The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

STUDY QUESTION: Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy? SUMMARY ANSWER: Sperm PLCζ levels in both mouse and humans correlate with measures of ideal embryogenesis whereby minimal levels seem to be required to result in successful pregnancy. WHAT IS KNOWN ALREADY: While causative factors underlying male infertility are multivariable, cases are increasingly associated with the efficacy of oocyte activation, which in mammals occurs in response to specific profiles of calcium (Ca2+) oscillations driven by sperm-specific PLCζ. Although sperm PLCζ abrogation is extensively linked with human male infertility where oocyte activation is deficient, less is clear as to whether sperm PLCζ levels or localization underlies cases of defective embryogenesis and failed pregnancy following fertility treatment. STUDY DESIGN, SIZE, DURATION: A cohort of 54 couples undergoing fertility treatment were recruited at the assisted reproductive technology laboratory at the King Faisal Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The recruitment criteria for males was a minimum sperm concentration of 5×106 sperm/ml, while all female patients had to have at least five oocytes. Sperm PLCζ analysis was performed in research laboratories, while semen assessments were performed, and time-lapse morphokinetic data were obtained, in the fertility clinic as part of routine treatment. The CRISPR/Cas9 system was concurrently used to induce indels and single-nucleotide mutations within the Plcζ gene to generate strains of Plcζ mutant mice. Sperm PLCζ was evaluated using immunofluorescence and immunoblotting with an antibody of confirmed consistent specificity against PLCζ. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated PLCζ profiles in sperm samples from 54 human couples undergoing fertility treatment in the context of time-lapse morphokinetic analysis of resultant embryos, correlating such profiles to pregnancy status. Concurrently, we generated two strains of mutant Plcζ mice using CRISPR/Cas9, and performed IVF with wild type (WT) oocytes and using WT or mutant Plcζ sperm to generate embryos. We also assessed PLCζ status in WT and mutant mice sperm in the context of time-lapse morphokinetic analysis and breeding outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: A significant (P ≤ 0.05) positive relationship was observed between both PLCζ relative fluorescence and relative density with the times taken for both the second cell division (CC2) (r = 0.26 and r = 0.43, respectively) and the third cell division (S2) (r = 0.26). Examination of localization patterns also indicated significant correlations between the presence or absence of sperm PLCζ and CC2 (r = 0.27 and r = -0.27, respectively; P ≤ 0.025). Human sperm PLCζ levels were at their highest in the ideal times of CC2 (8-12 h) compared to time ranges outside the ideal timeframe (<8 and >12 h) where levels of human sperm PLCζ were lower. Following assignment of PLCζ level thresholds, quantification revealed a significantly higher (P ≤ 0.05) rate of successful pregnancy in values larger than the assigned cut-off for both relative fluorescence (19% vs 40%, respectively) and relative density (8% vs 54%, respectively). Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported (Kashir et al., 2021) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones. LIMITATIONS, REASONS FOR CAUTION: The human population examined was relatively small, and should be expanded to examine a larger multi-centre cohort. Infertility conditions are often multivariable, and it was not possible to evaluate all these in human patients. However, our mutant Plcζ mouse experiments do suggest that PLCζ plays a significant role in early embryo development. WIDER IMPLICATIONS OF THE FINDINGS: We found that minimal levels of PLCζ within a specific range were required for optimal early embryogenesis, correlating with increased pregnancy. Levels of sperm PLCζ below specific thresholds were associated with ineffective embryogenesis and lower pregnancy rates, despite eliciting successful fertilization in both mice and humans. To our knowledge, this represents the first time that PLCζ levels in sperm have been correlated to prognostic measures of embryogenic efficacy and pregnancy rates in humans. Our data suggest for the first time that the clinical utilization of PLCζ may stand to benefit not just a specific population of male infertility where oocyte activation is completely deficient (wherein PLCζ is completely defective/abrogated), but also perhaps the larger population of couples seeking fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): J.K. is supported by a faculty start up grant awarded by Khalifa University (FSU-2023-015). This study was also supported by a Healthcare Research Fellowship Award (HF-14-16) from Health and Care Research Wales (HCRW) to J.K., alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST) for J.K. and A.M.A. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

ASTL is mutated in female infertility.

