A novel frameshift variant of LMX1A that leads to autosomal dominant non-syndromic sensorineural hearing loss: functional characterization of the C-terminal domain in LMX1A.

Non-syndromic sensorineural hearing loss (NSHL) is a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. There is, at present, no curative treatment for genetic hearing loss (HL). Early molecular diagnosis of progressive disorders and elucidation of the causes and pathomechanisms are essential for developing therapeutic strategies. Here, we identified a novel rare frameshift variant of LMX1A (c.915dup), which resulted in the C-terminal-altered and -truncated LMX1A (p.Val306Cysfs*32). This C-terminal frameshift mutation co-segregated with autosomal dominant (AD) NSHL in a four-generation Chinese family, suggesting that the LMX1A non-missense mutation is also contributed to ADNSHL. In this family, the affected individuals exhibited the variable auditory phenotypes ranging from profound congenital deafness at birth or to mild/moderate HL in adulthood. We also found that the embryonic cells carrying with the heterozygous variant significantly expressed several upregulated HL-associated genes at transcriptional level. In vitro splicing assay suggested that the LMX1A mRNA with c.915dup did not cause nonsense-mediated decay and was translated into a truncated LMX1A. In addition, electrophoresis mobility shift assay and luciferase assays have shown that the highly conserved C-terminal domain (amino acid 306-382) of the LMX1A was required for regulating the protein-DNA interaction and transactivation in vitro. Furthermore, apoptosis assays suggested that the C-terminal domain of the LMX1A was important for mediating apoptosis in the cochlear hair cells. Our work provided the multiline of the evidence to support that non-missense mutation of LMX1A leads to ADNSHL and the C-terminal domain of LMX1A is important for mediating transcriptional activity and associated with promoting apoptosis in the cells.

Bi-allelic variants in ASTL cause abnormal fertilization or oocyte maturation defects.

Fertilization is a fundamental process of development, and the blocking mechanisms act at the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, permeating and fusing after fertilization. In clinical practice, some couples undergoing recurrent IVF failures that mature oocytes had abnormal fertilization for unknown reason. Ovastacin encoded by ASTL cleave the ZP protein ZP2 and play a key role in preventing polyspermy. Here, we identified bi-allelic variants in ASTL that are mainly characterized by fertilization problems in humans. All four independent affected individuals had bi-allelic frameshift variants or predicted damaging missense variants, which follow a Mendelian recessive inheritance pattern. The frameshift variants significantly decreased the quantity of ASTL protein in vitro. And all missense variants affected the enzymatic activity that cleaves ZP2 in mouse egg in vitro. Three knock-in female mice (corresponding to three missense variants in patients) all show subfertility due to low embryo developmental potential. This work presents strong evidence that pathogenic variants in ASTL cause female infertility and provides a new genetic marker for the diagnosis of fertilization problems.

Comprehensive preimplantation genetic testing for balanced insertional translocation carriers.

BACKGROUND: Balanced insertional translocations (BITs) can increase the risk of infertility, recurrent miscarriages or neonatal birth defects due to chromosomal imbalances in gametes. However, studies on preimplantation genetic testing (PGT) for patients carrying BITs are inadequate. METHODS: A preimplantation genetic genotyping and haplotype analysis approach was developed and implemented in this study. Genome-wide SNP genotyping was performed, followed by core family-based haplotype analysis. The balanced insertion segments in euploid embryos were inferred from the haplotypes inherited from the carrier parent. RESULTS: A total of 10 BIT carrier couples were enrolled in our study. 15 in vitro fertilisation cycles were conducted, resulting in 73 blastocysts biopsied and subjected to PGT analysis. Among these, 20 blastocysts displayed rearrangement-related imbalances, 13 exhibited de novo aneuploidies, 15 presented a complex anomaly involving both imbalances and additional aneuploidies, while 25 were euploid. Within the euploid embryos, 12 were balanced carrier embryos and 13 were non-carrier embryos. To date, eight non-carrier and one carrier embryos have been transferred, resulting in seven clinical pregnancies. All pregnancies were recommended to perform prenatal diagnosis, our date revealed complete concordance between fetal genetic testing results and PGT results. Presently, five infants have been born from these pregnancies, and two pregnancies are still ongoing. CONCLUSION: The proposed method facilitates comprehensive chromosome screening and the concurrent identification of balanced insertions or normal karyotypes in embryos. This study offers an effective and universally applicable strategy for BIT carriers to achieve a healthy pregnancy and prevent the transmission of BITs to their offspring.

