Mitochondrial metabolic substrate utilization in granulosa cells reflects body mass index and total follicle stimulating hormone dosage in in vitro fertilization patients.

PURPOSE: To utilize a novel mitochondrial function assay with pooled granulosa cells to determine whether mitochondrial function would differ by patient demographics and embryo development. METHODS: This was a prospective pilot study in a hospital-based assisted reproductive program and public university. Mitochondrial metabolic substrate utilization was assessed in pooled granulosa cells from 40 women undergoing in vitro fertilization during 2018 and 2019. RESULTS: Assessment of mitochondrial substrate metabolism in pooled granulosa cells revealed higher citric acid, L-malic acid, and octanoyl-L-carnitine utilization with higher body mass index (BMI). Utilization of citric acid, cis-aconitic acid, D-alpha-keto-glutaric acid, L-glutamine, and alanine plus glycine was significantly lower as total dosage of FSH administered increased. Utilization of glycogen was significantly higher in patients with a higher percentage of fertilized oocytes. D-alpha-keto-glutaric acid utilization was significantly lower in patients with a higher percentage of good 8-cell embryos. L-glutamine utilization was significantly lower, with a higher percentage of blastocyst formation. Mitochondrial metabolic scores (MMS), which reflect overall mitochondrial activity of the granulosa pool, were significantly higher in patients with higher BMI and with greater numbers of mature oocytes retrieved. MMS in granulosa decreased as total FSH dose administered increased. CONCLUSIONS: Granulosa cell utilization of substrates feeding into the citric acid cycle changed with total FSH dosage and BMI. Fertilization rate, 8-cell embryo quality, and blastocyst formation also associated with different energy substrate usage. Mitochondrial substrate utilization by granulosa cells from individual follicles could be further developed into a useful diagnostic tool.

Life Interrupted: The Nature and Consequences of Cryostasis.

Cryopreservation and associated cryostorage has become a well-established technique in both basic and clinical science. When the potentially lethal consequences of freezing itself are ameliorated, existence at cryogenic temperatures seems to be a form of true viable stasis that can persist for long periods of time. Natural cryopreservation and revival after long-term periods in cryostasis is reality in many species. While some evidence exists for imperfections in artificial cryopreservation protocols and storage, these protocols are for the most part successful and compatible with efficient restoration of vitality in a variety of biomaterial after freezing. Clinical protocols in use for cryopreserving and storing gametes and embryos in human-assisted reproduction are similarly well proven and supported by a large body of basic science and clinical outcome data.

Micromanipulation in assisted reproductive technology.

Micromanipulation describes a set of tools and techniques for cellular microsurgery and manipulation. Micromanipulation techniques have played an important role in basic research and the development of clinical techniques in assisted reproductive technology. This work provides a review of the development and current practices involving micromanipulation in the human clinical assisted reproduction laboratory.

Follicular fluid placental growth factor is increased in polycystic ovarian syndrome: correlation with ovarian stimulation.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is characterized by increased ovarian angiogenesis and vascularity. Accumulating evidence indicates that vascular endothelial growth factor (VEGF) is increased in PCOS and may play an important role in these vascular changes and the pathogenesis of this disease. Placental growth factor (PlGF), a VEGF family member, has not been previously characterized in PCOS women. We investigated levels and temporal expression patterns of PlGF and its soluble receptor sFlt-1 (soluble Fms-like tyrosine kinase) in serum and follicular fluid (FF) of women with PCOS during controlled ovarian stimulation. METHODS: This was a prospective cohort study of 14 PCOS women (Rotterdam criteria) and 14 matched controls undergoing controlled ovarian stimulation. Serum was collected on day 3, day of hCG and day of oocyte retrieval. FF was collected on retrieval day. PlGF, sFlt-1 and anti-mullerian hormone (AMH) protein concentrations were measured using ELISA. Since sFlt-1 binds free PlGF, preventing its signal transduction, we calculated PlGF bioavailability as PlGF/sFlt-1 ratio. RESULTS: Serum PlGF and sFlt-1 levels were constant throughout controlled ovarian stimulation, and no significant differences were observed in either factor in PCOS women compared with non-PCOS controls at all three measured time points. However, FF PlGF levels were increased 1.5-fold in PCOS women compared with controls (p < 0.01). Moreover, FF PlGF correlated positively with number of oocytes retrieved and the ovarian reserve marker anti-mullerian hormone (AMH) and negatively with age. In addition, FF sFlt-1 levels were decreased 1.4-fold in PCOS women compared to controls (p = 0.04). PlGF bioavailability in FF was significantly greater (2-fold) in PCOS women compared with non-PCOS controls (p < 0.01). CONCLUSIONS: These data provide evidence that FF PlGF correlates with ovarian stimulation and that its bioavailability is increased in women with PCOS undergoing controlled ovarian stimulation. This suggests that PlGF may play a role in PCOS pathogenesis and its angiogenic dysregulation.

