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.

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.

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.

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.

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.

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.

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.

Phospholipase C zeta profiles are indicative of optimal sperm parameters and fertilisation success in patients undergoing fertility treatment.

BACKGROUND: Oocyte activation is driven by intracellular calcium (Ca2+ ) oscillations induced by sperm-specific PLCζ, abrogation of which causes oocyte activation deficiency in humans. Clinical PLCζ investigations have been limited to severe male infertility conditions, while PLCζ levels and localisation patterns have yet to be associated with general sperm viability. MATERIALS AND METHODS: PLCζ profiles were examined within a general population of males attending a fertility clinic (65 patients; aged 29-53), examining PLCζ throughout various fractions of sperm viability. Male recruitment criteria required a minimum sperm count of 5 × 106 spermatozoa/mL, while all female patients included in this study yielded at least five oocytes for treatment. Sperm count, motility and semen volume were recorded according to standard WHO reference guidelines and correlated with PLCζ profiles examined via immunoblotting and immunofluorescence. Appropriate fertility treatments were performed following routine clinical standard operating protocols, and fertilisation success determined by successful observation of second polar body extrusion. RESULTS AND DISCUSSION: Four distinct PLCζ patterns were observed at the equatorial, acrosomal + equatorial regions of the sperm head, alongside a dispersed pattern, and a population of spermatozoa without any PLCζ. Acrosomal + equatorial PLCζ correlated most to sperm health, while dispersed PLCζ correlated to decreased sperm viability. Total levels of PLCζ exhibited significant correlations with sperm parameters. PLCζ variance corresponded to reduced sperm health, potentially underlying cases of male sub-fertility and increasing male age. Finally, significantly higher levels of PLCζ were exhibited by cases of fertilisation success, alongside higher proportions of Ac + Eq, and lower levels of dispersed PLCζ. CONCLUSIONS: PLCζ potentially represents a biomarker of sperm health, and fertilisation capacity in general cases of patients seeking fertility treatment, and not just cases of repeated fertilisation. Further focused investigations are required with larger cohorts to examine the full clinical potential of PLCζ.

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.

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.

Cell-free DNA levels of twins and sibling pairs indicate individuality and possible use as a personalized biomarker.

Cell-free DNA (cfDNA) in the human blood circulation has been under investigation since its initial observation in 1948. Plasma cfDNA is known to be significantly elevated in diseased people. Due to possible variation in the population, evaluating cfDNA as a non-invasive biomarker at disease onset alone may not be sensitive enough to accurately diagnose diseases, particularly early stage cancers on a personal level. To understand the factors that define the cfDNA levels on the personal level and for better use as a non-invasive biomarker, we isolated cfDNA from the plasma of healthy individuals with varying degrees of genetic and/or environmental similarities (monozygotic twins, dizygotic twins, sibling pairs, and unrelated individuals) as well as from patients with varying stages of breast and ovarian cancer undergoing treatment. Cell-free DNA levels were quantified by a fluorometer (ng/ml) and/or real-time PCR (copies/ml). The associations between individuals with various degrees of genetic and/or environmental similarities and their plasma cfDNA levels were evaluated. The ACE model (A = additive genetic, C = common environment, and E = specific environmental factors) was used to determine the proportion of each factor on the cfDNA levels. We found a high correlation (r = 0.77; p < 0.0001) in plasma cfDNA levels between monozygotic twins (n = 39). However, the correlation was gradually reduced to moderate (r = 0.47; p = 0.016) between dizygotic twins (n = 13) and low correlation (r = 0.28; p = 0.043) between sibling pairs (n = 26). The ACE model analysis showed that the plasma cfDNA level of a given healthy individual is influenced both by genetic and the environmental components in similar proportions (53% and 47%, respectively; A = 53%, C = 22.5%, E = 24.5%). Moreover, while age had no effect, gender significantly influenced the individual's plasma cfDNA level. As expected, cfDNA levels were significantly higher in both breast (n = 26) (p<0.0001) and ovarian (n = 64) (p<0.0001) cancer patients compared to the healthy individuals. Our study demonstrated that both genome and environmental factors modulate the individual's cfDNA level suggesting that its diagnostic sensitivity may be improved only if the person's cfDNA level is known prior to disease presentation.

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.

Couples exposure to phthalates and its influence on in vitro fertilization outcomes.

