Autophagy activity is increased in the cumulus cells of women with poor ovarian response.

OBJECTIVE: To evaluate the autophagy status of cumulus cells (CCs) in women with poor ovarian response (POR). MATERIALS AND METHODS: CCs were divided into normal ovarian response (NOR) group and POR group. The ultrastructure of autophagy was analyzed by transmission electron microscopy (NOR: n = 18, POR: n = 26). The mRNA and protein of autophagy markers were detected by Quantitative real-time polymerase chain reaction (NOR: n = 15, POR: n = 19) and Western blotting (NOR: n = 41, POR: n = 38), respectively. RESULTS: Transmission electron microscopy demonstrated abundant autophagosomes and even autophagic death in the POR group. There were no differences in LC3 and P62 mRNA expression between the two groups (p > 0.05). The BCL2 mRNA expression was lower in the POR group (p < 0.05). Moreover, the LC3 II/I ratio and the P62 protein expression were significantly higher in the POR group (p < 0.05). CONCLUSIONS: Autophagy in CCs of POR women is activated and the autophagic flux is blocked. The up-regulation of autophagy in CCs may be related to the pathogenesis of POR.

Effect of noninvasive embryo viability testing versus conventional IVF on the live birth rate in IVF/ICSI patients: a study protocol for a double-blind, multicenter, randomized controlled trial.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) was demonstrated to be superior to conventional IVF in reducing the incidence of miscarriage and abnormal offspring after the first embryo transfer (ET). PGT-A requires several embryo trophectoderm cells, but its negative impacts on embryo development and long-term influence on the health conditions of conceived children have always been a concern. As an alternative, noninvasive PGT-A (niPGT-A) approaches using spent blastocyst culture medium (SBCM) achieved comparable accuracy with PGT-A in several pilot studies. The main objective of this study is to determine whether noninvasive embryo viability testing (niEVT) results in better clinical outcomes than conventional IVF after the first embryo transfer. Furthermore, we further investigated whether niEVT results in higher the live birth rate between women with advanced maternal age (AMA, > 35 years old) and young women or among patients for whom different fertilization protocols are adopted. METHODS: This study will be a double-blind, multicenter, randomized controlled trial (RCT) studying patients of different ages (20-43 years) undergoing different fertilization protocols (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]). We will enroll 1140 patients at eight reproductive medical centers over 24 months. Eligible patients should have at least two good-quality blastocysts (better than grade 4 CB). The primary outcome will be the live birth rate of the first embryo transfer (ET). Secondary outcomes will include the clinical pregnancy rate, ongoing pregnancy rate, miscarriage rate, cumulative live birth rate, ectopic pregnancy rate, and time to pregnancy. DISCUSSION: In this study, patients who undergo noninvasive embryo viability testing (niEVT) will be compared to women treated by conventional IVF. We will determine the effects on the pregnancy rate, miscarriage rate, and live birth rate and adverse events. We will also investigate whether there is any difference in clinical outcomes among patients with different ages and fertilization protocols (IVF/ICSI). This trial will provide clinical evidence of the effect of noninvasive embryo viability testing on the clinical outcomes of the first embryo transfer. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR) Identifier: ChiCTR2100051408. 9 September 2021.

The effect of luteinizing hormone changes in GnRH antagonist protocol on the outcome of controlled ovarian hyperstimulation and embryo transfer.