Characterization of women with elevated antimüllerian hormone levels (AMH): correlation of AMH with polycystic ovarian syndrome phenotypes and assisted reproductive technology outcomes.

OBJECTIVE: Serum Antimüllerian hormone (AMH) levels are elevated in polycystic ovarian syndrome and have been shown to be useful in its diagnosis. However, the clinical significance of extremely high AMH levels is understudied. We aimed to characterize a population of women with elevated AMH (>5 ng/mL). STUDY DESIGN: This was a retrospective cohort study of 134 women presenting to our fertility clinic for infertility evaluation and treatment who were found to have random serum AMH over 5 ng/mL. Women were divided into 3 groups according to AMH: 5-10 ng/mL, >10-14 ng/mL, and >14 ng/mL. Endocrine characteristics, polycystic ovarian syndrome (PCOS) phenotypes, fertilization rate, implantation rate, clinical pregnancy, and multiple pregnancy rates were compared between groups. RESULTS: AMH ranged between 5 to 48 ng/mL. Greater than 97% of women with ultrahigh AMH (>10 ng/mL) had PCOS. In addition, women with AMH >10 ng/mL had greater prevalence of polycystic ovarian morphology and oligoamenorrhea than women with AMH 5-10 ng/mL. Moreover, serum AMH correlated positively with luteinizing hormone, total testosterone, and dehydroepiandrosterone sulfate. Furthermore, AMH showed strong predictive ability for the presence of amenorrhea (area under the curve, 0.87; 95% confidence interval, 0.80-0.92; P < .0001). Despite similar age and mean number of transferred embryos, women with AMH >10 ng/mL showed higher rates of ovarian hyperstimulation syndrome and clinical pregnancy rates compared with women with AMH 5-10 ng/mL. CONCLUSION: These data characterize a population of women with elevated AMH levels, demonstrating that the vast majority of women with AMH >10 ng/mL have PCOS. Increased AMH levels correlated with PCOS severity and are associated with greater ovarian stimulation and higher clinical pregnancy rates following assisted reproductive technology.

Angiopoietin-1 and angiopoietin-2 are altered in polycystic ovarian syndrome (PCOS) during controlled ovarian stimulation.

Polycystic ovarian syndrome (PCOS) ovaries are characterized by increased angiogenesis and hypervascularity. While angiopoietin-1 (Ang-1) and its antagonist, angiopoietin-2 (Ang-2), are essential for ovarian function and angiogenesis, the levels of Ang-1 and Ang-2 in PCOS are unknown. This was a prospective cohort study of 14 PCOS women and 14 matched controls undergoing controlled ovarian stimulation (COS). Serum was collected on day 3, hCG and retrieval days. Follicular fluid (FF) was collected on retrieval day. Serum Ang-1 and Ang-2 levels were constant throughout COS, but serum Ang-1 levels were increased at all time points in PCOS women compared with controls (p < 0.05). No differences between groups were found in serum Ang-2 levels or FF Ang-1 levels. However, FF Ang-2 levels were increased almost 2-fold in PCOS women compared with controls (p < 0.01), and correlated positively with number of oocytes retrieved (r = 0.65, p < 0.0001). This study is the first to provide evidence of an alteration in the Ang-1/Ang-2 system in PCOS women. The biological role of Ang-2 in promoting capillary leakage, the increased Ang-2 FF level in PCOS, and its correlation with number of oocytes suggest that Ang-2 may play an important role in the increased risk of ovarian hyperstimulation in PCOS.

Transforming growth factor-ß1 and its receptor soluble endoglin are altered in polycystic ovary syndrome during controlled ovarian stimulation.

OBJECTIVE: To evaluate the relationship between transforming growth factor (TGF)-ß1 and its receptor, soluble endoglin (sENG), in the serum and follicular fluid of women with polycystic ovarian syndrome (PCOS) compared with that of non-PCOS normal ovulating women during controlled ovarian stimulation (COS). DESIGN: Prospective case-control study. SETTING: Academic-affiliated assisted reproductive technology unit. PATIENT(S): Fourteen PCOS and 14 matched non-PCOS control women undergoing COS. INTERVENTION(S): Serum was collected on day 3 (baseline), day of hCG, and day of retrieval. Follicular fluid (FF) was collected on day of oocyte retrieval. ELISA was performed to determine TGF-ß1 and sENG protein levels. MAIN OUTCOME MEASURE(S): Serum and FF levels of TGF-ß1 and sENG. RESULT(S): Serum TGF-ß1 did not change significantly during COS but was increased in PCOS compared with non-PCOS women on day 3 and days of hCG administration and oocyte retrieval. Serum sENG increased after hCG administration only in the non-PCOS control group. In addition, serum sENG was decreased in PCOS compared with non-PCOS control women on the days of hCG and retrieval. Accordingly, the bioavailability of TGF-ß1 (TGF-ß1/sENG ratio) was increased in women with PCOS compared with non-PCOS controls at all three time points. No differences in either factor were noted in FF between groups. CONCLUSION(S): The increased TGF-ß1 bioavailability in PCOS is not only due to increased TGF-ß1 levels but also to decreased levels of its receptor, sENG. These data suggest that increased TGF-ß1 bioavailability may contribute to the pathogenesis of PCOS and its increased risk for ovarian hyperstimulation.