Female infertility is a relatively common phenotype with a growing number of single gene causes although these account for only a minority of cases. Here, we report a consanguineous family in which adult females who are homozygous for a truncating variant in ASTL display markedly reduced fertility in a pattern strikingly similar to Astl-/- female mice. ASTL encodes ovastacin, which is known to trigger zona pellucida hardening (ZPH) as part of the cortical reaction upon fertilization. ZPH is required for normal early embryonic development and its absence can be caused by pathogenic variants in other zona pellucida proteins that result in a similar infertility phenotype in humans and mouse. This is the first report of ASTL-related infertility in humans and suggests that the inclusion of ASTL in female infertility gene panels is warranted.

Recurrent spontaneous oocyte activation causes female infertility.

PURPOSE: Spontaneous oocyte activation (SOA) is a recently classified phenomenon characterized by the presence of a single pronucleus immediately following oocyte retrieval, without the apparent involvement of sperm. SOA currently remains poorly understood in humans, with no clear genetic or pathological factor(s). Herein, we report two separate cases of recurrent spontaneous oocyte activation, investigating potential avenues to identify causative etiology. METHODS: Two patients with several cycles with SOA have undergone further genetic and embryologic investigation to reveal underlying causes for SOA and provide a treatment if possible. RESULTS: One case was a patient with recurrent pregnancy loss and the other was diagnosed as unexplained infertility. In the first case, 61 out of 69 oocytes retrieved exhibited SOA in five cycles while in the second case 44 out of 49 oocytes exhibited SOA in five cycles. Oocytes were injected with sperm; embryo development and presence of paternal contribution were investigated. No pregnancy is ensued following embryo transfer in both patients. Time-lapse imaging of embryogenesis from the second case did not reveal even momentary second pronucleus appearance. We also performed clinical whole exome sequencing for both patients but did not identify any disease-causing variant. CONCLUSION: Patients with SOA suffer from infertility. Our results indicate that more investigation is required to understand the etiology of SOA in humans concentrating on the molecular mechanisms that underpin regulation of oocyte activation and calcium dynamics need to be investigated to fully understand, and perhaps in the future rectify, recurrent SOA.

A genomics approach to females with infertility and recurrent pregnancy loss.

Infertility affects 10% of reproductive-age women and is extremely heterogeneous in etiology. The genetic contribution to female infertility is incompletely understood, and involves chromosomal and single-gene defects. Our aim in this study is to decipher single-gene causes in infertile women in whom endocrinological, anatomical, and chromosomal causes have been excluded. Our cohort comprises women with recurrent pregnancy loss and no offspring from spontaneous pregnancies (RPL, n = 61) and those who never achieved clinical pregnancy and were referred for in vitro fertilization [primary infertility (PI), n = 14]. Whole-exome sequencing revealed candidate variants in 14, which represents 43% of those with PI and 13% of those with RPL. These include variants in previously established female infertility-related genes (TLE6, NLRP7, FSHR, and ZP1) as well as genes with only tentative links in the literature (NLRP5). Candidate variants in genes linked to primary ciliary dyskinesia (DNAH11 and CCNO) were identified in individuals with and without systemic features of the disease. We also identified variants in genes not previously linked to female infertility. These include one homozygous variant each in CCDC68, CBX3, CENPH, PABPC1L, PIF1, PLK1, and REXO4, which we propose as candidate genes for infertility based on their established biology or compatible animal models. Our study expands the contribution of single genes to the etiology of PI and RPL, improves the precision of disease classification at the molecular level, and offers the potential for future treatment and development of human genetics-inspired fertility regulators.

Female Infertility Caused by Mutations in the Oocyte-Specific Translational Repressor PATL2.

Infertility is a relatively common disorder of the reproductive system and remains unexplained in many cases. In vitro fertilization techniques have uncovered previously unrecognized infertility phenotypes, including oocyte maturation arrest, the molecular etiology of which remains largely unknown. We report two families affected by female-limited infertility caused by oocyte maturation failure. Positional mapping and whole-exome sequencing revealed two homozygous, likely deleterious variants in PATL2, each of which fully segregates with the phenotype within the respective family. PATL2 encodes a highly conserved oocyte-specific mRNP repressor of translation. Previous data have shown the strict requirement for PATL2 in oocyte-maturation in model organisms. Data gathered from the families in this study suggest that the role of PATL2 is conserved in humans and expand our knowledge of the factors that are necessary for female meiosis.