BACKGROUD: To investigate the effect of Luteinizing hormone (LH) level changes on the outcomes of controlled ovarian hyperstimulation (COH) and embryo transfer (ET) in gonadotropin-releasing hormone antagonist (GnRH-ant) protocol. METHODS: A total of 721 patients undergoing GnRH-ant protocol COH for the first IVF/ICSI cycles were retrospectively analyzed. COH process were divided into 2 stages, before (stage 1) and after (stage 2) the GnRH-ant initiation, and each with 5 groups basing on LH levels: LH decreased more than 50% (groups A1, A2), decreased 25-50% (groups B1, B2), change less than 25% (groups C1, C2), increased 25-50% (groups D1, D2), and increased more than 50% (groups E1, E2). RESULTS: There were no significant differences among groups of stage1 regarding COH and ET outcomes. For stage 2, the more obvious the decrease of LH level, the more the number of oocytes retrieved, mature oocytes, fertilized oocytes, embryos cleavaged and the numbers of embryo available (P < 0.05), but without significant differences regarding ET outcomes. We also found the freeze-all rate in Group A2 was higher (P < 0.001). CONCLUSION: LH level changes before GnRH-ant addition were not related to COH and ET outcomes. LH level changes after the addition of GnRH-ant were related to the outcome of COH, and no significant differences were found relating to ET outcomes.

The role of spermatozoa-zona pellucida interaction in selecting fertilization-competent spermatozoa in humans.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.

Identification of Sialyl-Lewis(x)-Interacting Protein on Human Spermatozoa.

Capacitated spermatozoa initiate fertilization by binding to the zona pellucida (ZP). Defective spermatozoa-ZP binding causes infertility. The sialyl-Lewis(x) (SLeX) sequence is the most abundant terminal sequence on the glycans of human ZP glycoproteins involving in spermatozoa-ZP binding. This study aimed to identify and characterize the SLeX-binding proteins on human spermatozoa. By using affinity chromatography followed by mass spectrometric analysis, chromosome 1 open reading frame 56 (C1orf56) was identified to be a SLeX-binding protein of capacitated spermatozoa. The acrosomal region of spermatozoa possessed C1orf56 immunoreactive signals with intensities that increased after capacitation indicating translocation of C1orf56 to the cell surface during capacitation. Treatment with antibody against C1orf56 inhibited spermatozoa-ZP binding and ZP-induced acrosome reaction. Purified C1orf56 from capacitated spermatozoa bound to human ZP. A pilot clinical study was conducted and found no association between the percentage of capacitated spermatozoa with C1orf56 expression and in vitro fertilization (IVF) rate in assisted reproduction treatment. However, the percentage of C1orf56 positive spermatozoa in the acrosome-reacted population was significantly (P < 0.05) lower in cycles with a fertilization rate < 60% when compared to those with a higher fertilization rate, suggesting that C1orf56 may have functions after ZP-binding and acrosome reaction. A larger clinical trial is needed to determine the possible use of sperm C1orf56 content for the prediction of fertilization potential of sperm samples.

ATP mediates the interaction between human blastocyst and endometrium.

OBJECTIVE: Embryo implantation needs a reciprocal interaction between competent embryo and receptive endometrium. Adenosine triphosphate (ATP) produced by stressed or injured cells acts as an important signalling molecule. This study aims to investigate whether adenosine triphosphate (ATP) plays an important role in the dialogue of human blastocyst-endometrium. MATERIALS AND METHODS: The concentration of lactate was analysed in culture medium from human embryos collected from in vitro fertilization patients. Extracellular ATP was measured by ATP Bioluminescent Assay Kit. Ishikawa cells and T-HESCs were treated with ATP, ATP receptor antagonist, ATP hydrolysis enzyme or inhibitors of ATP metabolic enzymes. The levels of gene expression were evaluated by real-time PCR and immunoassay. RESULTS: We showed that injured human endometrial epithelial cells could rapidly release ATP into the extracellular environment as an important signalling molecule. In addition, blastocyst-derived lactate induces the release of non-lytic ATP from human endometrial receptive epithelial cells via connexins. Extracellular ATP stimulates the secretion of IL8 from epithelial cells to promote the process of in vitro decidualization. Extracellular ATP could also directly promote the decidualization of human endometrial stromal cells via P2Y-purinoceptors. More importantly, the supernatants of injured epithelial cells clearly induce the decidualization of stromal cells in time-dependent manner. CONCLUSION: Our results suggest that ATP should play an important role in human blastocyst-endometrium dialogue for the initiation of decidualization.

