The press-assisted fusion scheme significantly mitigates the quantity of HVJ-E required in mitochondrial replacement techniques.

In brief: The impact of HVJ-E employed in mitochondrial replacement techniques (MRTs) on embryonic development remains uncertain. This study has exhibited the influence of HVJ-E utilized in MRTs on embryonic development and has devised a novel HVJ-E-induced fusion approach to curtail the amount of HVJ-E employed in MRTs. Abstract: Mitochondrial replacement techniques (MRTs) provide a viable option for women carrying pathogenic mitochondrial DNA (mtDNA) variants to conceive disease-free offspring with a genetic connection. In comparison to electrofusion, HVJ-E-induced fusion has been identified as the most promising approach for clinical translation of MRTs due to its absence of electrical interference. However, despite confirmation of the absence of RNA activity in HVJ-E, a reduction in blastocyst quality has been observed in various MRTs studies utilizing the HVJ-E-induced fusion scheme. Recent investigations have revealed a dose-dependent elevation of reactive oxygen species (ROS) levels in various cancer cells incubated with HVJ-E. However, the impact of HVJ-E as a sole determinant on embryonic development in MRTs remains unverified. This investigation establishes that the augmented concentration of HVJ-E utilized in the conventional HVJ-E fusion protocol is an autonomous variable that influences embryonic development in MRTs. This effect may be attributed to amplified DNA damage resulting from heightened levels of ROS in reconstructed embryos. To mitigate the presence of HVJ-E in reconstructed zygotes while maintaining optimal fusion efficiency in MRTs, a novel HVJ-E-induced fusion approach was devised, namely, press-assisted fusion. This technique offers potential advantages in reducing detrimental factors that impede embryo development in MRTs.

A delayed ovulation of progestin-primed ovarian stimulation (PPOS) by downregulating the LHCGR/PGR pathway.

Progestin-primed ovarian stimulation (PPOS) is a new ovulation stimulation protocol, and its role in ovulation and regulatory mechanism is unclear. The clinical PPOS protocol was simulated in mice. The ovulated oocytes, estradiol, progesterone, and luteinizing hormone (LH) levels were analyzed at different hours after trigger. mRNA extraction and real-time PCR, hematoxylin and eosin staining, and immunofluorescence of ovaries were used to explore the involved signaling pathways. The PPOS group had a delayed ovulation at 12.5 h after trigger. Its suppressed LH level reduced the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) on the preovulatory follicles before trigger and significantly decreased the following progesterone synthesis, blood progesterone level, and progesterone receptor (PGR) expression within 4-6 h after trigger. Furthermore, the important ovulatory genes regulated by PGR including ADAMTS-1, VEGF-A, and EDN2 were downregulated, ultimately delaying the ovulation. PPOS suppresses the LH level before trigger and decreases the synthesis of progesterone after trigger, thus delaying the ovulation by downregulating the LHCGR-PGR pathway.

Metabolomics analysis of follicular fluid in ovarian endometriosis women receiving progestin-primed ovary stimulation protocol for in vitro fertilization.

This study aimed to investigate the metabolite profile and inflammatory state of follicular fluid (FF) in women with stage III-IV ovarian endometriosis (OE) who underwent in vitro fertilization (IVF). A cohort of 20 consecutive patients with OE were recruited and received progestin-primed ovary stimulation (PPOS) protocol (study group), while another 20 OE patients received one-month ultra-long term protocol (control group) for IVF in this prospective, nonrandomized study. FF samples were obtained from dominant follicles during oocyte retrieval, and liquid chromatography-mass spectrometry (LC-MS) was used to investigate the metabolites profile of FF. Results showed that significant increases in the levels of proline, arginine, threonine, and glycine in patients who received PPOS protocol compared to the control group (P < 0.05). A panel of three metabolites (proline, arginine, and threonine) was identified as specific biomarkers of OE patients using PPOS protocol. Additionally, levels of interleukin-1ß, regulated on activation, normal T cell expressed and secreted, and tumor necrosis factor-α markedly decreased in women who received PPOS protocol compared to the control group (P < 0.05). In conclusion, PPOS protocol regulates the metabolism of several amino acids in the FF, which may play critical roles in the oocyte development and blastocyst formation, and their specific mechanism should be further elucidated.

Optogenetic stimulation of Kiss1ARC terminals in the AVPV induces surge-like luteinizing hormone secretion via glutamate release in mice.