This prospective study examined the associations between the levels of eight urinary phthalate metabolites in 599 couples and in vitro fertilization (IVF) outcomes. We used log-binomial multivariate regression to estimate relative risks (RR) for the association between phthalate concentration and IVF binary outcomes (fertilization rate >50%, biochemical pregnancy, clinical pregnancy and live birth) for each woman after adjusting the model for the concentration in a male partner and each relevant confounders. RR was expressed per unit increase in log-transformed urinary metabolite concentration. The percentage of bis-2-ethylhexyl phthalate (DEHP) metabolites excreted as mono-2-ethylhexyl phthalate (MEHP) was calculated as %MEHP. Urinary MEHP in women was associated with an increased risk of biochemical pregnancy (RR = 1.35; p = 0.04), failed clinical pregnancy (RR = 1.56; p = 0.006) and live birth (RR = 1.54; p = 0.011). An increase in monoethyl phthalate was associated with a high risk of failed clinical pregnancy (RR = 1.25; p = 0.03) and live birth (RR = 1.35; p = 0.006). An increase in %MEHP was associated with an increase in the risk of biochemical pregnancy (RR = 1.55; p = 0.05), failed clinical pregnancy (RR = 1.73; p = 0.02) and live birth (RR = 1.65; p = 0.046). Our results demonstrated that exposure to some phthalates may adversely affect IVF outcomes, particularly when couples' exposure was jointly modeled, emphasizing the importance of a couple-based approach in assessing fertility outcomes. The associations between IVF outcomes and DEHP metabolites were stronger in women whose %MEHP was >75th percentile which may be due to their less efficient metabolism and excretion of DEHP and/or MEHP.

The relationships between urinary phthalate metabolites, reproductive hormones and semen parameters in men attending in vitro fertilization clinic.

Evidence from previous studies has shown that phthalates may play a role in male reproductive function; however, results are still inconclusive, and the mechanism remains unclear. In this study, we first assessed whether exposure to phthalates is associated with altered reproductive hormones and semen parameters in 599 men attending an in vitro fertilization clinic. Secondly, we evaluated whether reproductive hormones could play a mediating role in the association between phthalates and sperm parameters. Eight phthalate metabolites were measured in two different spot urine samples: mono­n­butyl phthalate, mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP), monobenzyl phthalate, and four oxidative metabolites of di­(2­ethylhexyl) phthalate (DEHP) [i.e., mono­(2­ethylhexyl) phthalate (MEHP), mono­(2­ethyl­5­hydroxyhexyl) phthalate (MEHHP), mono­(2­ethyl­5­oxohexyl) phthalate (MEOHP), and mono­(2­ethyl­5­carboxypentyl) phthalate (MECPP)]. Semen parameters (concentration, volume, motility, and morphology) and reproductive hormones, i.e., follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone, estradiol (E2), testosterone (TEST) and prolactin (PROL) were also determined and considered the main study outcomes. Separate multivariate linear regression was used to assess associations between levels of each urinary phthalate metabolite, molar sum of DEHP metabolites (∑DEHP), percentage of MEHP to ∑DEHP (%MEHP), and each outcome (natural log-transformed). Inverse associations were observed between TEST and MiBP (ß = -0.099), FSH and MEHHP (ß = -0.087), and PROL and MEOHP (ß = -0.102), while a positive relationship was seen between E2 and MEP (ß = 0.098). %MEHP was associated positively with FSH (ß = 0.118) and LH (ß = 0.099), but negatively with TEST/LH (ß = -0.086) and TEST/E2 (ß = -0.109). Sperm concentration was associated positively with MECPP (ß = 0.131), MEHHP (ß = 0.117), MEOHP (ß = 0.107) and ∑DEHP (ß = 0.111), but negatively with %MEHP (ß = -0.135). All p-values were <0.05. Sobel's test indicated that FSH mediated significantly up to 60% of the positive relationship between sperm concentration and MEHHP, while FSH and LH mediated respectively 15 and 12% of the inverse association between sperm concentration and %MEHP. Further research on this topic is warranted.

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.

Intramuscular progesterone (Gestone) versus vaginal progesterone suppository (Cyclogest) for luteal phase support in cycles of in vitro fertilization-embryo transfer: patient preference and drug efficacy.

