Total and bioavailable 25-hydroxyvitamin D is not associated with improved sexual dysfunction following vitamin D supplementation in women with polycystic ovarian syndrome: a pilot study.

BACKGROUND: Vitamin D (Vit D) deficiency has been linked to symptoms of polycystic ovary syndrome (PCOS), yet little is known about Vit D supplementation as a treatment for sexual dysfunction (SDy) in women with PCOS. AIM: To explore the implications of serum total 25-hydroxyvitamin D (25[OH]D) and bioavailable 25[OH]D (bio-25[OH]D) status and replacement on women with PCOS and SDy. METHODS: Reproductive-age women with PCOS who were not desiring fertility were eligible provided that they also had SDy, as assessed by the Female Sexual Function Index (FSFI), and were without severe depression, as evaluated by the Beck Depression Inventory II (BDI-II). Participants were given the recommended dietary allowance of Vit D (600 IU daily) plus hormonal contraception (HC; cyclic ethinyl estradiol/drospirenone) or no HC for 6 months. Comparisons between groups were analyzed by chi-square test and t-test, and Pearson's correlation coefficient analyzed correlations between FSFI with demographics, BDI-II, androgen levels, and total and bio-25[OH]D. OUTCOMES: The outcomes included SDy (FSFI <26.55), total and serum bio-25[OH]D levels, and total and free testosterone. RESULTS: A total of 42 women without severe depression completed the FSFI, with 28 (66.7%) having SDy. All FSFI domains, including arousal, lubrication, orgasm, and pain, were significantly lower as compared with women without SDy, with no associations with respect to demographics, total and free testosterone, or total and bio-25[OH]D. Vit D replacement was initiated with HC (n = 18) or no HC (n = 10), and for those completing the study, FSFI improved (score >26.55) in 61% (11/18) regardless of the treatment group. A time-treatment effect showed a significant change for the domain of orgasm, suggesting that HC had more of an impact than Vit D replacement. Improvement in sexual function as a dichotomous variable was not associated with age, body mass index, other demographics, total and free testosterone, total and bio-25[OH]D, or HC use. CLINICAL IMPLICATIONS: Due to the prevalence of SDy in women with PCOS, efficacious treatment options are necessary. STRENGTHS AND LIMITATIONS: This study is the first to analyze the effect of Vit D supplementation on SDy in women with PCOS. Limitations included the small number of participants who completed the study, thus limiting meaningful conclusions and generalizability. CONCLUSION: Vit D status was not associated with SDy and BDI-II. While HC may have played a role, standard Vit D supplementation could not account for the noted improvement in FSFI in women with PCOS.

Effect of trophectoderm biopsy for PGT-A on live birth rate per embryo in good prognosis patients.

PURPOSE: To determine if blastocyst trophectoderm biopsy for PGT-A is associated with an increased rate of live birth per embryo in good prognosis IVF patients at a single center. METHODS: We performed a retrospective cohort study of good prognosis embryo transfer cycles at a single center from 1/1/2017 to 12/31/2019. We evaluated the rate of live birth per embryo with and without PGT-A for transfer of embryos in two groups of good prognosis patients: embryos from donor oocytes and embryos from autologous oocytes with maternal age less than 35 years at oocyte retrieval. Two-sided Fisher's exact tests were used for comparisons between groups. RESULTS: After transfer of embryos created from donor oocytes the live birth rate per euploid embryo was 70.6% (24/34) compared to 34.3% (35/102) for untested embryos for a rate difference of 36.3% (95% CI 18.4-54.1%, p < 0.01). After transfer of embryos created from autologous oocytes with maternal age less than 35 years at oocyte retrieval the live birth rate per euploid embryo was 70.0% (49/70) compared to 52.5% (53/101) for untested embryos for a rate difference of 17.5% (95% CI 3.0-32.0%, p = 0.03). CONCLUSIONS: In good prognosis patients at our center the live birth rate per euploid blastocyst was higher than for untested blastocysts.

Effect of age and morphology on sustained implantation rate after euploid blastocyst transfer.