Kisspeptin neurons are mainly located in the arcuate (Kiss1ARC, vis-à-vis the GnRH pulse generator) and anteroventral periventricular nucleus (Kiss1AVPV, vis-à-vis the GnRH surge generator). Kiss1ARC send fibre projections that connect with Kiss1AVPV somata. However, studies focused on the role of Kiss1ARC neurons in the LH surge are limited, and the role of Kiss1ARC projections to AVPV (Kiss1ARC→AVPV) in the preovulatory LH surge is still unknown. To investigate its function, this study used optogenetics to selectively stimulate Kiss1ARC→AVPV and measured changes in circulating LH levels. Kiss1ARC in Kiss-Cre-tdTomato mice were virally infected to express channelrhodopsin-2 proteins, and optical stimulation was applied selectively via a fibre optic cannula in the AVPV. Sustained 20 Hz optical stimulation of Kiss1ARC→AVPV from 15:30 to 16:30 h on proestrus effectively induced an immediate increase in LH reaching peak surge-like levels of around 8 ng/ml within 10 min, followed by a gradual decline to baseline over about 40 min. Stimulation at 10 Hz resulted in a non-significant increase in LH levels and 5 Hz stimulation had no effect in proestrous animals. The 20 Hz stimulation induced significantly higher circulating LH levels on proestrus compared with diestrus or estrus, which suggested that the effect of terminal stimulation is modulated by the sex steroid milieu. Additionally, intra-AVPV infusion of glutamate antagonists, AP5+CNQX, completely blocked the increase on LH levels induced by Kiss1ARC→AVPV terminal photostimulation in proestrous animals. These results demonstrate for the first time that optical stimulation of Kiss1ARC→AVPV induces an LH surge-like secretion via glutamatergic mechanisms. In conclusion, Kiss1ARC may participate in LH surge generation by glutamate release from terminal projections in the AVPV.

The comparison of two different protocols ultra-long versus medroxyprogesterone acetate in women with ovarian endometriosis: a prospective randomized controlled trial.

BACKGROUND: This study aimed to investigate the medroxyprogesterone acetate (MPA) + HMG protocol vs ultra-long gonadotrophin releasing hormone (GnRH) agonist protocol in patients with advanced ovarian endometriosis who received in vitro fertilization (IVF). METHODS: Three hundred patients with advanced ovary endometriosis who underwent IVF were included, and embryological and clinical outcomes were assessed between March 2017 and September 2017. Patients were divided into MPA + HMG group and 1-month ultra-long GnRHa protocol group. RESULTS: Lower hMG dose and shorter medication time were found in the MPA + HMG group than in the GnRHa group (P < 0.05). Follicle to-Oocyte Index was significantly different between MPA + HMG group and GnRHa group (P < 0.001). No differences were found in the ovary response and numbers of mature oocytes, fertilized oocytes and viable embryos. The clinical pregnancy and live birth outcomes were similar between MPA + HMG group and GnRHa group, and these outcomes were independent of fresh or frozen embryo transfer in the GnRHa protocol group. There were no significant differences in the time to embryo transfer, medical cost and adverse effects. CONCLUSION: The number of oocytes retrieved and pregnancy outcomes after MPA + HMG protocol are similar to those after ultra-long GnRHa protocol in women with ovarian endometriosis. MPA + HMG protocol may be an alternative to ultra-long GnRHa protocol for IVF in ovary endometriosis patients. Trial registration The trial was registered in the Chinese Clinical Trial Registry (ChiCTR-INR-17010924) In conclusion, the administration of MPA in COH showed similar number of oocytes retrieved, no premature LH surge, and similar pregnancy and live birth outcomes in patients with advanced ovarian endometriosis undergoing IVF/ICSI as compared to the one-month long protocol. The use of MPA in COH appears to be promising although many questions remain to be elucidated, including the dose and time of progestin priming as well as its possible influence on the oocyte development potential and microenvironment. Given their good tolerability, few metabolic influence, and low cost, progestogens provide a novel alternative to the conventional protocol for patients with endometriosis.

Impact of uterine malformations on pregnancy and neonatal outcomes of IVF/ICSI-frozen embryo transfer.