A genomics approach to male infertility.

PURPOSE: Male infertility remains poorly understood at the molecular level. We aimed in this study to investigate the yield of a "genomics first" approach to male infertility. METHODS: Patients with severe oligospermia and nonobstructive azoospermia were investigated using exome sequencing (ES) in parallel with the standard practice of chromosomal analysis. RESULTS: In 285 patients, 10.5% (n = 30) had evidence of chromosomal aberrations while nearly a quarter (n = 69; 24.2%) had a potential monogenic form of male infertility. The latter ranged from variants in genes previously reported to cause male infertility with or without other phenotypes in humans (24 patients; 8.4%) to those in novel candidate genes reported in this study (37 patients; 12.9%). The 33 candidate genes have biological links to male germ cell development including compatible mouse knockouts, and a few (TERB1 [CCDC79], PIWIL2, MAGEE2, and ZSWIM7) were found to be independently mutated in unrelated patients in our cohort. We also found that male infertility can be the sole or major phenotypic expression of a number of genes that are known to cause multisystemic manifestations in humans (n = 9 patients; 3.1%). CONCLUSION: The standard approach to male infertility overlooks the significant contribution of monogenic causes to this important clinical entity.

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases.

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

Exposure to phthalates in couples undergoing in vitro fertilization treatment and its association with oxidative stress and DNA damage.

This prospective study of 599 couples seeking fertility treatment and who were recruited between 2015 and 2017 was conducted to (a) explore the associations between phthalate exposure and in vitro fertilization (IVF) outcomes; and (b) examine the implication of oxidative stress as a mediator of these. We measured eight phthalate metabolites in two spot urine samples; oxidative stress biomarkers such as malondialdehyde, 8-hydroxy-2-deoxyguanosine, hydrogen peroxide, catalase (CAT), and total antioxidant capacity in follicular fluid and seminal plasma. We also examined DNA damage in sperm and granulosa cells. Couples were exposed to a broad range of phthalate compounds and seven metabolites were detected in over 94% of the urine samples, whereas monobenzyl phthalate was found in only 24% of women and 26% of men. Our results showed high levels of seven urinary phthalate metabolites (except monobenzyl phthalate) and a notable increase in many oxidative stress markers in both follicular fluid and seminal plasma. However, their associations with exposure were rather limited. Multivariate binomial regression modeling showed higher levels of follicular CAT levels reduced the probability of fertilization rate (≤ 50%) [Adjusted relative risk (RRadj) = 0.52, p = 0.005] and unsuccessful live birth (RRadj = 0.592, p = 0.023). We observed a 46% decrease in the probability of clinical pregnancy in association with an elevated percentage of DNA in the tail (RRadj = 0.536, p = 0.04). There was a 32% and 22% increase in the probability of clinical pregnancy and unsuccessful live birth associated with higher levels of mono-(2-ethylhexyl) phthalate (RRadj = 1.32, p = 0.049) and monoethyl phthalate (RRadj = 1.22, p = 0.032) in women, respectively. In contrast, the probability of clinical pregnancy reduced by 20% with higher levels of mono-(2-ethyl-5-carboxypentyl) phthalate (RRadj = 0.797, p = 0.037) and 19.6% with mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) (RRadj = 0.804, p = 0.041) in men. Other oxidative stress biomarkers or urinary phthalate metabolites showed suggestive relationships with certain IVF outcomes. Lastly, our results demonstrated that elevated levels of CAT in follicular fluid might have a positive impact on fertilization rate ≥ 50% and successful live birth. CAT seems to play a potential role in mediating the relationship between the risk of poor fertilization rate and MEOHP and mono-isobutyl phthalate. Additional data are required to understand the clinical implications of oxidative stress and its contribution to the reproductive toxicity of phthalate exposure.

The extent and predictors of phthalate exposure among couples undergoing in vitro fertilization treatment.

