of the female population.