STUDY QUESTION: What is the impact of uterine malformations on reproductive and neonatal outcomes of IVF/ICSI-frozen embryo transfer? SUMMARY ANSWER: Unification defective uteri are associated with poorer neonatal outcomes including higher preterm delivery rate and lower birthweight, and septate uteri are associated with worse fertility outcomes including higher miscarriage and lower live birth rates (LBRs). WHAT IS KNOWN ALREADY: Several studies have investigated the negative effects of uterine malformations on pregnancy outcomes. However, an all-round and definitive conclusion has not been reached yet owing to the relatively low incidence of the disease and the heterogeneity of study populations, especially among women undergoing ART. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study including 411 women with congenital uterine anomalies and 14 936 women with a normal uterus who underwent first frozen-thawed embryo transfer cycles of IVF/ICSI from July 2008 to August 2019. We compared reproductive outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductive outcomes of women with uterine malformations were studied through three propensity score-matched comparisons (patients with bicorporeal uterus, septate uterus and hemi-uterus [bicorporeal, septate and hemi-uterus groups, respectively] along with corresponding control groups without uterine malformations). We also compared pregnancy and neonatal outcomes, and performed subgroup analysis addressing didelphus, bicornuate uteri and septate uteri before and after surgery independently. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to the matched control group, women with a bicorporeal uterus had a significantly lower LBR (24.4% versus 34.8%, odds ratio (OR) 0.61 [95% CI: 0.37, 1.00], P = 0.048). The incidence of miscarriage and preterm delivery was higher but not statistically significant (29.0% versus 18.1%, OR 1.85 [95% CI: 0.82, 4.19], P = 0.135; 22.6% versus 9.9%, OR 2.64 [95% CI: 1.07, 6.52], P = 0.063, respectively). In addition, the bicorporeal group had a significantly lower gestational age, higher caesarean rate and lower birthweight than bicorporeal control. Women with a septate uterus had comparable clinical pregnancy rates to controls (43.3% versus 49.9%, OR 0.77 [95% CI: 0.57, 1.04], P = 0.091), increased miscarriage rates (23.5% versus 13.0%, OR 2.05 [95% CI: 1.18, 3.58], P = 0.010) and lower LBRs (29.4% versus 42.2%, OR 0.57 [95% CI: 0.41, 0.79], P = 0.001). In both singleton and twins pregnancies, pregnancy and neonatal outcomes were comparable between women with a septate uterus and control. Women with a hemi-uterus had a tendency for lower clinical pregnancy rate (36.8% versus 42.3%, OR 0.80 [95% CI: 0.52, 1.21], P = 0.287) and LBR (29.8% versus 33.1%, OR 0.86 [95% CI: 0.55, 1.34], P = 0.502), compared to women without malformations. The incidences of miscarriage and preterm delivery, respectively, were 16.7% versus 16.6% (OR 1.01 [95% CI: 0.41, 2.47], P = 0.989), and 9.5% versus 11.4% (OR 0.82 [95% CI: 0.27, 2.51], P = 1) in women with a hemi-uterus as compared to control. LIMITATIONS, REASONS FOR CAUTION: This was a single-centre, retrospective study in which neonatal data were extracted from parental questionnaires. The information on uteri septum type and surgery methods was poorly presented, with limited detail. In patients with uterine malformations, the number of babies with birth defects and twin pregnancies was relatively small, limiting the power of the study. WIDER IMPLICATIONS OF THE FINDINGS: Compared to patients with a normal uterus, women with uterine malformation have poorer reproductive outcomes. Pregnant women with a uterine anomaly need to be managed as high-risk pregnancies and followed with appropriate obstetric review. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Ministry of Technology (2018YFC1003000), the Elite Team Project of Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JY201801), Shanghai Sailing Program (21YF1423200) and the Fundamental Research Program Funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYZZ117). B.W.M. is supported by an NHMRC Investigatorgrant (GNT1176437). B.W.M. reports consultancy (with stock options) for ObsEva. B.W.M. has received research funding from Ferring and Merck. The authors declare no other competing interests. TRIAL REGISTRATION NUMBER: N/A.

Progesterone affects clinic oocyte yields by coordinating with follicle stimulating hormone via PI3K/AKT and MAPK pathways.

Introduction: As an effective inhibitor of premature ovulation, progestin was introduced to a novel ovarian stimulation regimen for infertility treatment. However, the local action of progestin on the ovary and its effect on clinical outcomes have not been described. Objectives: The influence of progesterone administration on clinical oocyte outcomes and the mechanisms involved in the coordination of progesterone and follicle stimulating hormone (FSH) on follicle growth and oocyte yields were investigated. Methods: Clinical outcomes of patients undergoing ovarian stimulation for in vitro fertilization were analyzed. The murine ovarian stimulation model and follicle culture system were used to evaluate the effects of progesterone on oocyte yield, follicle development, granular cell proliferation, and hormone secretion. Phospho-specific protein microarrays were used to explore involved signaling pathways. Results: Progesterone decreased clinical oocyte yields, and yields were rescued with an increased dose of human menopausal gonadotropin. Administration of progesterone inhibited murine granular cell proliferation and reduced the growth rate of follicles; both of which were rescued by FSH. The phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) were identified as pivotal signaling pathways to integrate progesterone into the FSH signaling network in granular cells. Conclusion: Progesterone inhibited granular cell proliferation and antral follicle growth during ovarian stimulation, and subsequently influenced oocyte outcomes in the clinical setting. Progesterone coordinated with FSH to regulate follicle growth through PI3K/AKT and MAPK signaling pathways. These findings advance our knowledge regarding the ovarian response to gonadotropins during progestin-primed ovarian stimulation and create an opportunity to manipulate individual oocyte yields.

Quercetin prevents primordial follicle loss via suppression of PI3K/Akt/Foxo3a pathway activation in cyclophosphamide-treated mice.