RESEARCH QUESTION: What impact does maternal age and embryo morphology have on sustained implantation rates of euploid blastocysts? DESIGN: This was a retrospective analysis of sustained implantation rates of euploid blastocysts stratified by maternal age and morphology. The primary analysis included 208 embryo transfers with a total of 229 embryos transferred from January 2017 through August 2020. RESULTS: For all ages the sustained implantation rates for day 5 good quality blastocysts were higher than for day 5 fair, day 5 poor and day 6 blastocysts. At a maternal age of 36 years the best-fit sustained implantation rates were 86% for day 5 good quality blastocysts, 64% for day 5 fair, 63% for day 5 poor, and 51% for all day 6 blastocysts analysed as one group. When controlling for morphology and day of biopsy, there were higher sustained implantation rates for euploid embryos of younger patients compared with older patients. The best-fit sustained implantation rates for age 33 compared to age 39 years were 86% versus 80% for day 5 good, 71% versus 62% for day 5 fair, 59% versus 55% for day 5 poor, and 81% versus 46% for all day 6. CONCLUSIONS: There was a clinically significant higher sustained implantation rate at all ages for euploid day 5 good quality embryos compared with day 5 fair, day 5 poor and day 6 embryos.

Examining pre-term birth and cesarean section rates in gestational carrier pregnancies.

PURPOSE: This study sought to report on the route and gestational age at delivery of gestational carrier (GC) pregnancies with respect to the GCs' prior obstetric history. METHODS: A retrospective analysis of all GC pregnancies from one of the largest surrogacy agencies in California between 2008 and 2018 was performed. Available demographic data and obstetric history, including a history of prior cesarean section (CS) and preterm birth (PTB), were collected for each GC and correlated to outcomes of the index GC pregnancy. Primary outcomes for the index GC pregnancies included delivery route and gestational age at delivery. RESULTS: Eight-hundred-thirty-six GCs were included in our analysis. 319 (38.2%) delivered via CS, and 517 (61.8%) delivered vaginally. 60 (18.8%) of the CS deliveries were due to multifetal gestation. Primary CS rate in singleton GC pregnancies was 38.5%. In women without a history of CS, neither age, BMI, interpregnancy interval, prior parity, nor year of delivery impacted the primary singleton CS rate (all, P > 0.05). Of GCs with a history of a prior CS (n = 350, 41.9%), 218 (62.3%) had a vaginal delivery after CS (VBAC) and 132 (37.7%) had a repeat CS. Women who had successful VBACs were significantly younger than those who had repeat CS (mean 33.7 vs. 35.2 years, P = .003). BMI was lower in patients who had a VBAC compared to those that had a repeat CS (mean BMI 24.6 vs. 25.5, P = 0.074), although this did not reach statistical significance. In GCs with a history of CS, interpregnancy interval, year of delivery, prior parity, and multiple gestation in the index GC pregnancy did not impact mode of delivery. VBAC rates did not change over the study period (P = 0.757). Overall PTB rate was 15.1%. Most PTB in GC pregnancies were in those with a history of PTB, and PTB was more likely in singletons rather than multifetal gestations (76.7% in singletons vs. 30% in multiples) in patients with history of PTB (P < 0.001). Those with no history of PTB and who carried multiples had a low rate of PTB; in fact, in this group, only 1 out of 35 patients had a PTB with multiples. CONCLUSIONS: Both primary CS and PTB rates in singleton GC pregnancies are higher than national averages. CS rates are independent of age, BMI, and interpregnancy interval. In GCs with a history of a CS, VBAC rates well exceed national averages and are higher in younger GCs with a lower BMI. PTB rates are impacted primarily by the GCs obstetric history. In those GCs without a history of PTB, rates of PTB are low, even in those with a multifetal gestation.

What is the optimal timing of intracytoplasmic sperm injection (ICSI) after EGG retrieval? A randomized controlled trial.

PURPOSE: To determine if oocyte denudation and ICSI at 36.5 versus 39 h post HCG and/or Lupron trigger (2.5 h versus 5 h post-oocyte retrieval) influences fertilization and blastulation rates in good prognosis couples METHODS: We performed a prospective, randomized controlled trial of 12 patients undergoing IVF with ICSI at an academic fertility center, resulting in 206 MII oocytes analyzed. At time of retrieval, patients with more than 10 oocytes retrieved had their oocytes randomized into two groups-one group for oocyte denudation and ICSI at 36.5 h post HCG and/or Lupron trigger and the other group for these procedures at 39 h post HCG and/or Lupron trigger (2.5 and 5 h after oocyte retrieval). Primary outcomes were fertilization and blastulation rates. RESULTS: No difference was observed in fertilization rate, total blastulation rate, or day of blastulation based on timing of denudation and ICSI (all p > 0.05). Multiple regression analyses for fertilization and blastulation controlling for age and BMI revealed no difference in fertilization based on time of ICSI (p = 0.38, 0.71, respectively). A conditional logistic regression to account for multiple oocytes derived from each patient also found no difference in fertilization or blastulation based on timing of ICSI, even when controlling for age and BMI (p = 0.47, 0.59, respectively). CONCLUSION(S): In good prognosis couples, we observed no difference in fertilization or blastulation rates based on timing of ICSI within the currently accepted 2- to 6-h window post-retrieval based on a 34-h trigger. The oocyte appears to have a physiological tolerance for fertilization during this window of time, and variability in the timing of ICSI during this window is unlikely to have an impact on cycle outcome.