Bi-allelic missense variants in MEI4 cause preimplantation embryonic arrest and female infertility.

Preimplantation embryonic arrest is an important pathogenesis of female infertility, but little is known about the genetic factors behind this phenotype. MEI4 is an essential protein for DNA double-strand break formation during meiosis, and Mei4 knock-out female mice are viable but sterile, indicating that MEI4 plays a crucial role in reproduction. To date, MEI4 has not been found to be associated with any human reproductive diseases. Here, we identified six compound heterozygous and homozygous MEI4 variants-namely, c.293C > T, p.(Ser98Leu), c.401C > G, p.(Pro134Arg), c.391C > G, p.(Pro131Ala), c.914A > T, p.(Tyr305Phe), c.908C > G, p.(Ala303Gly), and c.899A > T, p.(Gln300Leu)-in four independent families that were responsible for female infertility mainly characterized by preimplantation embryonic arrest. In vitro, we found that these variants reduced the interaction between MEI4 and DNA. In vivo, we generated a knock-in mouse model and demonstrated that female mice were infertile and were characterized by developmental defects during oogenesis. Our findings reveal the important roles of MEI4 in human reproduction and provide a new diagnostic marker for genetic counseling of clinical infertility patients.

Live Birth with or without Preimplantation Genetic Testing for Aneuploidy.

BACKGROUND: Embryo selection with preimplantation genetic testing for aneuploidy (PGT-A) may improve pregnancy outcomes after initial embryo transfer. However, it remains uncertain whether PGT-A improves the cumulative live-birth rate as compared with conventional in vitro fertilization (IVF). METHODS: In this multicenter, randomized, controlled trial, we randomly assigned subfertile women with three or more good-quality blastocysts to undergo either PGT-A or conventional IVF; all the women were between 20 and 37 years of age. Three blastocysts were screened by next-generation sequencing in the PGT-A group or were chosen by morphologic criteria in the conventional-IVF group and then were successively transferred one by one. The primary outcome was the cumulative live-birth rate after up to three embryo-transfer procedures within 1 year after randomization. We hypothesized that the use of PGT-A would result in a cumulative live-birth rate that was no more than 7 percentage points higher than the rate after conventional IVF, which would constitute the noninferiority margin for conventional IVF as compared with PGT-A. RESULTS: A total of 1212 patients underwent randomization, and 606 were assigned to each trial group. Live births occurred in 468 women (77.2%) in the PGT-A group and in 496 (81.8%) in the conventional-IVF group (absolute difference, -4.6 percentage points; 95% confidence interval [CI], -9.2 to -0.0; P<0.001). The cumulative frequency of clinical pregnancy loss was 8.7% and 12.6%, respectively (absolute difference, -3.9 percentage points; 95% CI, -7.5 to -0.2). The incidences of obstetrical or neonatal complications and other adverse events were similar in the two groups. CONCLUSIONS: Among women with three or more good-quality blastocysts, conventional IVF resulted in a cumulative live-birth rate that was noninferior to the rate with PGT-A. (Funded by the National Natural Science Foundation of China and others; ClinicalTrials.gov number, NCT03118141.).

Analysis of assisted reproductive outcomes for gynecologic cancer survivors: a retrospective study.

OBJECTIVE: To examine the reproductive outcomes of assisted reproductive technology (ART) in gynecologic cancer patients and to assess maternal and neonatal complications. METHODS: Women diagnosed with gynecologic cancer who underwent their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment between 2013 and 2021 at Shanghai Ji Ai Genetics and IVF Institute were included in this study. Infertile women without any history of cancer were matched to the cancer group. The primary outcome was the cumulative live birth rate. Baseline and follow-up data were compared between groups using Student's t-tests for normally distributed variables and with Chi-square test for categorical variables. A propensity score-based patient-matching approach was adopted to ensure comparability between individuals with and without specific cancer type. RESULTS: A total of 136 patients with a history of gynecologic cancer and 241 healthy infertile controls were included in this study. Endometrial cancer constituted 50.70% of the cases and cervical cancer constituted 34.60% of the cases. The cancer group exhibited significantly shorter duration of stimulation, lower levels of estradiol, lower number of retrieved oocytes, day-3 embryos, and blastocysts compared to the control group (P < 0.05). The cumulative live birth rate of the gynecologic cancer group was significantly lower than that of the control group (36.10% vs. 60.50%, P < 0.001). Maternal and neonatal complications did not significantly differ between the groups (P > 0.05). The endometrial cancer and cervical cancer groups showed significantly lower cumulative live birth rates than their matched controls (38.60% vs. 64.50%, P = 0.011 and 24.20% vs. 68.60%, P < 0.001, respectively). CONCLUSIONS: These findings highlight the decreased occurrence of pregnancy and live birth in female gynecologic cancer patients undergoing ART, particularly in endometrial cancers and cervical cancers. These findings have important implications for counseling and managing gynecologic cancer patients undergoing ART.