BACKGROUND: Chemotherapy improves the survival rates of patients with various cancers but often causes some adverse effects, including ovarian damage, characterised by a decrease in primordial follicle stockpiles. Recent studies have revealed that chemotherapy may stimulate the PI3K signalling pathway, thereby resulting in accelerated primordial follicle activation and a decreased ovarian reserve. Quercetin is an inhibitor of the PI3K pathway; however, its protective effects against chemotherapy-induced follicle loss in mice have not been established. In this study, the effects of quercetin in a mouse model of cyclophosphamide-induced ovarian dysfunction were investigated. METHODS: C57BL/6 female mice were used for the study. Paraffin sections of mouse ovaries (n = 30 mice) were stained with haematoxylin and eosin for differential follicle counts. Apoptosis (n = 5 mice per group) was evaluated by TUNEL assay. Immunohistochemical staining for ki67 and Foxo3a (n = 5 mice per group) was performed to evaluate the activation of primordial follicles. The role of the PI3K signalling pathway in the ovaries (n = 45 mice) was assessed by western blotting. RESULTS: Quercetin attenuated the cyclophosphamide-induced reduction in dormant primordial follicles. Analysis of the PI3K/Akt/Foxo3a pathway showed that quercetin decreased the phosphorylation of proteins that stimulate follicle activation in cyclophosphamide-induced ovaries. Furthermore, quercetin prevented cyclophosphamide-induced apoptosis in early growing follicles and early antral follicles, maintained anti-Müllerian hormone levels secreted by these follicles, and preserved the quiescence of the primordial follicle pool, as determined by intranuclear Foxo3a staining. CONCLUSIONS: Quercetin attenuates cyclophosphamide-induced follicle loss by preventing the phosphorylation of PI3K/Akt/Foxo3a pathway members and maintaining the anti-Müllerian hormone level through reduced apoptosis in growing follicles. Accordingly, quercetin is expected to improve fertility preservation and the prevention of endocrine-related side effects of chemotherapy.

Different dendritic domains of the GnRH neuron underlie the pulse and surge modes of GnRH secretion in female mice.

The gonadotropin-releasing hormone (GnRH) neurons exhibit pulse and surge modes of activity to control fertility. They also exhibit an unusual bipolar morphology comprised of a classical soma-proximal dendritic zone and an elongated secretory process that can operate as both a dendrite and an axon, termed a 'dendron'. We show using expansion microscopy that the highest density of synaptic inputs to a GnRH neuron exists at its distal dendron. In vivo, selective chemogenetic inhibition of the GnRH neuron distal dendron abolishes the luteinizing hormone (LH) surge and markedly dampens LH pulses. In contrast, inhibitory chemogenetic and optogenetic strategies targeting the GnRH neuron soma-proximal dendritic zone abolish the LH surge but have no effect upon LH pulsatility. These observations indicate that electrical activity at the soma-proximal dendrites of the GnRH neuron is only essential for the LH surge while the distal dendron represents an autonomous zone where synaptic integration drives pulsatile GnRH secretion.

Impacts of medroxyprogesterone acetate on oocytes and embryos: matched case-control study in women with stage III-IV ovarian endometriosis undergoing controlled ovarian hyperstimulation for in vitro fertilization.

BACKGROUND: This study investigated the effects of medroxyprogesterone acetate (MPA) on the oocytes and embryos in patients with advanced endometriosis who had a normal ovarian reserve and tubal infertility and received controlled ovarian hyperstimulation (COH) and explored the characteristics and pregnancy outcomes in subsequent frozen-thawed embryo transfer (FET) cycles. METHODS: In this prospective controlled study, 150 advanced endometriosis patients involving 150 in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) cycles and 163 FET cycles and 150 age-matched tubal infertility patients requiring 150 IVF/ICSI cycles and 115 FET cycles were recruited. Patients with endometriosis were sub-grouped into surgery group (n=102) (they were diagnosed with ovarian endometriomas and underwent 102 IVF/ICSI and 115FET cycles) and aspiration group (n=48) [they had ovarian "chocolate" cysts (>3 cm) that were aspirated and underwent 48 IVF/ICSI and 74 FET cycles]. RESULTS: Lower oocyte retrieval rate was noted in the endometriosis group than in the control group. Similar oocyte yield and peak estrogen (E2) level were found in two groups. The rates of mature oocyte, fertilization, cleavage, high-quality embryo, viable embryo, cancellation, implantation, and clinical pregnancy were similar between two groups. A higher oocyte yield was observed in the EMS cyst group than in the surgery group. CONCLUSIONS: The ovary response, oocytes, embryos and pregnancy outcome were not influenced by the advanced endometriosis and the use of MPA and also independent of endometrioma or cyst surgery.

Dynorphin and GABAA Receptor Signaling Contribute to Progesterone's Inhibition of the LH Surge in Female Mice.