Phthalates are chemicals used as plasticizers and solvents in many consumer products but are suspected of disrupting the endocrine system and are known for their reproductive/developmental health risks. This study examined the extent and predictors of phthalate exposure among 599 couples undergoing in vitro fertilization. A questionnaire was administered to obtain sociodemographic, health, and lifestyle data, and two spot urine samples were collected from the couples to analyze eight phthalate metabolites, cotinine (COT) as a smoking index, and creatinine to adjust for urine dilution. Seven phthalate metabolites were detected in > 94% of the urine samples, and monobenzyl phthalate (MBzP) was found in 24% of the women and 26% of their male partners. Median phthalate levels were highest for monoethyl phthalate (MEP), at 333.26 µg/l in women and 290 µg/l in male partners, and lowest for MBzP, at 1.17 µg/l in women and 1.14 µg/l in male partners. Correlation coefficients of ≥ 0.4 between the women and their male partners for the eight urinary phthalate metabolites may indicate a shared source of exposure. A multivariate regression model was used to assess the association between predictors and each urinary phthalate metabolite. Several potential predictors for the variations in specific urinary phthalate metabolites were identified, including the body mass index, age, socioeconomic status, and regional distribution for both women and their male partners but with slightly different patterns. Women with a history of breastfeeding, using bottled water for cooking and storing food in plastic bags had lower MEP (8.7%), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) (9.2%), and both mono-iso-butyl phthalate and MECPP (8.2 and 8.1%). A history of contraceptive use was associated with an increase in MECPP (8.7%), mono-(2-ethyl-5-hydroxyhexyl) phthalate (11.4%), mono-(2-ethyl-5-oxohexyl) phthalate (7.6%), and the molar sum of bis (2-ethylhexyl) phthalate metabolites (8.9%). Urinary COT levels were associated with an increase of 10-16% in all urinary metabolites in women but of only 10.5% in mono-(2-ethylhexyl) phthalate in male partners. More than 95% of the couples reported the use of cosmetics, perfumes, and personal-care products, but we were not able to find associations with urinary phthalate metabolites, perhaps due to their short half-lives. MEP levels associated with the use of household cleaning products were 11.2% higher in male partners. Our levels were generally higher than those reported elsewhere, perhaps due to different lifestyles, cultural practices, dietary habits, use of personal-care products, and governmental legislation.

A retrospective analysis of the treatment results of 1557 patients with acute cholecystitis.

OBJECTIVE: To analyse outcomes of variable management strategies for the treatment of Acute Cholecystitis in relation to morbidity, mortality and conversion to open surgery. METHODS: The retrospective study was conducted at Firat University Hospital, Turkey and comprised records of Acute Cholecystitis patients admitted between 2005 and 2011. Patients were divided into subgroups according to admission time as well as American Society of Anaesthesiologists score. The outcomes of early cholecystectomy, interval cholecystectomy, delayed cholecystectomy, 'cooling-off' therapy and percutaneous cholecystostomy were evaluated. Mortality, morbidity, and conversion to open surgery were calculated as measures of success. Data was analysed using SPSS. RESULTS: Of the 1557 patients, 1052(67.6%) were female. The overall mean age was 42.4±14.7 years. Success rates of 'cooling-off' therapy and percutaneous cholecystostomy were 89.3% and 96.3%, respectively. The conversion rate following delayed cholecystectomy was 30%, which was higher than that of both early and interval cholecystectomy (0.2% and 0%, respectively; p<0.001 each). Mortality and morbidity rates of delayed cholecystectomy (57.1% and 7.1%, respectively) were also significantly higher than early and interval cholecystectomy (5% and 0.1%; 5.6 and 0%, respectively). CONCLUSIONS: Early laparoscopic cholecystectomy and interval cholecystectomy shared similar outcomes and rates of efficacy. Percutaneous cholecystostomy was a successful treatment option for high-risk patients, while delayed cholecystostomy correlated to the highest rates of conversion to open surgery, mortality and morbidity.

Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro.

PURPOSE: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. METHODS: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. RESULTS: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. CONCLUSION: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.

Gene expression profiling of granulosa cells from PCOS patients following varying doses of human chorionic gonadotropin.