[Updated application of artificial intelligence in reproductive medicine].

With the rapid development of precision medicine and big data application, artificial intelligence (AI) has become a frontier technology in medical research. AI can be applied to the clinical diagnosis and treatment of reproductive diseases, prediction of pregnancy outcomes for infertile patients via the multi-layer neural network, and identification of the embryos with more developing potential from a series of embryo images by extraction of their texture features. AI can also provide medical workers with more accurate diagnosis and individualized treatment of reproductive diseases and help the patients better predict their reproductivity. This article presents an overview of the status quo, existing problems and future development of the application of AI in reproductive medicine.

Predictive value of initial serum human chorionic gonadotropin levels for pregnancies after single fresh and frozen blastocyst transfer.

As one of the earliest markers for predicting pregnancy outcomes, human chorionic gonadotropin (hCG) values have been inconclusive on reliability of the prediction after frozen and fresh embryo transfer (ET). In this retrospective study, patients with positive hCG (day 12 after transfer) were included to examine the hCG levels and their predictive value for pregnancy outcomes following 214 fresh and 1513 vitrified-warmed single-blastocyst transfer cycles. For patients who got clinical pregnancy, the mean initial hCG value was significantly higher after frozen cycles than fresh cycles, and the similar result was demonstrated for patients with live births (LB). The difference in hCG value existed even after adjusting for the potential covariates. The area under curves (AUC) and threshold values calculated by receiver operator characteristic curves were 0.944 and 213.05 mIU/mL for clinical pregnancy after fresh ET, 0.894 and 399.50 mIU/mL for clinical pregnancy after frozen ET, 0.812 and 222.86 mIU/mL for LB after fresh ET, and 0.808 and 410.80 mIU/mL for LB after frozen ET with acceptable sensitivity and specificity, respectively. In conclusion, single frozen blastocyst transfer leads to higher initial hCG values than single fresh blastocyst transfer, and the initial hCG level is a reliable predictive factor for predicting IVF outcomes.

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

Study question: Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? Summary answer: ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. What is known already: A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. Study design, size, duration: The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Participants/materials, setting, methods: Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Main results and the role of chance: Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa. Large scale data: N/A. Limitations, reasons for caution: The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted. Wider implications of the findings: Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction. Study funding/competing interest(s): This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests.

Metastasis-Associated in Colon Cancer-1 Associates With Poor Prognosis and Promotes Cell Invasion and Angiogenesis in Human Cervical Cancer.

OBJECTIVE: The aim of this study is to investigate the clinicopathologic significance and potential role of metastasis-associated in colon cancer-1 (MACC1) in the progression of cervical cancer. METHODS: MACC1 expression was examined in cervical cancer cell lines, 6 matched cervical cancer tissues, and adjacent noncancerous tissues using Western blotting and real-time reverse transcriptase polymerase chain reaction. MACC1 protein expression and localization were determined in 181 paraffin-embedded archived cervical cancer samples using immunohistochemistry. Statistical analyses were applied to evaluate the clinicopathologic significance. The effects of MACC1 on cell migration, invasion, and angiogenesis were examined using migration assay, wound healing assay, 3-dimensional morphogenesis assay, and chicken chorioallantoic membrane assay. Western blotting was performed to examine the impact of MACC1 on the Akt and nuclear factor κB signaling pathways. RESULTS: Both protein and messenger RNA levels of MACC1 was up-regulated in cervical cancer cell lines and cervical cancer tissues, as compared with normal tissues. High MACC1 expression was detected in 96 (53%) of 181 of the cervical cancer tissues. In addition, high MACC1 expression correlated significantly with aggressiveness of cervical cancer, including International Federation of Gynecology and Obstetric stage (P = 0.001), pelvic lymph node metastasis (P = 0.004), recurrence (P = 0.037), and poor survival (P = 0.001). Moreover, enforced expression of MACC1 in cervical cancer cell lines significantly enhanced cell migration, invasion, and angiogenesis. Conversely, knockdown of MACC1 caused an inhibition of cell migration, invasion, and angiogenesis. Up-regulation of MACC1 increased, but knockdown of MACC1 decreased the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. Furthermore, enforced expression of MACC1 could enhance, but knockdown of MACC1 could reduce AKT and nuclear factor κB pathway activity. CONCLUSIONS: Our findings suggest that MACC1 protein, as a valuable marker of cervical cancer prognosis, plays an important role in the progression of human cervical cancer cells.