Progesterone can block estrogen-induced luteinising hormone (LH) surge secretion and can be used clinically to prevent premature LH surges. The blocking effect of progesterone on the LH surge is mediated through its receptor in the anteroventral periventricular nucleus (AVPV) of the hypothalamus. However, the underlying mechanisms are unclear. The preovulatory LH surge induced by estrogen is preceded by a significant reduction in hypothalamic dynorphin and gamma-aminobutyric acid (GABA) release. To test the detailed roles of dynorphin and GABA in an LH surge blockade by progesterone, ovariectomized and 17ß-estradiol capsule-implanted (OVX/E2) mice received simultaneous injections of estradiol benzoate (EB) and progesterone (P) or vehicle for 2 consecutive days. The LH level was monitored from 2:30 pm to 8:30 pm at 30-minute intervals. Progesterone coadministration resulted in the LH surge blockade. A continuous microinfusion of the dynorphin receptor antagonist nor-BNI or GABAA receptor antagonist bicuculline into the AVPV from 3:00 pm to 7:00 pm reversed the progesterone-mediated blockade of the LH surge in 7 of 9 and 6 of 10 mice, respectively. In addition, these LH surges started much earlier than the surge induced by estrogen alone. However, 5 of 7 progesterone-treated mice did not show LH surge secretion after microinfusion with the GABAB receptor antagonist CGP-35348. Additionally, peripheral administration of kisspeptin-54 promotes LH surge-like release in progesterone treated mice. These results demonstrated that the progesterone-mediated suppression of the LH surge is mediated by an increase in dynorphin and GABAA receptor signaling acting though kisspeptin neurons in the AVPV of the hypothalamus in female mice.

Association between peak serum estradiol level during controlled ovarian stimulation and neonatal birthweight in freeze-all cycles: a retrospective study of 8501 singleton live births.

STUDY QUESTION: Is there an association between peak serum estradiol (E2) level during controlled ovarian stimulation (COS) and neonatal birthweight in freeze-all cycles? SUMMARY ANSWER: Peak serum E2 level during ovarian stimulation is not associated with neonatal birthweight in freeze-all cycles. WHAT IS KNOWN ALREADY: Supraphysiologic E2 levels during COS have been demonstrated to generate a suboptimal peri-implantation endometrial environment and thus lead to adverse neonatal outcomes in fresh embryo transfer cycles. Previous experimental studies also suggested a potential influence of superovulation on oocyte epigenetic programming, but whether it translates into altered phenotypes of fetal growth and development remains unclear in clinical practice. By segmenting the process of COS and embryo transfer, the freeze-all policy provides a novel model to investigate the sole impact of ovarian stimulation on oocytes after ruling out the effects of hyperestrogenic milieu on endometrium in fresh cycles. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of 8501 patients who underwent their first COS cycles with a freeze-all strategy and delivered live-born singletons in subsequent frozen-thawed embryo transfer cycles from January 2007 to December 2016 at a tertiary-care academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were categorized into six groups according to E2 level on trigger day in regular increments of 1000 pg/mL: <1000, 1000-1999, 2000-2999, 3000-3999, 4000-4999 and ≥5000 pg/mL. Univariable and multivariable linear regression and logistic regression analysis were performed to assess the independent association between peak E2 level and measures of neonatal birthweight including absolute birthweight, Z-score, low birthweight (LBW) and small-for-gestational age (SGA). MAIN RESULTS AND THE ROLE OF CHANCE: The six groups did not differ significantly in birthweight, Z-score or the incidence of LBW and SGA. Compared with the E2 <1000 pg/mL group, the adjusted mean difference (95% confidence interval [CI]) of stratified higher E2 groups was 17.2 (-31.0-65.5), 12.3 (-35.9-60.5), -4.1 (-51.9-43.7), -0.6 (-48.9-47.8) and -3.6 (-50.0-42.8) g for birthweight, and 0 (-0.11-0.10), 0.02 (-0.08-0.12), 0.04 (-0.06-0.14), -0.01 (-0.11-0.10) and -0.04 (-0.14-0.06) for Z-score, respectively. Regarding the outcomes of LBW and SGA, no increased risks were observed in each E2 category, with the adjusted odds ratio (95% CI) being 1.21 (0.68-2.16), 1.0 (0.58-1.90), 0.90 (0.50-1.63), 0.93 (0.51-1.69) and 1.08 (0.61-1.90) for LBW, and 0.97 (0.58-1.64), 1.06 (0.63-1.77), 0.77 (0.46-1.31), 0.71 (0.41-1.22) and 1.00 (0.60-1.65) for SGA, respectively. LIMITATIONS, REASONS FOR CAUTION: The study was retrospective in design, and other unknown confounding factors may not be included for adjustment. Furthermore, the generalization of the study finding could be limited to some extent by the majority of double cleavage-stage embryo transfer and difference in birthweight reference percentiles between Chinese and other populations. WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggest that the hyperestrogenic milieu during COS does not seem to pose adverse effects on neonatal birthweight after frozen-thawed embryo transfer, which provides reassuring information for high ovarian responders in freeze-all cycles concerning their offspring's health. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the National Key Research and Development Program of China (SQ2018YFC100163) and National Natural Science Foundation of China (81571397, 81771533). The authors declare no conflict of interest.

Novel mutations in PLCZ1 cause male infertility due to fertilization failure or poor fertilization.

