Prevalence of pathogenic variants and digenic disease in patients diagnosed with normosmic hypogonadotropic hypogonadism/Kallmann Syndrome.

BACKGROUND: Hypogonadotropic hypogonadism (HH) is due to impaired gonadotropin releasing hormone (GnRH) action resulting in absent puberty and infertility. At least 44 genes have been identified to possess genetic variants in 40-50% of nHH/KS, and 2-20% have presumed digenic disease, but not all variants have been characterized in vitro. HYPOTHESIS: The prevalence of pathogenic (P)/likely pathogenic (LP) variants in monogenic and digenic nHH/KS is lower than reported. DESIGN: Cross-sectional study. SETTING: University Research Laboratory. SUBJECTS: 158 patients with nHH/KS. METHODS: Exome sequencing (ES) was performed and variants were filtered for 44 known genes using Varsome and confirmed by Sanger Sequencing. MAIN OUTCOME MEASURES: P/LP variants in nHH/KS genes. RESULTS: ES resulted in >370,000 variants, from which variants in 44 genes were filtered. Thirty-one confirmed P/LP variants in 10 genes (ANOS1, CHD7, DUSP6, FGFR1, HS6ST1, KISS1, PROKR2, SEMA3A, SEMA3E, TACR3), sufficient to cause disease, were identified in 30/158 (19%) patients. Only 2/158 (1.2%) patients had digenic variant combinations: a male with hemizygous ANOS1 and heterozygous TACR3 variants and a male with heterozygous SEMA3A and SEMA3E variants. Two patients (1.2%) had compound heterozygous GNRHR (autosomal recessive) variants-one P and one variant of uncertain significance (VUS). Five patients (3.2%) had heterozygous P/LP variants in either GNRHR or TACR3 (both autosomal recessive), but no second variant. CONCLUSION: Our prevalence of P/LP variants in nHH/KS was 19%, and digenicity was observed in 1.2%. These findings are less than those previously reported, and probably represent a more accurate estimation since VUS are not included.

Investigation of subfertility in the female Nsmf knockout mouse.

OBJECTIVE: To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. DESIGN: Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. SETTING: University research laboratory. PATIENTS: None; mice were used. INTERVENTIONS: Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. MAIN OUTCOME MEASURES: Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. RESULTS: We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. CONCLUSIONS: Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Sperm DNA fragmentation on the day of fertilization is not associated with embryologic or clinical outcomes after IVF/ICSI.

PURPOSE: To evaluate if sperm DNA fragmentation (SDF) in the sample used for intracytoplasmic sperm injection (ICSI) impacts outcomes after euploid blastocyst transfer. METHODS: Prospective cohort study of couples undergoing IVF with preimplantation genetic testing for aneuploidy from December 2014-June 2017. Sperm collected on the day of ICSI was analyzed for SDF using the sperm chromatin structure assay (SCSA®). Semen analysis parameters, embryologic outcomes, and clinical outcomes after euploid blastocyst transfer were compared between groups with DNA fragmentation index (DFI) ≤ 15% and DFI > 15% using Mann-Whitney U, t tests, and generalized linear mixed effects models. RESULTS: Two hundred thirty-four patients were included. One hundred seventy-nine men had DFI ≤ 15% (low DFI group) and 55 men had DFI > 15% group (high DFI group). Total motile sperm and sperm concentration were significantly lower in the group with DFI > 15% vs. DFI ≤ 15%. There was no difference in fertilization (86.3 vs. 84.2%, adjusted OR (95% CI) 0.86 (0.63-1.18)), blastulation (49.5 vs. 48.8%, adjusted OR 1.02 (0.75-1.36)), or euploidy (55.7 vs. 52.1%, adjusted OR 0.96 (0.7-1.31)) between the low and high DFI groups, respectively. Clinical outcomes were similar between low and high DFI groups, including implantation rate (68.8 vs. 79.8%), ongoing pregnancy rate (65.9 vs. 72.6%), and miscarriage rate (4.2 vs. 8.8%), respectively. CONCLUSION: Sperm DNA fragmentation on the day of ICSI is not associated with embryologic or clinical outcomes after euploid blastocyst transfer. Increasing levels of SDF are associated with low sperm concentration and total motile sperm count.