Atrial fibrillation and obesity among older Chinese: the Guangzhou Biobank Cohort Study.

BACKGROUND: Atrial fibrillation (AF) is the most common form of arrhythmia, and its prevalence is increasing. Few studies have examined its association with obesity as defined by different criteria, particularly in developing countries. We investigated the association between atrial fibrillation (AF) and obesity indices among older Chinese. METHODS: We conducted a community-based nested case control study using cross-sectional data of 5882 men and 14,548 women aged 50 or above from Phases I and II (September 2003 to May 2006) of the Guangzhou Biobank Cohort Study (GBCS). AF cases were identified by the 12-lead body surface electrocardiogram. RESULTS: 159 AF cases (65 men and 94 women) were identified from 19,964 participants with ECG records. 9249 participants with other abnormal ECG findings were excluded, resulting in a case control comparison on 159 AF cases and 10,369 controls. After multivariate adjustment, BMI (adjusted odds ratio (OR) 1.06 per kg/m(2), 95% confidence interval (CI)=1.01-1.11) and waist circumference (adjusted OR 1.02 per cm (1.00-1.04)) were significant risk factors. The adjusted OR per Z-score [(individual value-mean)/standard deviation] for BMI and waist circumference was 1.21 (1.03-1.41) and 1.18 (1.01-1.38) respectively. CONCLUSIONS: This is the first report showing that both general and central obesity are associated with increased risk of AF in an Eastern population with much lower level of obesity than in the West. As both AF and obesity are increasing in developing countries, the results should have important public health implications.

[Screening for the microdeletions of azoospermia factor on the Y chromosome in male infertile patients from Guangzhou].

OBJECTIVE: To investigate the relationship between microdeletion of azoospermia factor (AZF) and male infertility. METHODS: Multiplex PCR was used to detect Y chromosome microdeletion in AZFa, AZFb and AZFc in 103 cases of idiopathic azoospermia, 72 cases of severe idiopathic oligozoospermia, and 60 healthy male controls. RESULTS: No microdeletion was found in 60 controls. Y chromosome microdeletion was found in 19 of 175 azoospermia patients, the total prevalence rate of microdeletion was 10.9%. There were 15 cases (11 for azoospermia, 4 for severe oligozoospermia) in AZFc (8.6%), 3 cases (1 for azoospermia, 2 for severe oligozoospermia) in AZFb+c (1.7%), 1 case (azoospermia) in AZFa+b+c (0.6%). According to statistics, the difference of microdeletion rate between two groups was significant(P < 0.01). CONCLUSION: Y chromosome microdeletions is an important reason of azoospermia. Screening of Y chromosome microdeletions for azoospermia patients before intracytoplasmic sperm injection treatment is essential.

[The clinical application of whole chromosome painting probes in preimplantation genetic diagnosis for translocation carriers].