STUDY QUESTION: Do sperm-specific phospholipase C zeta (PLCZ1) mutations account for male infertility due to fertilization failure? SUMMARY ANSWER: Six novel mutations and one reported mutation in PLCZ1 were identified in five of 14 independent families characterized by fertilization failure or poor fertilization, suggesting that these mutations may be responsible for fertilization failure in men exhibiting primary infertility. WHAT IS KNOWN ALREADY: PLCZ1 is essential for the induction of intracellular calcium (Ca2+) oscillations and the initiation of oocyte activation during mammalian fertilization. However, genetic evidence linking PLCZ1 mutations with male infertility remains limited. STUDY DESIGN, SIZE, DURATION: Fourteen unrelated primary infertility patients were recruited into this study from January 2016 to December 2018; the patients exhibited total fertilization failure or poor fertilization, as evidenced by ICSI and sperm-related oocyte activation deficiencies identified in mouse oocyte activation assays. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genomic DNA samples were extracted from the peripheral blood of patients. The whole exons of PLCZ1 were sequenced by Sanger sequencing. The PLCZ1 sequences were aligned by CodonCode software to identify rare variants. The ExAC database was used to search for the frequency of corresponding mutations. The pathogenicity of identified variants and their possible effects on the protein were assessed in silico. PLCZ1 protein levels in semen samples were evaluated by western blotting. Oocyte activation ability was assessed by the injection of wild-type and mutant PLCZ1 cRNAs into human mature metaphase II (MII) oocytes in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: We identified six novel mutations and one reported mutation in PLCZ1 among five affected individuals. In addition to four novel missense mutations, two new types of genetic variants were identified, including one in-frame deletion and one splicing mutation. Western blot analysis revealed that PLCZ1 protein expression was not observed in the semen samples from the five affected patients. Microinjection with the PLCZ1 cRNA variants was performed, and a significant decrease in the percentage of pronuclei was observed for four novel missense mutations and one novel in-frame deletion mutation, suggesting that these mutations have a deleterious influence on protein function. By artificial oocyte activation treatment, the fertilization failure phenotypes of four affected patients were successfully rescued and three healthy babies were delivered. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We screened only the whole exons of PLCZ1. Additional possible mutations in the non-coding region of PLCZ1 should be further studied. WIDER IMPLICATIONS OF THE FINDINGS: Our study not only further confirms the important role of PLCZ1 in human fertilization but also expands the mutational spectrum of PLCZ1 associated with male infertility, which provides a basis for assessing genetic variation in PLCZ1 as a potential diagnostic marker for infertile men suffering from fertilization failure. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Foundation of China (81 571 486 and 81 771 649). All authors have no conflicts of interest to declare.

Novel FSHR variants causing female resistant ovary syndrome.

BACKGROUND: Pathogenic variants of follicle-stimulating hormone receptor (FSHR) are known to cause amenorrhea and infertility in women. However, only a limited number of pathogenic FSHR variants have been reported, and few reports described detailed characteristics of patients with pathogenic FSHR variants. METHODS: The affected siblings and both parents were subjected to whole-genome exon sequencing. Transient transfection of HEK 293T cells was performed with constructed vectors. The cellular localization of the FSHR protein was evaluated using confocal microscopy, and cyclic adenosine monophosphate (cAMP) production was detected with a cAMP ELISA kit. RESULTS: A Chinese family with two siblings carrying compound heterozygous pathogenic variants of FSHR: c.182T>A (p.Ile61Asn) and c.2062C>A (p.Pro688Thr). Both siblings had amenorrhea, infertility, and resistance to gonadotropin (Gn) stimulation but showed high anti-Müllerian hormone levels and early antral follicles. Molecular dynamics simulations of the FSHR variants revealed significant changes in structural characteristics and electrostatic potential. In vitro analysis indicated that the p.Ile61Asn variant lacked cell surface localization and completely abolished the cAMP second messenger response. The p.Pro688Thr variant retained cell surface localization but caused decreased FSH-induced cAMP production. CONCLUSION: We found two novel pathogenic FSHR variants causing resistant ovarian syndrome. This study expands the genotypic spectrum of pathogenic FSHR variants and our knowledge of phenotype-genotype correlations.

Association between the number of oocytes retrieved and neonatal outcomes after freeze-all IVF cycles.