PURPOSE: Human chorionic gonadotrophin (hCG) has been used to induce ovulation and oocyte maturation. Although the most common dose of hCG used in IVF is 10,000 IU, there are reports that suggest 5,000 IU is sufficient to yield similar results. The objective of this study is to evaluate the dose dependent differences in gene expression of granulosa cells following various doses of hCG treatment. METHODS: Patients with polycystic ovarian syndrome (PCOS) were stimulated for IVF treatment. The hCG injection was either withheld or given at 5,000 or 10,000 IU. Granulosa cells from the follicular fluids have been collected for RNA isolation and analyzed using Affymetrix genechip arrays. RESULTS: Unsupervised hierarchical clustering based on whole gene expression revealed two distinct groups of patients in this experiment. All untreated patients were clustered together whereas hCG-treated patients separated to a different group regardless of the dose. A large number of the transcripts were similarly up- or down-regulated across both hCG doses (2229 and 1945 transcripts, respectively). However, we observed dose-dependent statistically significant differences in gene expression in only 15 transcripts. CONCLUSIONS: Although hCG injection caused a major change in the gene expression profile of granulosa cells, 10,000 IU hCG resulted in minimal changes in the gene expression profiles of granulosa cells as compared with 5,000 IU. Thus, based on our results, we suggest the use of 10,000 IU hCG should be reconsidered in PCOS patients.

Antigen Unmasking Is Required to Clinically Assess Levels and Localisation Patterns of Phospholipase C Zeta in Human Sperm.

Mammalian oocyte activation is initiated by intracellular calcium (Ca2+) oscillations, driven by the testis-specific phospholipase C zeta (PLCζ). Sperm PLCζ analysis represents a diagnostic measure of sperm fertilisation capacity. The application of antigen unmasking/retrieval (AUM) generally enhanced the visualisation efficacy of PLCζ in mammalian sperm, but differentially affected the PLCζ profiles in sperm from different human males. It is unclear whether AUM affects the diagnosis of PLCζ in human sperm. Herein, we examined whether the application of AUM affected the correlation of PLCζ profiles with sperm parameters and fertilisation capacity. PLCζ fluorescence levels and localisation patterns were examined within the sperm of males undergoing fertility treatment (55 patients aged 29-53) using immunofluorescence in the absence/presence of AUM. The changes in PLCζ profiles following AUM were examined in relation to sperm health and fertilisation outcome. AUM enhanced the observable levels and specific localisation patterns of PLCζ in relation to both optimal sperm parameters and fertilisation outcome, without which significant differences were not observed. The extent of the change in levels and localisation ratios of PLCζ was also affected to a larger degree in terms of the optimal parameters of sperm fertility and fertilisation capacity by AUM. Collectively, AUM was essential to accurately assesses PLCζ in human sperm in both scientific and clinical contexts.

Global Transcriptional Profiling of Granulosa Cells from Polycystic Ovary Syndrome Patients: Comparative Analyses of Patients with or without History of Ovarian Hyperstimulation Syndrome Reveals Distinct Biomarkers and Pathways.

Ovarian hyperstimulation syndrome (OHSS) is often a complication of polycystic ovarian syndrome (PCOS), the most frequent disorder of the endocrine system, which affects women in their reproductive years. The etiology of OHSS is multifactorial, though the factors involved are not apparent. In an attempt to unveil the molecular basis of OHSS, we conducted transcriptome analysis of total RNA extracted from granulosa cells from PCOS patients with a history of OHSS (n = 6) and compared them to those with no history of OHSS (n = 18). We identified 59 significantly dysregulated genes (48 down-regulated, 11 up-regulated) in the PCOS with OHSS group compared to the PCOS without OHSS group (p-value < 0.01, fold change >1.5). Functional, pathway and network analyses revealed genes involved in cellular development, inflammatory and immune response, cellular growth and proliferation (including DCN, VIM, LIFR, GRN, IL33, INSR, KLF2, FOXO1, VEGF, RDX, PLCL1, PAPPA, and ZFP36), and significant alterations in the PPAR, IL6, IL10, JAK/STAT and NF-κB signaling pathways. Array findings were validated using quantitative RT-PCR. To the best of our knowledge, this is the largest cohort of Saudi PCOS cases (with or without OHSS) to date that was analyzed using a transcriptomic approach. Our data demonstrate alterations in various gene networks and pathways that may be involved in the pathophysiology of OHSS. Further studies are warranted to confirm the findings.

First successful application of preimplantation genetic diagnosis and haplotyping for congenital hyperinsulinism.

