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Objective: To investigate the impact of late follicular phase progesterone (P) elevation in relation to ovarian response on cycle outcome., Design: Cohort study. The progesterone-to-follicle index (PFI) was calculated by dividing the blood P by the number of follicles ≥14 mm. The clinical pregnancy rate was calculated against the range of PFI values and blood P levels., Setting: In vitro fertilization unit., Patient(s): A heterogenous population undergoing IVF with pituitary suppression and gonadotropin stimulation resulting in 3-15 follicles ≥14 mm and blood P≤10 nmol/L on hCG day and resulting in fresh embryo transfer., Intervention(s): None., Main Outcome Measure(s): Association of blood P and PFI with clinical pregnancy rate., Result(s): Data were retrieved for 8,649 IVF cycles in normal responders. The (reverse) odd ratios for pregnancy were 1.112 (95% confidence interval [CI], 1.077-1.165) for blood P and 4.104 (95% CI, 3.188-5.284) for the PFI. Elevated P levels were associated with a lower pregnancy rate only when they reached the >93rd percentile. The PFI was inversely and linearly related to the pregnancy rate for the whole range of values., Conclusion(s): A late increase in P level is detrimental if it is a consequence of increased P production per follicle (high PFI) but not if it is a consequence of additional follicular recruitment. The PFI enables clinicians to differentiate these conditions., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the impact of oocyte aspiration, denudation, and sperm injection timing in relation to oocyte hCG exposure time on intracytoplasmic sperm injection (ICSI) outcome., Design: Cohort study., Setting: Tertiary medical center., Patient(s): A total of 614 consecutive ICSI cycles were performed in 421 patients aged <38 years with at least three aspirated oocytes and no more than three previous treatments., Intervention(s): Gonadotropin-releasing hormone agonist or GnRH antagonist suppression; oocyte pickup (OPU)-hCG interval more/less than 36 hours; OPU-denudation interval more/less than 2 hours; denudation-ICSI interval more/less than 1 hour., Main Outcome Measure(s): Fertilization, embryo transfer, and pregnancy rates., Result(s): Late OPU was associated with more available embryos than early OPU and significantly higher rates of fertilization (66.0% ± 22.8% vs. 61.8% ± 24.3%), ET (99.5% vs. 96.2%), and pregnancy (47.2% vs. 35.4%). This advantage was more pronounced in GnRH agonist cycles. The length of incubation before or after denudation had no effect, regardless of OPU timing. On logistic stepwise regression, OPU timing was the only significant independent predictor of pregnancy (odds ratio 1.6, 95% confidence interval 1.17-2.29)., Conclusion(s): The timing of OPU has a predominant effect on ICSI success, especially in GnRH agonist cycles. Delaying oocyte denudation or sperm injection does not compensate for insufficient postpriming exposure to the follicular environment., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the in vitro effect of neurotrophin 4 (NT-4) on follicular assembly in human fetal ovaries., Design: Human ovarian tissue from fetuses at 19-20 gestational weeks was placed in organ culture for two weeks with NT-4. Control groups were cultured with a neutralizing antibody against NT-4., Setting: Infertility unit at an university-affiliated tertiary medical center., Patient(s): Four patients who underwent pregnancy terminations at 19-20 gestational weeks., Intervention(s): None., Main Outcome Measure(s): Histologic findings of follicular assembly., Result(s): Follicular assembly was significantly increased in the specimens cultured with NT-4 than in the uncultured specimens, the samples cultured without NT-4, and samples cultured with the neutralizing antibody. In the second week of culture, additional follicular assembly was promoted in the presence of 100 ng/mL NT-4 but not with 10 ng/mL NT-4., Conclusion(s): This is the first report showing that NT-4 seems to promote human follicular assembly in fetal ovaries, probably in a dose-dependent manner. Follicular assembly is regulated by multiple signals, and additional studies on the effects of other growth factors in combination with NT-4 are warranted., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF alpha 1 (GFR-alpha1) and tyrosine kinase receptor for rearranged during transfection (RET), in human ovaries at the protein and mRNA levels., Design: Samples were prepared for immunohistochemical staining for GDNF, GFR-alpha1, and RET and in situ hybridization for the mRNA of GFR-alpha1. The mRNA expression of two GDNF isoforms and two RET isoforms was investigated by reverse transcription polymerase chain reaction., Setting: Infertility unit at a university-affiliated tertiary medical center., Patient(s): Fifteen patients who underwent pregnancy terminations at 21-35 gestational weeks and 28 girls/women aged 5-39 years who underwent ovarian laparoscopies., Intervention(s): None., Main Outcome Measure(s): Laboratory analysis of human ovarian specimens., Result(s): The GDNF protein was detected in oocytes of all samples. Granulosa cells (GCs) stained for GDNF in a portion of fetal samples and in all samples from girls/women. The proteins for GFR-alpha1 and RET were detected in both oocytes and GCs with weak staining for the long RET isoform. GFR-alpha1 mRNA transcripts were detected in oocytes and GCs from all samples. The mRNA transcripts for the two GDNF isoforms and the two RET isoforms were detected in all fetal and adult specimens., Conclusion(s): The presence of the receptors of GDNF in the GCs of human primordial follicles suggests that GDNF may be involved in the regulation of primordial follicular