STUDY QUESTION: Is there any association between the number of oocytes retrieved and neonatal outcomes following IVF/ICSI treatment for patients using a freeze-all strategy? SUMMARY ANSWER: There was no increased risk of adverse neonatal outcomes in cycles with high number of oocytes retrieved (≥ 16) compared to those with 10-15 oocytes retrieved in freeze-all cycles. WHAT IS KNOWN ALREADY: Recent studies have found that there is an increased risk of preterm birth (PTB, <37 weeks gestation) and low birth weight (LBW, <2500 g) following IVF in women with a high number (>20) of oocytes retrieved in fresh embryo transfer (ET) cycles. Other studies have found that there is an association between the number of oocytes retrieved and placenta praevia. However, the association between the number of oocytes retrieved and neonatal outcomes when using a freeze-all strategy is unknown. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 14 170 women with singleton deliveries achieved by a freeze-all strategy performed between November 2006 and December 2017 in China. Only the first delivery from one episode of ovarian stimulation was included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Only cycles using a freeze-all strategy performed during the study period and resulting in singleton live births were included. Patients were categorized into five groups according to the number of oocytes retrieved: 1-3, 4-9, 10-15, 16-20 or >20 oocytes. In univariate and multivariate logistic regression analysis of the association between ovarian response and the outcomes of PTB, early PTB, LBW and other neonatal outcomes, the 10 to 15 oocyte category was used as a reference and other four groups were analysed as dummy variables. Multiple linear regression analysis was used to evaluate possible associations of birth weight z-scores and the number of oocytes retrieved (analysed as a continuous variable) with other confounding factors. MAIN RESULTS AND THE ROLE OF CHANCE: After adjusted for confounding factors, no significant differences were observed in the risk of PTB (P = 0.837), LBW (P = 0.974), early PTB (P = 0.341), very LBW (P = 0.848), congenital malformation (P = 0.916) and other adverse neonatal outcome among patients with different number of oocytes retrieved. There was a higher risk of early PTB among women with a poor ovarian response (1-3 oocytes) compared with women with a normal response (10-15 oocytes) (1.5% vs 0.8%), crude odds ratio (OR): 2.001, 95% CI: 1.159-3.465, P = 0.013. However, the difference was not significant after adjusting for confounders, adjusted OR: 1.753, 95% CI: 0.997-3.081, P = 0.051. LIMITATIONS, REASONS FOR CAUTION: Data on some known confounders such as smoking and medical history of gestational diabetes mellitus and preeclampsia were lacking. As with any retrospective study, unknown confounders may affect outcomes. WIDER IMPLICATIONS OF THE FINDINGS: In the freeze-all cycles, there was no association between number of oocytes retrieved and adverse neonatal outcomes. This is a reassuring finding for both clinicians and patients who are planning to use freeze-all cycles for a variety of indications. STUDY FUNDING/COMPETING INTEREST(S): Grants from the National Natural Science Foundation of China (NSFC) (31770989 to Y.W.) and the Shanghai Ninth People's Hospital Foundation of China (JYLJ030 to Y.W.). None of the authors have any conflicts of interest to declare.

Pregnancy and Live Birth In Women With Pathogenic LHCGR Variants Using Their Own Oocytes.

CONTEXT: The LH/chorionic gonadotropin receptor (LHCGR) is mainly expressed in gonads and plays important roles in estradiol production, ovulation, and luteal formation. Women with pathogenic LHCGR variants suffer from infertility, and successful fertility treatments for such women have never been reported. OBJECTIVE: The purpose of this study was to determine whether women with pathogenic LHCGR variants can achieve successful pregnancies through in vitro fertilization. DESIGN: Three women with LH resistance and infertility and their parents underwent exome sequencing. The biochemical characteristics and functional effects of LHCGR mutation were assessed in transfected human embryonic kidney -293T cells and primary granulosa cells. RESULTS: All affected women harbored pathogenic LHCGR variants. The LHCGR variants lacked cell surface localization and signal transduction abilities in vitro and in vivo. After dual triggering and prolonging the interval between triggering and oocyte pick-up, all three patients achieved oocytes and high-quality embryos. After frozen embryo transfer, one woman successfully birthed twins, and one woman successfully birthed a live boy. Apart from difficulties in oocyte retrieval, no obvious abnormalities in fertilization or during embryo development and pregnancy were identified in these patients. CONCLUSIONS: This study is, to our knowledge, the first to report successful assisted reproductive treatment of women with pathogenic LHCGR variants using their own oocytes. Our results supported that defects in LHCGR disrupted ovulation but had no effect on fertilization and embryo development.

A pannexin 1 channelopathy causes human oocyte death.

Connexins and pannexins are two protein families that play an important role in cellular communication. Pannexin 1 (PANX1), one of the members of pannexin family, is a channel protein. It is glycosylated and forms three species, GLY0, GLY1, and GLY2. Here, we describe four independent families in which mutations in PANX1 cause familial or sporadic female infertility via a phenotype that we term "oocyte death." The mutations, which are associated with oocyte death, alter the PANX1 glycosylation pattern, influence the subcellular localization of PANX1 in cultured cells, and result in aberrant PANX1 channel activity, ATP release in oocytes, and mutant PANX1 GLY1. Overexpression of a patient-derived mutation in mice causes infertility, recapitulating the human oocyte death phenotype. Our findings demonstrate the critical role of PANX1 in human oocyte development, provide a genetic explanation for a subtype of infertility, and suggest a potential target for therapeutic intervention for this disease.

Decrease in preovulatory serum estradiol is a valuable marker for predicting premature ovulation in natural/unstimulated in vitro fertilization cycle.