Developmental potential of immature human oocytes aspirated after controlled ovarian stimulation.

PURPOSE: Intracytoplasmic sperm injection (ICSI) for initially immature oocytes that mature in vitro is controversial and practice varies widely. While it may increase the number of usable embryos, it may also be time-intensive and potentially low-yield. This study sought to elucidate which patients may benefit from ICSI of initially immature oocytes that matured in vitro. METHODS: A retrospective study comparing fertilization, cleavage, blastulation, and embryo usage rates between sibling initially immature and mature oocytes that underwent ICSI between 2015 and 2019 was performed. Outcomes of initially immature oocytes were stratified by initial maturation stage, timing of progression to metaphase II (MII) in vitro, percentage of mature oocytes in the cycle, and female age. RESULTS: Ten thousand eight hundred seventeen oocytes from 889 cycles were included. Of 3137 (29.0%) initially immature oocytes, 418 (13.3%) reached MII later on the day of retrieval (day 0) and 1493 (47.6%) on day 1. Overall, embryos originating from initially immature oocytes had lower cleavage and blastulation rates compared to those from initially mature oocytes (P<0.05, all groups). However, embryos from oocytes that matured later on day 0 comprised a unique subset that had clinically similar cleavage (75% vs 80%, RR 0.93, P=0.047) and blastulation rates (41% vs 50%, RR 0.81, P=0.024) compared to initially mature oocytes. Women with low percentages of mature oocytes in the cycle overall and women ≥40 in cleavage cycles derived the highest relative benefit from the use of immature oocytes. CONCLUSION: ICSI of immature oocytes, particularly those that mature later on the day of retrieval, may improve numbers of usable embryos. This study supports routine reassessment of immature oocytes for progression to MII and ICSI on day 0. An additional reassessment on day 1 may also be of use in older women or those with low percentage of mature oocytes.

Transfer the best and biopsy the rest? Blastocyst euploidy rates differ by morphology and day of biopsy.

PURPOSE: To determine if blastocyst euploidy rates differ by embryo morphology or day of biopsy. METHODS: We performed a retrospective analysis of euploidy rates based on patient age, overall embryo morphology grade (good, fair, or poor), and day of biopsy (days 5, 6, or 7) for blastocysts undergoing preimplantation genetic testing for aneuploidy (PGT-A). Our primary analysis included 904 embryos from oocytes age 33-39 years at retrieval. RESULTS: In our primary analysis, euploidy rates were higher for good quality embryos than poor (64% vs. 48%, p < 0.01) and for fair quality embryos than poor (61% vs. 48%, p < 0.01). There was no significant difference in the euploidy rate between good and fair quality embryos (64% vs. 61%, p = 0.56). Embryos biopsied on day 5 were more likely to be euploid than embryos biopsied on day 6 (59% vs. 50%, p < 0.01) or day 7 (59% vs. 37%, p < 0.01). There was no significant difference in the euploidy rate between day 6 and day 7 embryos (50% vs. 37%, p = 0.07). CONCLUSION: PGT-A may be more useful in cycles where a lower euploidy rate is expected based on age at oocyte retrieval, embryo morphology, and day of biopsy. There may be little benefit to biopsy of embryos with a high euploidy rate. Young patients with one or more good quality day 5 embryos may benefit from a "transfer the best fresh and biopsy the rest" strategy.

Long-term imatinib diminishes ovarian reserve and impacts embryo quality.