Congenital hyperinsulinism is the most common cause of persistent hypoglycaemia in infancy. Early surgical intervention is usually required to prevent brain damage. The prevention of the transmission to the offspring is important in families carrying the mutated gene. Preimplantation genetic diagnosis (PGD) is an early genetic testing procedure for couples at risk of transmitting inherited diseases. A 36-year-old Saudi woman married to her first cousin with four affected children was referred for PGD. The hyperinsulinism disease was caused by a novel homozygous mutation in the KCNJ11 gene, an arginine 301 to proline (R301P) substitution.PGD was achieved by whole genome amplification followed by mutation detection combined with short tandem repeat identifier analysis in the first cycle and with haplotyping in the second cycle. The first and second cycles resulted in the births of healthy twin girls and a boy, respectively. As far as is known, this is the first application of PGD to hyperinsulinism. A feasible strategy including whole genome amplification followed by direct mutation detection combined with haplotyping is described.Utilizing haplotyping increases the efficiency of PGD diagnosis as well as confirming the genetic diagnosis. It reveals the parental origin of each inherited chromosome.

Desalination and Detoxification of Textile Wastewater by Novel Photocatalytic Electrolysis Membrane Reactor for Ecosafe Hydroponic Farming.

In this study, a novel photoelectrocatalytic membrane (PECM) reactor was tested as an option for the desalination, disinfection, and detoxification of biologically treated textile wastewater (BTTWW), with the aim to reuse it in hydroponic farming. The anionic ion exchange (IEX) process was used before PECM treatment to remove toxic residual dyes. The toxicity evaluation for every effluent was carried out using the Vibrio fischeri, Microtox® test protocol. The disinfection effect of the PECM reactor was studied against E. coli. After PECM treatment, the 78.7% toxicity level of the BTTWW was reduced to 14.6%. However, photocatalytic desalination during treatment was found to be slow (2.5 mg L-1 min-1 at 1 V potential). The reactor demonstrated approximately 52% COD and 63% TOC removal efficiency. The effects of wastewater reuse on hydroponic production were comparatively investigated by following the growth of the lettuce plant. A detrimental effect was observed on the lettuce plant by the reuse of BTTWW, while no negative impact was reported using the PECM treated textile wastewater. In addition, all macro/micronutrient elements in the PECM treated textile wastewater were recovered by hydroponic farming, and the PECM treatment may be an eco-safe wastewater reuse method for crop irrigation.

Oxidative stress and DNA damage status in couples undergoing in vitro fertilization treatment.

This study examined the status of oxidative stress in 599 couples undertaking in vitro fertilization (IVF) treatment and its association with reproductive hormones, smoking, and outcomes. Oxidative stress biomarkers such as malondialdehyde, 8-hydroxy-2-deoxyguanosine, hydrogen peroxide (H2O2), catalase (CAT), and total antioxidant capacity (TAC) were determined in follicular fluid and seminal plasma. Tail moment (TM) was used to evaluate DNA damage in the sperm and granulosa cells. Reproductive hormones in serum and cotinine (COT) in urine, follicular fluid, and seminal plasma samples were determined. Separate multivariate linear regression was used to assess associations between levels of each oxidative stress biomarker and each hormone and smoking parameter (modeled as natural log-transformed). The findings indicate that some oxidative stress and DNA damage biomarkers played a role in disrupting certain reproductive hormones in women and their male partners either by overproducing reactive oxygen species or reducing antioxidant defense capacity. Although women were nonsmokers, COT levels > 50 and 10 µg/L in urine and follicular were observed in 5.7 and 1.7%, respectively. Levels of follicular fluid COT were positively associated with H2O2 and TM. We used log-binomial multivariate regression to estimate relative risks for the association between oxidative stress/DNA damage and IVF binary outcomes (fertilization rate > 50%, biochemical pregnancy, clinical pregnancy, and live birth). An increase in the CAT levels of follicular fluid was associated with a 48 and 41% decrease in the risk of poor fertilization rate (≤50%) and unsuccessful live birth, respectively. After the models were adjusted for hormonal factors, the associations remained the same, except that the elevated TAC in follicular fluid became significantly associated with a decrease of 42% in the risk of poor fertilization rate (≤50%). The higher antioxidant activity (CAT and TAC) in follicular fluid might positively impact specific IVF outcomes. LAY SUMMARY: Oxidative stress occurs when antioxidant molecules are insufficient in the body to destroy free radicals that can damage the cells, proteins and DNA, causing different health conditions, including infertility. The role of oxidative stress in female infertility has not received as much attention as male infertility, and research is still limited. This study explored whether the overproduction of free radicals can impact the success of in vitro fertilization (IVF) treatment using several biological markers such as hydrogen peroxide, catalase, and total antioxidant capacity. Our findings revealed that the high antioxidant levels in the fluid surrounding the egg were linked with a high fertilization rate. Additionally, oxidative stress status in couples was associated negatively with several reproductive hormones and smoking status. Biomarkers of oxidative stress and DNA damage might have potential applications in evaluating IVF patients' clinical characteristics such as causes of infertility, hormonal profile, fertilization rate, implantation and live birth.