BACKGROUND: The requirement for luteal phase support (LPS) in stimulated IVF cycles is well established, however drug choice, and route of administration and duration of use are not. This report evaluates patients' preference and satisfaction by using either vaginal or intramuscular (IM) progesterone (P) supplementation for luteal phase support after in vitro fertilization and embryo transfer (IVF-ET). METHODS: It is a prospective cohort study done in a reproductive and infertility unit in a tertiary care hospital from March 2013 through February 2015 for four hundred and nine patients undergoing IVF-ET. Patients were allowed to choose either vaginal or IM P for LPS. Patient preference and satisfaction, as well as differences in clinical pregnancy rates between the two groups were assessed at one or two time points throughout the study. RESULTS: There were no statistically significant differences in the patients' characteristics and clinical outcomes between the two groups. There were 88 pregnancies (38.8%) among patients treated with vaginal p and 62 pregnancies (34%) among IM P patients. Average satisfaction score at the pregnancy test and ultrasound (U/S) visits was similar between both groups. CONCLUSIONS: Patients' satisfaction and pregnancy rates were similar between vaginal and IM P supplementation.

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.

Hepatitis B and C virus prevalence in couples attending an in vitro fertilization clinic in a tertiary care hospital in Saudi Arabia: comparison with ten years earlier.

BACKGROUND: Viral hepatitis B (HBV) and C (HCV) are a major public health problem in Saudi Arabia. Recent data has indicated a major reduction in viral hepatitis prevalence in Saudi population. However, there is limited data for infertile Saudi couples. OBJECTIVES: To determine the prevalence of HCV and HBV attending an in vitro fertilization (IVF) clinic in Saudi Arabia between 2012 and 2015 to compare with the prevalence 10 years earlier in the same center. DESIGN: Retrospective prevalence study. SETTING: Tertiary care center in Riyadh. PATIENTS AND METHODS: Data on the prevalence of HBV and HCV was collected on all couples seen at the IVF unit between 2002-2005 and 2012-2015. MAIN OUTCOME MEASURE(S): Prevalence of HBV and HCV. RESULTS: In 4442 patients during 2002-2005 and 5747 patients during 2012-2015, the prevalence of HBV was significantly less in 2012-2015 compared with 2002-2005 (1.67% [97 patients] vs 4.7% [210 patients], P < .0001), respectively, but HCV prevalence was similar for the two periods (0.7% for both periods) (P=.887). The hepatitis B seroprevalence rate was higher in males compared to females during 2002-2005 (6.3% vs 3.1%) (P < .0001) and 2012-2015 (2.4% vs 1.1% ) (P < .0001), respectively. CONCLUSION: The significant drop in HBV prevalence was most likely due to the introduction of the vaccination program in 1989, while reasons for HCV prevalence remaining unchanged are unclear. LIMITATION: No data on confounding factors that may have affected the prevalence.

Association of blood groups with ovarian reserve and outcome of in vitro fertilization treatment.

BACKGROUND: The association between ABO blood groups and ovarian reserve in infertile patients has been a point of controversy. OBJECTIVES: The aim of this study was to assess the correlation of certain blood groups with ovarian reserve and response to treatment in patients undergoing infertility treatment. DESIGN: Retrospective medical record review. SETTING: Infertility clinic in the assisted reproductive technology (ART) unit at King Faisal Specialist Hospital and Research Center, Riyadh Saudi Arabia. PATIENTS AND METHODS: All patients under 40 years of age who attended the infertility clinic at a tertiary care centre in 2010 and underwent in vitro fertilization (IVF) treatment in 2010 and 2011 were divided into groups according to blood type, and clinical parameters were compared. MAIN OUTCOME MEASURE(S): The association between blood groups and ovarian reserve using day 3 luteinzing hormone (LH) and follicular stimulating hormone (FSH) levels, and antral follical count (AFC). RESULTS: In 424 patients who underwent 566 IVF cycles, age, LH, FSH and AFC were similar among the different blood groups (P=.9, .1, .5, respectively). with controlled ovarian stimulation, no difference was observed among the four groups in menopausal gonadotrophin (hMG) dose or the duration of stimulation. The number of oocytes retrieved, fertilization rate, cleavage rate, and number of embryos transferred were similar. There was no difference in the cancellation rate or pregnancy rate among the groups. CONCLUSION: There was no significant association between blood type and ovarian reserve or response during IVF treatment in our population. LIMITATIONS: Anti-Mullerian hormone levels are best correlated with ovarian reserve testing. Unavailability of AMH levels. Retrospective design.