Detection of cryptic balanced chromosomal rearrangements using high-resolution optical genome mapping.

BACKGROUND: Chromosomal rearrangements have profound consequences in diverse human genetic diseases. Currently, the detection of balanced chromosomal rearrangements (BCRs) mainly relies on routine cytogenetic G-banded karyotyping. However, cryptic BCRs are hard to detect by karyotyping, and the risk of miscarriage or delivering abnormal offspring with congenital malformations in carrier couples is significantly increased. In the present study, we aimed to investigate the potential of single-molecule optical genome mapping (OGM) in unravelling cryptic chromosomal rearrangements. METHODS: Eleven couples with normal karyotypes that had abortions/affected offspring with unbalanced rearrangements were enrolled. Ultra-high-molecular-weight DNA was isolated from peripheral blood cells and processed via OGM. The genome assembly was performed followed by variant calling and annotation. Meanwhile, multiple detection strategies, including FISH, long-range-PCR amplicon-based next-generation sequencing and Sanger sequencing were implemented to confirm the results obtained from OGM. RESULTS: High-resolution OGM successfully detected cryptic reciprocal translocation in all recruited couples, which was consistent with the results of FISH and sequencing. All high-confidence cryptic chromosomal translocations detected by OGM were confirmed by sequencing analysis of rearrangement breakpoints. Moreover, OGM revealed additional complex rearrangement events such as inverted aberrations, further refining potential genetic interpretation. CONCLUSION: To the best of our knowledge, this is the first study wherein OGM facilitate the rapid and robust detection of cryptic chromosomal reciprocal translocations in clinical practice. With the excellent performance, our findings suggest that OGM is well qualified as an accurate, comprehensive and first-line method for detecting cryptic BCRs in routine clinical testing.

A prospective study to evaluate whether serum kisspeptin is a marker predictive of the first-trimester miscarriage of women who conceive in IVF.

PURPOSE: Kisspeptin is an emerging biomarker for the discrimination of viable pregnancy. The aim of the study is to determine whether serum kisspeptin can predict the first-trimester miscarriage and compare it with serum HCG in the prediction of the first-trimester miscarriage. METHODS: This study is a prospective case-control design including 178 women who had experienced early miscarriage (n = 21) and viable single pregnancy (n = 157), following frozen-thawed embryo transfer (FET) from May to December 2019. Serum samples on 14 days, 21 days, and 28 days after FET were collected for kisspeptin and HCG detection. TRIAL REGISTRATION NUMBER: NCT03940495. RESULTS: On day 21 after FET, serum kisspeptin levels were significantly lower in the early miscarriage group [0.260 (0.185-0.375)] vs in the viable single-pregnancy group [0.370 (0.280-0.495)] (p = 0.005). Similar results were shown on day 28 after FET, the serum kisspeptin levels were significantly lower in the early miscarriage group [0.270 (0.200-0.330)] vs in the viable single pregnancy group [0.670 (0.455-1.235)] (p < 0.001). But on day 14 after FET, serum kisspeptin levels were comparable in the early miscarriage group [0.260 (0.210-0.325)] and in the viable single-pregnancy group [0.280 (0.215-0.340)] (p = 0.551). Serum kisspeptin levels on days 21 and 28 have a poor predictive value of miscarriage compared with serum HCG levels. [Day 21: AUC = 0.687 (kisspeptin) and 0.816 (HCG); Day 28: AUC = 0.896 (kisspeptin) and 0.909 (HCG)]. CONCLUSIONS: Serum kisspeptin on day 14 failed to discriminate between miscarriage and ongoing pregnancies, and days 21 and 28 had poor predictive values of miscarriage.