Anti-Müllerian hormone as an independent predictor of twin versus singleton pregnancy in fresh cycles.

This study evaluated anti-Müllerian hormone (AMH) as a possible predictor of twin pregnancy in women undergoing fresh cycles who had more than one embryo transferred. A retrospective study was performed of 139 patients undergoing fresh non-donor cycles which resulted in either singleton or twin pregnancy between 2009 and 2010 in this fertility clinic. Random serum AMH and other clinically relevant variables were compared. For further analysis, the population was stratified by age (<34 and ⩾34 years). Random serum AMH concentrations were 1.4-times greater in women conceiving twins compared with singletons (P=0.03). In women aged ⩾34, the AMH concentration in twins was 1.8-fold greater than singletons (P=0.001). Multivariate analysis demonstrated that AMH was an independent predictor of twins. ROC curve analysis showed that AMH had a significant predictive ability for twin pregnancy in women aged ⩾34 (AUC 0.67, P=0.01). In contrast, in women aged <34, AMH was not different between twin and singleton pregnancies. In summary, random serum AMH is an independent predictor of twin gestation when more than one embryo is transferred in women aged ⩾34. Considering a woman's AMH before transferring more than one embryo may assist in reducing the incidence of twins. Transferring multiple embryos to increase the chance of pregnancy and live birth rate is a common practice among assisted reproduction providers but often results in a high proportion of multiple pregnancies. Identifying factors predictive of multiple pregnancies is of paramount importance for the successful development of strategies to minimize multiple gestations. Anti-Müllerian hormone (AMH) has been shown to be closely correlated with a woman's egg reserve. Aside from the strong association of serum AMH concentration with quantitative ovarian response, serum AMH concentration has been associated with qualitative aspects of assisted reproduction such as embryo quality and pregnancy rates. Our objective was to evaluate AMH as a possible predictor of twin pregnancy in women undergoing fresh cycles who had more than one embryo transferred. Our study included 139 patients undergoing fresh non-donor cycles which resulted in either a singleton or twin pregnancy between 2009 and 2010 in our fertility clinic. Random serum AMH concentrations were compared. For further analysis, the population was divided into two groups (age <34 and ⩾34). AMH concentrations were 1.4-times greater in women conceiving twins compared with singletons. In women aged ⩾34, the AMH concentration in twins was 1.8-fold greater than singleton pregnancies. AMH was an independent predictor of twins with a significant predictive ability for twin pregnancy in women aged ⩾34. In contrast, in women aged <34, AMH was not different between twin and singleton pregnancies. In conclusion, considering a woman's AMH before transferring more than one embryo may assist in reducing the incidence of twins.

Fixing oocytes? A bovine model provides new hope.

In a previous issue of Reproductive BioMedicine Online, Chiaratti and co-workers presented a bovine model for ooplasmic transfer, which demonstrated a positive effect on early development. Developmental deficits resulting from artificial treatment of recipient eggs with a toxic compound were ameliorated by the addition of small volumes of healthy donor cytoplasm. This model provides an important advance in the understanding of ooplasmic effects in early development and addresses issues about the prior human trials in this area.

Association of abnormal morphology and altered gene expression in human preimplantation embryos.

OBJECTIVE: We set out to characterize the expression of nine genes in human preimplantation embryos and determine whether abnormal morphology is associated with altered gene activity. DESIGN: Reverse transcription and real-time polymerase chain reaction were used to quantify the expression of multiple genes in each embryo. The genes studied have various important cellular roles (e.g., cell cycle regulation, DNA repair, and apoptosis). SETTING: Research laboratory working closely with a clinical IVF practice. PATIENT(S): Over 50 embryos were donated by infertile patients (various etiologies). Among these, all major stages of preimplantation development and a variety of common morphologic abnormalities were represented. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantification of mRNA transcripts. RESULT(S): We detected an association between certain forms of abnormal morphology and disturbances of gene activity. Cellular fragmentation was associated with altered expression of several genes, including TP53, suggesting that fragmenting blastomeres are suffering stress of a type monitored by p53, possibly as a consequence of suboptimal culture conditions. CONCLUSION(S): Appropriate gene expression is vital for the regulation of metabolic pathways and key developmental events. Our data indicates a possible causal relationship between changes in gene expression and the formation of clinically relevant abnormal embryo morphologies. We hypothesize that embryos with expression profiles characteristic of good morphology and appropriate for their developmental stage have the greatest potential for implantation. If confirmed, this could lead to a new generation of preimplantation genetic diagnosis (PGD) tests for assessing embryo viability and predicting implantation potential.