activation., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the expression of vascular endothelial growth factor A (VEGF-A) and that its two receptors (VEGFR1, VEGFR2) in human preantral follicles., Design: Immunohistochemical, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) study of the expression of the VEGF-A system in human ovaries., Setting: Major tertiary-care academic center., Patient(s): Twenty-two patients who underwent pregnancy terminations at 21-35 gestational weeks and 29 girls/women aged 5-39 years who underwent ovarian laparoscopies., Intervention(s): None., Main Outcome Measure(s): Laboratory analysis of human ovarian specimens., Result(s): Immunhistochemistry and in situ hybridization revealed the expression of the proteins and mRNA transcripts for VEGFR1 and VEGFR2 in oocytes, granulosa cells, and stroma cells from fetuses and girls/women. The protein for VEGF-A was detected immunohistochemically in oocytes, granulosa cells, and stroma cells from fetuses and girls. VEGF-A and VEGFR1 proteins were expressed more strongly than VEGFR2. VEGF-A(121), VEGF-A(165), and VEGF-A(189) isoforms were identified by RT-PCR in the ovarian samples from fetuses and women., Conclusion(s): The presence of the VEGF-A receptors, particularly in the granulosa cells, suggests that VEGF-A might be involved in proliferation initiation of primordial follicles or play an as yet unknown role in human preantral follicles., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To compare the development of human fetal follicles from late-gestational-age fetuses frozen-thawed gradually and slowly with dimethylsulfoxide (DMSO) or 1,2 propanediol (PROH) and sucrose after renal grafting into follicle stimulating hormone-treated immunodeficient mice., Design: Controlled histologic study of grafted human fetal ovaries., Setting: Major tertiary care academic center., Patient(s): Eleven women undergoing pregnancy termination at 22 to 33 gestational weeks., Intervention(s): None., Main Outcome Measure(s): Microscopic morphometric analysis and immunohistochemistry for proliferating cell nuclear antigen (PCNA)., Result(s): Only follicles from samples frozen-thawed with PROH developed to secondary and antral stages 4 to 6 months after grafting, with PCNA expression in their granulosa cells. However, the number of surviving/developing follicles per section was very low (4-25 per graft), compared with 71 to 406 follicles in pretransplantation samples. Graft recovery was very high, with similar rates for transplants frozen-thawed with PROH and DMSO. Normal ovarian structure after grafting was identified only in the PROH frozen-thawed grafts. In deteriorated grafts, frozen-thawed with either DMSO or PROH, net-like hollows replaced follicles, whereas tubule-like structures were only identified in DMSO frozen-thawed grafts., Conclusion(s): This is the first report of the development of late-pregnancy-stage human fetal follicles in immunodeficient mice. PROH freezing-thawing supported development and survival better than DMSO. However, the low follicular survival points to the urgent need for efficient methods to enhance vascularization rate and prevent ischemia.

Objective: To evaluate whether basic fibroblast growth factor (FGF) benefits the in vitro development of human primordial follicles., Design: Human ovarian tissue was placed in organ culture for 4 weeks with basic FGF and either fetal calf serum or a serum-free combination. Control groups were cultured with a neutralizing antibody against basic FGF., Setting: Major tertiary care and referral academic centers., Patient(s): Fourteen women/girls undergoing various gynecological operations and two fetuses from women undergoing pregnancy terminations., Intervention(s): None., Main Outcome Measure(s): Follicular counts, immunohistochemistry for proliferating cell nuclear antigen and bromodeoxyuridine incorporation and measurement of 17beta E(2) production., Result(s): Only in the serum-free culture system was the number of developing follicles in samples cultured with basic FGF significantly higher than in uncultured specimens. The E(2) production increased significantly in the second week, and there was a significant reduction in E(2) secretion with the addition of the neutralizing antibody against basic FGF. The percentage of granulosa cells (GCs) that stained for proliferating cell nuclear antigen or bromodeoxyuridine was significantly higher in developing follicles than in primordial follicles, regardless of treatment., Conclusion(s): Basic FGF apparently plays a role in the E(2) production of early follicles. High doses of basic FGF enhanced follicular development in serum-free media.

Objective: To investigate the presence of growth hormone (GH) and its receptor (GH-R) in early developing follicles., Design: Immunocytochemical and in situ hybridization study., Setting: Major tertiary care and referral academic centers., Patient(s): Ten ovarian samples from adults/girls aged 6-38 years and from 10 fetuses of women undergoing second and third trimester pregnancy terminations., Intervention(s): None., Main Outcome Measure(s): Immunocytochemistry and in situ hybridization on paraffin sections of human ovaries from fetuses and women/girls., Result(s): The proteins and the mRNA transcripts for GH and GH-R were detected in oocytes, granulosa (GC), and stroma cells from both sources (fetuses and women/girls), with low staining intensity only in a portion of the fetal GC., Conclusion(s): This is the first report of the expression of GH-R in human ovaries from fetuses as well as women/girls and of GH in human fetal ovaries. Be that as it may, further experiments should be conducted to elucidate if indeed GH is involved in the initiation of human primordial follicular growth.