Mitochondrial replacement therapy.

PURPOSE OF REVIEW: The present study briefly reviews the history of mitochondrial replacement therapy (MRT); however, the focus is on recent advancements and future directions of the field. Specifically addressing societal and legal concerns and advances in MRT. RECENT FINDINGS: There continue to be new ethical debates surrounding MRT. In addition, there have been advancements in MRT techniques which could improve potential outcomes. Furthermore, advances in genetics continue to provide alternative approaches to treatment of many diseases, including alternatives to MRT. SUMMARY: MRT may be beneficial to eradicate a severely debilitating and often fatal disease. Despite significant supporting safety and efficacy, there are still many social and legal barriers to instituting MRT to clinical practice.

Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

Evidence that exposure to progesterone alone is a sufficient stimulus to cause a precipitous rise in the immunomodulatory protein the progesterone induced blocking factor (PIBF).

PURPOSE: To determine if exposure to progesterone alone is sufficient to increase the production of the immunomodulatory protein known as the progesterone induced blocking factor (PIBF). Also to determine what method of progesterone delivery or form of P best stimulates PIBF secretion. METHODS: Serum samples from patients with infertility and paid volunteers were evaluated for both PIBF and progesterone at various times during the follicular phase and the luteal phase in both natural cycles and cycles involving embryo transfer after endogenous and exogenous progesterone exposure and after various synthetic progestins. PIBF was measured by a non-commercial research ELISA assay. Comparisons were made of serum PIBF before and after exposure to progesterone, 17-hydroxyprogesterone, and oral contraceptives. PIBF was also measured before and after transfer of embryos. RESULTS: Progesterone alone without exposure to the fetal allogeneic stimulus was able to produce a marked increase in serum PIBF. Neither a synthetic progestin (19-nortestosterone derivative) nor 17-hydroxyprogesterone caused an increase in PIBF. Some PIBF is generally detected even in the follicular phase. CONCLUSIONS: A previous concept considered that an allogeneic stimulus, e.g., from the fetal semi-allograft, was necessary to induce de novo progesterone receptors in gamma delta T cells, which, in turn, when exposed to a high concentration of progesterone, would secrete high levels of PIBF. These data show that exposure to an allogeneic stimulus is not needed to cause a marked rise in PIBF, merely progesterone alone is sufficient.

Arthroscopic subscapularis repair.

As the largest and most powerful rotator cuff muscle, the subscapularis plays a critical role in proper shoulder function. The diagnosis of subscapularis tears is made based on history and physical examination and confirmed with imaging studies. Historically, subscapularis tears have been addressed in an open fashion. Recent literature has demonstrated successful all-arthroscopic repair of isolated subscapularis tears and anterosuperior rotator cuff tears. Successful arthroscopic treatment follows several critical steps: patient positioning, portal placement, obtaining adequate visualization and working space, identifying and mobilizing the subscapularis tendon, preparation of the tendon edge and lesser tuberosity, appropriate anchor placement, suture passage, and finally, secure knot-tying and fixation. Excellent functional and clinical outcomes following these steps have been demonstrated by various investigators. Additional research is needed, however, because direct comparison between all-arthroscopic and open subscapularis repair is limited, and neither technique has proved to be superior.

Control of lipid oxidation in extruded salmon jerky snacks.