PURPOSE: Tyrosine kinase inhibitors (TKIs) such as imatinib are commonly used chemotherapeutics, but the effects of long-term treatments on reproductive outlook for cancer survivors are unknown. The purpose of this study was to examine the effects of long-term imatinib treatments on follicle development and embryo quality. Since prospective studies are not possible in healthy humans, we have incorporated a commonly used mouse model. METHODS: Adult female mice were treated with daily IP injections of imatinib for 4-6 weeks. Liquid chromatography-mass spectrometry was used to measure imatinib in serum and ovarian tissues. At the end of treatments, females were superovulated and mated to yield fertilized embryos. Oocytes and embryos were collected from oviducts, assessed for development by microscopy, and fertilized embryos were cultured in vitro. Blastocysts were fixed and stained for differential cell counts. RESULTS: Long-term imatinib treatments caused a shift in follicle development, with imatinib-treated females having fewer primordial follicles, but an increase in primary and secondary follicles (P < 0.05). There was no effect on ovulation or fertilization rates. However, blastocysts from imatinib-treated females had fewer total cells (P < 0.05) and a significant shift from inner cell mass to increased trophectoderm cells. CONCLUSION: This pilot study indicates that long-term TKI treatments may have significant impact on ovarian reserve and embryo developmental capacity. More studies are needed in other model systems to determine the long-term impact of TKIs in patients. Knowing the potential effects of chemotherapeutics on reproductive outlook is critical for quality of life and more research is needed.

Autoimmune Regulator is required in female mice for optimal embryonic development and implantation†.

Autoimmune Regulator (AIRE) regulates central immune tolerance by inducing expression of tissue-restricted antigens in thymic medullary epithelial cells, thereby ensuring elimination of autoreactive T cells. Aire mutations in humans and targeted Aire deletion in mice result in multiorgan autoimmune disease, known in humans as autoimmune polyglandular syndrome type 1 (APS-1). APS-1 is characterized by the presence of adrenal insufficiency, chronic mucosal candidiasis, and/or hypoparathyroidism. Additionally, females often present with gonadal insufficiency and infertility. Aire-deficiency (KO) in mice results in oophoritis and age-dependent depletion of follicular reserves. Here, we found that while the majority of young 6-week-old Aire-KO females had normal follicular reserves, mating behavior, and ovulation rates, 50% of females experienced embryonic loss between gestation day (GD) 5.5 and 7.5 that could not be attributed to insufficient progesterone production or decidualization. The quality of GD0.5 embryos recovered from Aire KO mice was reduced, and when cultured in vitro, embryos displayed limited developmental capacity in comparison to those recovered from wild-type (WT) mice. Further, embryos flushed from Aire KO dams at GD3.5 were developmentally delayed in comparison to WT controls and had reduced trophoblastic outgrowth in vitro. We conclude that AIRE does not play a direct role in uterine decidualization. Rather, reduced fertility of Aire-deficient females is likely due to multiple factors, including oophoritis, delayed preimplantation development, and compromised implantation. These effects may be explained by autoimmune targeting of the ovary, embryo, or both. Alternatively, altered embryonic development could be due to a direct role for AIRE in early embryogenesis.

Sperm-oocyte contact induces outside-in signaling via PYK2 activation.

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm - oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline - rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm - oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2-/- oocytes and the pyk2-/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm - binding sites under conditions where sperm - oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm - oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Micro-RNAs involved in cellular proliferation have altered expression profiles in granulosa of young women with diminished ovarian reserve.

PURPOSE: The study aims to determine differences in micro-RNA (miRNA) expression in granulosa (GC) and cumulus cells (CC) between young women with diminished ovarian reserve (DOR) or normal ovarian reserve (NOR). Secondary objective was to identify downstream signaling pathways that could ultimately indicate causes of lower developmental competence of oocytes from young women with DOR. METHODS: The method of the study is prospective cohort study. RESULTS: Of the miRNA, 125 are differentially expressed in GC between DOR and NOR. Only nine miRNA were different in CC; therefore, we focused analysis on GC. In DOR GC, miR-100-5p, miR-16-5p, miR-30a-3p, and miR-193a-3p were significantly downregulated, while miR-155-5p, miR-192-5p, miR-128-3p, miR-486-5p, miR130a-3p, miR-92a-3p, miR-17-3p, miR-221-3p, and miR-175p were increased. This pattern predicted higher cell proliferation in the DOR GC. The primary pathways include MAPK, Wnt, and TGFbeta. CONCLUSIONS: The miRNA pattern identified critical functions in cell proliferation and survival associated with DOR. GC in women with DOR seems to respond differently to the LH surge.