OBJECTIVE: To make preimplantation genetic diagnosis (PGD) for female translocation carriers by analyzing first polar bodies (1PBs) with whole chromosome painting probe (WCP). METHODS: WCP was used in fluorescence in situ hybridization (FISH) analysis of 1PBs for four female Robertsonian carriers presented for PGD with 45 XX, der(13;14)(q10;q10) karyotype. All the patients underwent ovarian stimulation and during 6 h after oocyte retrieval 1PBs were biopsied and WCP were used in FISH. On day 3 after fertilization embryos diagnosed as normal or balanced were transferred. RESULTS: A total of 61 oocytes were collected in 4 PGD cycles. Of the 54 matured oocytes, 50 were biopsied and 45 were fixed successfully. Results were obtained in 40 1PBs. Overall, 74.1% (40/54) oocytes were diagnosed. The fertilization rate and good embryo rate were 64.8% (35/54) and 65.7% (23/35) respectively. Two clinical pregnancies were obtained. One patient delivered a normal female baby with karyotype 46, XX in June 2006. For another patient, the fetus spontaneously aborted at 9th week of pregnancy with karyotype of 45, X confirmed by amniotic villus diagnosis. CONCLUSION: WCP can differentiate normal, balanced and unbalanced oocytes accurately and can be used as an efficient PGD method for female carriers of translocation.

[Outcome of repeated epididymal sperm aspiration or testicular sperm extraction in azoospermic patients].

OBJECTIVE: To review the outcome of repeated percutaneous sperm aspiration (PESA) and testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI). METHODS: Forty-three cycles of 31 cases of azoospermic patients which underwent at least two PESA or TESE for ICSI from January 2001 to December 2002 were collected. The sperm retrieval, fertilization, implantation and clinical pregnancy were analyzed. RESULTS: Twenty-four cases underwent PESA and 7 cases underwent TESE. There were not any complications in these patients. Compared with the first cycle of 154 cases, the fertilization rate, implantation rate and clinical pregnancy rate were 78.39% vs 73.64%, 19.68% vs 18.38% and 34.88% vs 37.91%, respectively(P > 0.05). CONCLUSIONS: Repeated PESA or TESE is safe and well tolerated in azoospermic patients. Compared with the first cycle, the differences of repeated PESA or TESE cycles in fertilization rate, implantation rate and clinical pregnancy rate were not statistically significant.

[Effect of antisperm antibodies on assisted reproduction].

The formation of antisperm antibodies (AsAb) results from the disruption of the blood-testis barrier by a variety of mechanisms, which leads to exposure of immunogenic sperm antigens to the immune system and initiates an immune response. AsAb can impair the fusion of sperm and egg and even the embryo development, resulting in infertility. The etiology of AsAb, effect of AsAb on assisted reproduction and treatment of AsAb in the literature are reviewed in this article.

[Sperm retrieval methods and pregnancy outcome of 100 azoospermia patients].

OBJECTIVES: To review the retrospective treatment results of the azoospermia patients during January 2001 to January 2002 in the fertility center. METHODS: One hundred males attempted intracytoplasmic sperm injection (ICSI) cycle for treatment of azoospermia. All patients were undergone sperm retrieval by percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) while their wives received conventional ovarian hyperstimulation. The hormone levels, testicular histology, the rates of sperm retrieval, fertilization, implantation and pregnancy were analysed and evaluated. RESULTS: Sperm were retrieved by PESA in 76 of 100 (76%) and by TESE in 23 of 100 (23%) men of azoospermia. The fertilization rate, implantation rate and clinical pregnancy rate were 71.3%, 20.35% and 42.11% respectively in PESA group, and 75.18%, 22.05% and 41.60% respectively in TESA group. Thirty-two clinical pregnancies were achieved with 15 ongoing pregnancies and subsequent live delivery for 15 cases in PESA group, and 2 cases of miscarriage, while 10 clinical pregnancies were achieved with 6 ongoing pregnancies, 2 cases of live delivery and 2 cases of miscarriage in TESA group. One case failed to retrieve sperm by TESE and canceled. CONCLUSIONS: Hormonal levels and testicular histology are unable to predict which men with azoospermia will have sperm retrieved by PESA and TESE. PESA and TESE with ICSI are effective methods to treat azoospermia. There were no significant differences in fertilization, implantation and clinical pregnancy rate between two groups.