A shelf-life study was conducted to evaluate the effect of antioxidants on oxidative stability of extruded jerky-style salmon snacks. Deterioration of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) due to lipid oxidation is a major concern for this healthy snack. A control jerky with no added antioxidants and 4 jerkies with antioxidants (rosemary, mixed tocopherols, tertiary butylhydroquinone, and ascorbyl palmitate) added as 0.02% of the lipid content were extruded in duplicate in a Coperion ZSK-25 twin screw extruder. Salmon jerkies from each formulation were placed in 3 mil barrier pouches, flushed with nitrogen, and stored at 35 °C and 75% relative humidity. Lipid oxidation was evaluated as by peroxide value and malonaldehyde content. Other chemical analyses included total fatty acid composition, lipid content, moisture, water activity, pH, and salt. Astaxanthin and CIE L*, a*, b* color were also analyzed at 4-wk intervals. Rosemary inhibited peroxide formation better than did other antioxidants at week 8; no treatment inhibited malonaldehyde levels. All jerkies had lower astaxanthin levels after 8 wk, but rosemary-treated jerky had higher pigment concentrations than did the control at weeks 4 and 8. Protection of omega-3 lipids in these extruded jerkies must be improved to offer consumers a convenient source of these healthful lipids. Practical Application: Salmon flesh can be extruded to produce a jerky that provides 410 mg of omega-3 lipids per serving. Natural antioxidants such as rosemary should be added at levels over 0.02% of the lipid content to help control lipid oxidation. Astaxanthin and CIE a* values correlated well with lipid stability and could be used to monitor quality during storage if initial values are known.

Characteristics of formed Atlantic salmon jerky.

UNLABELLED: Smoked salmon (Salmo salar L.) processing may generate large amounts of small pieces of trimmed flesh that has little economic value. Opportunities exist to develop new added-value foods from this by-product. Brining was compared with dry salting for the production of formed salmon jerky-style strips that were then smoked. The formulations also contained brown sugar and potato starch. Salted samples had higher salt concentrations and required less force to break using a TA-XT2 Texture Analyzer. Brined samples contained more fat and were darker, redder and more yellow than the salted samples. Processing concentrated omega-3 fatty acids compared with raw salmon, and the brined jerky had the highest omega-3 fatty acid content. A panel of 57 consumers liked the appearance and aroma of both samples equally (approximately 6.7 for appearance and 6.3 for aroma on the 9-point hedonic scale. Higher acceptability scores for taste, texture, and overall quality were given to the brined product (6.7 to 6.9 against 6.2 to 6.3). PRACTICAL APPLICATION: Salmon trim from smoking facilities can be utilized to produce a jerky that is a good source of omega-3 fatty acids, simultaneously adding value and reducing the waste stream.

The new follow-on-biologics law: a section by section analysis of the patent litigation provisions in the Biologics Price Competition and Innovation Act of 2009.

An abbreviated pathway for the approval of biosimilar biological products, often called "follow-on biologics," has been enacted into law as part of the health care legislation recently passed by Congress and signed by the President. The subtitle of the health care bill establishing this approval pathway, the Biologics Price Competition and Innovation Act of 2009, includes many provisions governing the identification of patents relevant to a given biosimilar biological product and the assertion of those patents in infringement suits. This article provides a section-by-section analysis of the patent-related provisions of the new approval pathway for biosimilar biological products, and points out several ways in which the new law differs fundamentally from the Hatch-Waxman Act, which provides the approval pathway for generic versions of small molecule drugs.

Mutations define cross-talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: possible conservation of a signaling interface for coupling ATP hydrolysis to drug transport.

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K(m)((ATP)) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.

Raisin dietary fiber composition and in vitro bile acid binding.

Raisins are dried grapes that are popular shelf-stable snacks. Three commercially important types of raisins were studied: sun-dried (natural), artificially dried (dipped), and sulfur dioxide-treated (golden) raisins. Dietary fiber composition was analyzed by AACC method 32-25. Polysaccharides were hydrolyzed, and the resulting sugars were analyzed by colorimetric and gas chomatographic methods. Fructans were measured with a colorimetric kit assay. Total dietary fiber values agreed with published values, with pectins and neutral polysaccharides of mannose and glucose residues predominating. Dipped raisins had over 8% fructans. No fructans were found in fresh grapes. Raisin types varied in their ability to bind bile acids in vitro. Coarsely chopped raisins bound more bile than did finely chopped or whole raisins.