Blastulation timing is associated with differential mitochondrial content in euploid embryos.

PURPOSE: Preimplantation genetic screening (PGS) and assessment of mitochondrial content (MC) are current methods for selection of the best embryos for transfer. Studies suggest that time-lapse morphokinetics (TLM) may also be helpful for selecting embryos more likely to implant. In our study, we sought to examine the relationship between TLM parameters and MC to determine if they could be used adjunctively in embryo selection. We also examined the relationship between MC with ploidy and blastulation. METHODS: Cryopreserved human embryos at the zygote stage were thawed and cultured in a time-lapse system. Blastomere and trophectoderm biopsies were performed on days 3 and 6. Biopsied cells and all whole embryos from day 6 were analyzed for MC (ratio of mitochondrial to nuclear DNA) and ploidy using next-generation sequencing. RESULTS: In embryos, MC per cell declined between day 3 and day 6. While early cleavage parameters did not predict MC, embryos with longer blastulation timing had higher MC on day 6. Day 6 MC was lower in euploid vs. aneuploid embryos and lower in blastocysts vs. arrested embryos. CONCLUSIONS: A lower MC at the blastocyst stage was associated with euploid status and blastocyst formation, indicating better embryo quality compared to those with a higher MC. Higher MC in aneuploid and arrested embryos may be explained by slower cell division or degradation of genomic DNA over time. Blastulation timing may be helpful for selection of higher quality embryos. Combining blastulation timing and MC along with morphologic grading and euploid status may offer a new direction in embryo selection.

Stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation.

Follicular fluid within ovarian antral follicles contains numerous factors, which influence the development of a healthy oocyte including nucleic acids, steroids, proteins, and extracellular vesicles (EVs). Current evidence indicates that follicular EVs promote changes in cellular gene expression and support cumulus-oocyte complex expansion in vitro. In this study, we found EVs from different sized follicles differentially stimulate granulosa cell proliferation and this could be explained by both the differential contents associated, on or within the vesicles and by the preferential uptake of EVs dependent on follicle size from which they were isolated. Antibody array and inhibitor studies indicated that the Src, PI3K/Akt, and MAPK signaling pathways mediate the stimulatory effects of EVs on granulosa cell proliferation. This study demonstrates for the first time that EVs isolated from follicular fluid are capable of stimulating granulosa cell proliferation and that this stimulatory response is associated with the size of antral follicle from which the EVs originated. The study further also provides the first evidence that vesicles released by small antral follicles are preferentially taken up when compared to those isolated from large follicles, suggesting that vesicular surface proteins change during follicular maturation.

The role of angiogenic markers in adverse perinatal outcomes: fresh versus frozen embryo transfers.

PURPOSE: We aimed to investigate the angiogenic balance in fresh compared to frozen embryo transfers, and among neonates with adverse perinatal outcomes. METHODS: This was a retrospective cohort study. All IVF cycles resulting in a singleton live birth at a university academic fertility center from January 1, 2011, to December 31, 2013, were examined. Concentrations of sFLT-1 and PlGF were measured in previously frozen serum specimens collected during early gestation at approximately 5 weeks gestation. Patients completed an electronic survey to detail perinatal outcome. RESULTS: We identified 152 singleton live births (103 fresh, 49 frozen). Demographic characteristics were similar between the two groups. Ratios of sFlt-1:PlGF were not different between fresh and frozen transfers. Neonates from fresh cycles had a mean birth weight 202 g lighter (p = 0.01) than frozen cycles, after adjusting for gestational age. Among babies born with poor perinatal outcomes, there was a difference in sFlt-1:PlGF ratios after adjusting for race. In non-Asians, infants born small for gestational age (SGA) (< 10th percentile) had significantly higher sFLT-1:PLGF ratio, median ratio (0.21 vs 0.12, p = 0.016). CONCLUSIONS: Fresh transfers were associated with lower birth weight infants compared to frozen transfers. While there was no difference in sFlt-1:PlGF ratios between fresh and frozen transfers, these ratios were significantly lower in SGA infants, suggesting an imbalance in angiogenic markers during placentation.

Extracellular Vesicles from Bovine Follicular Fluid Support Cumulus Expansion.

Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation-inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present; however, no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on cumulus-oocyte complex (COC) expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3-5 mm) and large (>9 mm) antral bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy, and Western blot analysis. To test for bioactivity, mouse and bovine COCs were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression.