Combinatorial peptide library binding of mammalian spermatozoa identifies a ligand (HIPRT) in the axin protein: putative identification of a sperm surface axin binding protein and intriguing developmental implications.

The identification of components in cell-cell interactions is an important research goal in reproductive and developmental biology. Such interactions are critical to gamete development, fertilization, implantation and basic development. Several proteins involved with sperm-oocyte interaction and other developmentally important phenomena have been identified. However, these are obviously only a subset of the molecular components involved in such complex cell-cell interactions. One method that has been used to identify binding partners for particular molecular targets is the use of combinatorial libraries accessible on phage surfaces. For the most part, this technique has mainly been applied to screen specific target moieties. However, in some cases whole-cell screening has been attempted. This study describes the first report of screening intact, living mammalian gametes using a proprietary whole-cell combinatorial library binding and analysis protocol. Results from the first screening protocol of mouse spermatozoa strongly identified a putative sperm-binding ligand using proprietary bioinformatic analysis. This amino acid sequence (HIPRT) precisely corresponds with a previously characterized highly conserved protein-protein interaction site in the axin protein. This sequence is found within the binding site for a known sperm surface protein, glycogen synthase kinase-3. This result not only provides proof of the utility of this technique to identify cell surface ligands in mammalian gametes, but it also suggests a potential role for spermatozoa in facilitating developmental axis formation in mammalian embryos.

Expression profiles of individual human oocytes using microarray technology.

Microarray technology is a relatively new technique that provides the investigator with the ability to monitor and quantify the expression of thousands of genes simultaneously. This technological breakthrough has the potential to provide detailed insight into cellular processes involved in the regulation of gene expression. In this study, microarray methods were used to examine the expression of linearly amplified RNA from individual and pooled (n = 5) human oocytes. The amplification strategy consistently produced a complex representative cDNA population. A catalogue of 1361 transcripts expressed in human oocytes was identified, of which 406 have been independently confirmed using other methods.

Ooplasmic transfer: animal models assist human studies.

Ooplasmic transplantation is based on the premise that ooplasmic components are compromised in some individuals. In theory, the transfer of small amounts of healthy ooplasm can correct such deficits, allowing for improved development and implantation. The technique is based on a well-established background of experimental embryology demonstrating that cytoplasmic manipulation in oocytes and early embryos can be entirely compatible with normal development. Cytoplasm has been manipulated via karyoplast and cytoplast transfer and by cytoplasmic injection. Term development has been obtained following such manipulations in a variety of mammalian species. While some manipulative scenarios have exhibited compromised development, others have exhibited improved development. Developmental problems involving specific epigenetic and mitochondrial incompatibilities have been observed in a very limited subset of animal studies. These studies are based on genetic and physical models that have little relation to the actual substance of ooplasmic transplantation in the human. In fact, the majority of animal studies suggest that ooplasmic transplantation is well-founded and unlikely to result in negative developmental consequences. Furthermore, there are considerable physical, physiological and developmental differences between human and rodent eggs and embryos. These differences suggest that potentially negative issues raised by rodent results may not be relevant in the human.

Zona dissection by infrared laser: developmental consequences in the mouse, technical considerations, and controlled clinical trial.

Infrared laser systems are currently being marketed for application in clinical zona pellucida dissection. However, these systems have undergone only limited animal testing and minor clinical trials that lacked proper controls. Two of these systems have been evaluated in protocols that addressed potential detrimental effects on embryonic development in the mouse. Exaggerated large openings were made in the zona pellucida of 8-16 cell mouse embryos. Embryonic development and subsequent implantation and viability were assessed. A definite negative effect on these parameters was observed following the use of one of these systems. Following this animal trial, the second system was evaluated in a clinical trial for assisted hatching and embryo biopsy. Laser dissection was directly compared with the standard zona drilling using acidified Tyrode's solution. While no significant difference was evident between the two protocols, it was felt that laser dissection presented some problems in both consistency between operators and in the efficacy of subsequent manipulations such as blastomere biopsy and fragment removal. These results argue that laser zona dissection is far from a simple technique and should be carefully evaluated before any clinical application is made.