BACKGROUND: Premature ovulation occurs at a high rate in natural-cycle in vitro fertilization (IVF), and cycle cancellation further hampers the overall efficiency of the procedure. While lower levels of estradiol (E2) are observed in preovulatory follicles, it is unclear whether declines in E2 can be used as an effective marker of premature ovulation. METHODS: This retrospective analysis includes 801 natural/unstimulated IVF/ICSI cycles undergoing scheduled ovum pick-up (OPU) and 153 natural/unstimulated IVF/ICSI cycles undergoing emergency OPU at a university IVF center from May 2014 to February 2017. RESULTS: Among the 801 IVF/ICSI cycles undergoing scheduled OPU, preovulatory E2 levels increased by more than 10% in 403 (50.31%) cycles of the sample (Group A), while 192 (23.97%) cycles experienced a plateau (increased or decreased by 10%; Group B), and 206 (25.72%) cycles decreased by more than 10% (Group C). Group C had more patients who experienced premature LH surges, premature ovulation, as well as the fewest oocytes retrieved, frozen embryos, and top-quality embryos. A multivariate logistic regression analysis indicated that premature ovulation was associated with preovulatory E2/-1E2 ratio and premature LH surge. Moreover, preovulatory E2/-1E2 ratio served as a valuable marker for differentiating premature ovulation, with an AUC (area under the receiver operating curve) of 0.708 and 0.772 in cycles with premature LH surges and cycles without premature LH surges, respectively. Emergency OPU resulted in a significantly decreased rate of premature ovulation and increased number of frozen embryos. CONCLUSION: Decreases in preovulatory serum E2 was a valuable marker for premature ovulation in natural/unstimulated IVF cycle. Emergency OPU based on the preovulatory E2/-1E2 ratio decreased the rate of premature ovulation in cycles that experienced E2 decreases.

Elevated basal luteinizing hormone does not impair the outcome of human menopausal gonadotropin and medroxyprogesterone acetate treatment cycles.

The potential effects of high basal luteinizing hormone (LH) levels on human reproduction were controversial. To demonstrate the effects of elevated basal LH levels on the outcome of patients undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles, we performed a retrospective data analysis of 1011 polycystic ovarian syndrome (PCOS) patients treated with human menopausal gonadotropin and medroxyprogesterone acetate (hMG + MPA) protocol at our center between Nov. 2013 and Jun. 2017. PCOS patients with elevated basal LH levels had significantly higher LH exposure during the stimulation period. The group with LH ≥ 10 mIU/mL showed a lower mean total hMG dose used but higher numbers of oocytes retrieved, metaphase II oocytes, embryos and top-quality embryos developed than the groups with lower basal LH levels. Moreover, partial correlation analysis showed that the basal LH level was negatively correlated with the total hMG dose but positively correlated with the numbers of oocytes retrieved, metaphase II oocytes, embryos, and top-quality embryos. There were no significant differences in the rates of oocyte retrieval, fertilization, implantation, clinical pregnancy and miscarriage between the groups based on frozen embryo transfer (FET). We concluded that elevated basal LH level does not impair the final outcome of hMG + MPA-treated IVF/ICSI cycles in PCOS women.

Progestin-primed milder stimulation with clomiphene citrate yields fewer oocytes and suboptimal pregnancy outcomes compared with the standard progestin-primed ovarian stimulation in infertile women with polycystic ovarian syndrome.

BACKGROUND: Oral progestin has recently been used to prevent premature LH surges in ovarian stimulation, and this progestin-primed ovarian stimulation (PPOS) is effective and safe in patients with different ovarian reserves. The current data are lacking regarding how to individualize the gonadotropin dose and regimen for women with polycystic ovarian syndrome (PCOS). A retrospective cohort trial was performed to evaluate the efficacy of progestin-primed milder stimulation with clomiphene citrate (CC) compared to the standard progestin-primed ovarian stimulation (PPOS) protocol for infertile women with PCOS. METHODS: A total of 220 PCOS women were collected and classified into the study group (HMG 150 IU/d + CC 50 mg/d + MPA 10 mg/d) and control group (HMG 225 IU/d + MPA 10 mg/d). Ovulation was triggered by GnRH agonist 0.1 mg and hCG 1000 IU when dominant follicles matured. Viable embryos were cryopreserved for later transfer. The primary endpoint was the ongoing pregnancy rate. Secondary outcomes included the cycle characteristics and the live birth rate. RESULT(S): The study group consumed less HMG (1470.0 ± 360.1 IU vs 1943.8 ± 372.0 IU, P < 0.001) and harvested fewer oocytes than the control group (12.2 ± 7.4 vs 18.2 ± 9.7, P < 0.001). The study group showed a higher mid-follicular LH concentration (4.49 ± 2.49 mIU/ml vs 2.52 ± 2.09 mIU/ml, P < 0.05) but no endogenous LH surge. No between-group difference was found in the incidence of ovarian hyperstimulation syndrome (OHSS) (0.91% vs 0.91%, P > 0.05). The cumulative ongoing pregnancy rate and live birth rate per patient were lower but did not reach significance compared with the control group (71.8% vs 81.8 and 64.5% vs 75.5%, respectively, both P > 0.05). CONCLUSION(S): The milder PPOS with CC in PCOS women led to lower oocyte yields and suboptimal pregnancy outcomes compared to the standard PPOS treatment. The two regimens both achieved a low incidence of OHSS. The results from the CC combination regimen provide a new insight for developing a more patient-friendly protocol for PCOS women.