Role of focal adhesion kinase in oocyte-follicle communication.

Germ cells require communication with associated somatic cells for normal gametogenesis, as exemplified by an oocyte that interacts with granulosa cells via paracrine factors as well as gap junctions located at sites of contact between these two cell types. The objective of the present study was to define the mechanisms by which cell-cell contact with the oocyte is controlled and to determine the extent that the oocyte actively participates in this association. Proline-rich tyrosine kinase 2 (PTK2), a focal adhesion kinase, was found to be activated at sites of contact between the oocyte and trans-zonal cell processes from the surrounding granulosa cells. In order to determine the functional significance of oocyte-derived PTK2 signaling in oocyte-follicle communication, an oocyte-specific Ptk2 knockout was produced through a breeding strategy pairing a floxed Ptk2-CAT-eGFP mouse with the Zp3-Cre line. Since Ptk2-null mice never develop to birth, this represents the first opportunity to define the role of PTK2 in oocyte-follicle communication. Ablation of Ptk2 within the developing oocyte resulted in lower fertility with reduced numbers of pups, lower rates of blastocyst formation, and reduced cell numbers per blastocyst. Follicles containing Ptk2-null oocytes exhibited reduced oocyte diameter, reduced numbers of connexin 37 and 43 foci at the oocyte surface, and impaired dye coupling between oocyte and granulosa cells. These findings are consistent with a model in which PTK2 plays a critical role in establishing or maintaining oocyte-granulosa cell contacts that are essential for gap junction-mediated communication between granulosa cells and the oocyte.

PTK2b function during fertilization of the mouse oocyte.

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

Post-ovulatory aging of oocytes disrupts kinase signaling pathways and lysosome biogenesis.

Post-ovulatory aging of oocytes results in the progressive loss of fertilization and developmental competence. This degradation of oocyte quality has been the object of numerous investigations, primarily focused on individual signaling pathways which provide limited insight into the status of global signaling events. The purpose of the present investigation was to comprehensively assess broad patterns of signaling pathway activity during in vitro aging as an initial step in defining control points that can be targeted to prevent the reduction in oocyte quality during prolonged culture. An antibody microarray-based phospho-proteome analysis performed on oocytes before and after eight hours of culture revealed significant changes in the abundance or activation state of 43 proteins that function in a wide variety of protein kinase-mediated signaling pathways. Several of the most significantly affected kinases were studied by Western blot and confocal immunofluorescence to corroborate the array results. Prolonged culture resulted in global changes in the abundance and activity of protein kinases that regulate the response to calcium, stress, and cell-cycle control. Examination of intracellular structures revealed a previously unrecognized increase in the abundance of large autophogagic lysosomes, which correlates with changes in protein kinase pathways. These results provide insight into the stresses experienced by oocytes during culture and the diversity of responses that results from them. The observed increase in autophagy-related activity, together with the disruptions in calcium signaling, cell-cycle, and stress-response pathways, have the potential to negatively impact oocyte quality by interfering with the normal sequence of biochemical changes that constitute egg activation following fertilization.

Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

Anti-Müllerian hormone levels are associated with skeletal maturity in adolescent girls in the Fels Longitudinal Study.

The role of anti-Müllerian hormone (AMH), a potential marker of the hypothalamic-pituitary-ovarian axis, is not well established in adolescent females. Most studies use secondary sexual characteristics or chronological age as predictors for AMH. Skeletal maturity, an indicator of bone development, has not been examined to predict AMH. This study sought to examine patterns of change in AMH in relation to skeletal maturity. Demographics, anthropometry, hand-wrist radiographs, and cardiometabolic risk factors from 88 females (212 observations), between the ages of 8 to 18 years from the Fels Longitudinal Study were used in this study. AMH was analyzed using ELISA from stored frozen serum samples. Generalized linear mixed effect modeling was used. In the stepwise regression models, log-transformed AMH (AMHlog) was regressed on relative skeletal age as the skeletal maturity indicator (calculated as chronological age minus skeletal age) and adjusted for chronological age, adiposity, and cardiometabolic risk factors. Skeletal maturity significantly predicted lower AMHlog (ß= -0.073, SE=0.032, p=0.023). Glucose was significantly associated with decreases in AMHlog (ß= -0.008, SE=0.004, p=0.044). Chronological age modeled as a cubic function was not significant. AMH and skeletal maturity may provide correlated information on growth and pubertal status in adolescent females.