The effect of caffeine consumption on the success rate of pregnancy as well various performance parameters of in-vitro fertilization treatment.

BACKGROUND: This study evaluated the effect of caffeine consumption on the success rate of pregnancy and various in vitro fertilization (IVF) performance parameters. MATERIAL/METHODS: Serum and follicular fluid samples were collected from 619 women undergoing IVF treatment (2002-2003). Caffeine assessment was based on measuring the levels of caffeine in serum and follicular fluid and on the number of coffee or tea or caffeinated drinks consumed per day. RESULTS: A total of 97.3% of participants reported the consumption of caffeinated drinks such as coffee, tea and soft drinks. Their average caffeine consumption was 455.82 mg/day (range: 3.71-3561 mg/day). Coffee was the primary source of caffeine intake. The average caffeine levels in serum (0.913 µg/ml) were significantly higher than in follicular fluid (0.701 µg/ml). After controlling for various potential confounding variables, no association was found between coffee or tea consumption and the success rate of pregnancy. Looking at the effect of caffeine consumption on the IVF performance parameters, we found that the number of eggs decreased as the caffeine serum levels increased (P=0.011). An increase in coffee consumption was positively associated with the number of aborted pregnancy (P=0.007), while the number of good embryo decreased with high tea consumption (P=0.015). CONCLUSIONS: Though no association was seen between coffee or tea consumption and pregnancy rate, this study is the first to report that caffeine can reach the follicular fluid and there is a suggestive evidence of its possible harmful role on the consequences of reproductive process. This clearly warrants further investigation.

Primary hepatic actinomycosis.

OBJECTIVES: To present a case of primary hepatic actinomycosis. CLINICAL PRESENTATION: A-40-year-old man was admitted to the general surgery clinic with a 1-month history of abdominal pain and weight loss. Liver transaminase, bilirubin levels and white blood cell counts were increased. Abdominal ultrasound and CT revealed cystic lesions with necrotic debris involving the posterior segment of the right lobe of the liver and the medial segment of the left lobe. INTERVENTION: The patient underwent surgery under general anesthesia. On exploration, three cavities were found within the liver containing necrotic material. Surgical debridement and drainage was performed. Histopathological examination revealed actinomycotic colonies with a surrounding suppurative granulomatous reaction. The patient was treated with penicillin for 3 months. CONCLUSION: This case showed that histological examination of biopsy or surgical material or anaerobic cultures was needed for definitive diagnosis and that hepatic actinomycosis should be included in the differential diagnosis of solitary or multiple hypodense liver lesions.

Levels of DNA adducts in the blood and follicular fluid of women undergoing in vitro fertilization treatment and its correlation with the pregnancy outcome.

This study is designed to investigate the impact of DNA damage on pregnancy and fertilization rate outcome in a sub-sample of women undergoing IVF treatment. Blood and follicular fluid samples (n = 60) were analyzed for DNA adducts. While no BPDE-DNA adducts were detected, other unknown lipophilic adducts were seen in blood and follicular fluid. Women who failed to achieve pregnancy had higher DNA adducts in follicular fluid than those who succeeded (p < 0.05). Follicular fluid cotinine levels were associated with DNA adduct levels in blood and follicular fluid (p < 0.05). Evaluation of DNA damage resulting from oxidative stress could have a role in predicting IVF success rate.