ADGB variants cause asthenozoospermia and male infertility.

Asthenozoospermia is one of the main factors leading to male infertility, but the genetic mechanisms have not been fully elucidated. Variants in the androglobin (ADGB) gene were identified in an infertile male characterized by asthenozoospermia. The variants disrupted the binding of ADGB to calmodulin. Adgb-/- male mice were infertile due to reduced sperm concentration (< 1 × 106 /mL) and motility. Spermatogenesis was also abnormal, with malformation of both elongating and elongated spermatids, and there was an approximately twofold increase in apoptotic cells in the cauda epididymis. These exacerbated the decline in sperm motility. It is surprising that ICSI with testicular spermatids allows fertilization and eventually develops into blastocyst. Through mass spectrometry, we identified 42 candidate proteins that are involved in sperm assembly, flagella formation, and sperm motility interacting with ADGB. In particular, CFAP69 and SPEF2 were confirmed to bind to ADGB. Collectively, our study suggests the potential important role of ADGB in human fertility, revealing its relevance to spermatogenesis and infertility. This expands our knowledge of the genetic causes of asthenozoospermia and provides a theoretical basis for using ADGB as an underlying genetic marker for infertile males.

Homozygous Mutations in BTG4 Cause Zygotic Cleavage Failure and Female Infertility.

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.

Bi-allelic Missense Pathogenic Variants in TRIP13 Cause Female Infertility Characterized by Oocyte Maturation Arrest.

Normal oocyte meiosis is a prerequisite for successful human reproduction, and abnormalities in the process will result in infertility. In 2016, we identified mutations in TUBB8 as responsible for human oocyte meiotic arrest. However, the underlying genetic factors for most affected individuals remain unknown. TRIP13, encoding an AAA-ATPase, is a key component of the spindle assembly checkpoint, and recurrent homozygous nonsense variants and a splicing variant in TRIP13 are reported to cause Wilms tumors in children. In this study, we identified homozygous and compound heterozygous missense pathogenic variants in TRIP13 responsible for female infertility mainly characterized by oocyte meiotic arrest in five individuals from four independent families. Individuals from three families suffered from oocyte maturation arrest, whereas the individual from the fourth family had abnormal zygote cleavage. All displayed only the infertility phenotype without Wilms tumors or any other abnormalities. In vitro and in vivo studies showed that the identified variants reduced the protein abundance of TRIP13 and caused its downstream molecule, HORMAD2, to accumulate in HeLa cells and in proband-derived lymphoblastoid cells. The chromosome mis-segregation assay showed that variants did not have any effects on mitosis. Injecting TRIP13 cRNA into oocytes from one affected individual was able to rescue the phenotype, which has implications for future therapeutic treatments. This study reports pathogenic variants in TRIP13 responsible for oocyte meiotic arrest, and it highlights the pivotal but different roles of TRIP13 in meiosis and mitosis. These findings also indicate that different dosage effects of mutant TRIP13 might result in two distinct human diseases.

Loss of ACTL7A causes small head sperm by defective acrosome-acroplaxome-manchette complex.

BACKGROUND: Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms. METHODS: Nuclear morphology analysis was used to observe the sperm head shape of ACTL7A-mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a. RESULTS: The new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a-KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a-KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a-KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice. CONCLUSIONS: Loss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

Heterozygous loss-of-function variants in LHX8 cause female infertility characterized by oocyte maturation arrest.

PURPOSE: The genetic causes of oocyte maturation arrest leading to female infertility are largely unknown, and no population-based genetic analysis has been applied in cohorts of patients with infertility. We aimed to identify novel pathogenic genes causing oocyte maturation arrest by using a gene-based burden test. METHODS: Through comparison of exome sequencing data from 716 females with infertility characterized by oocyte maturation arrest and 3539 controls, we performed a gene-based burden test and identified a novel pathogenic gene LHX8. Splicing event was evaluated using a minigene assay, expression of LHX8 protein was assessed in HeLa cells, and nuclear subcellular localization was determined in both HeLa cells and mouse oocytes. RESULTS: A total of 5 heterozygous loss-of-function LHX8 variants were identified from 6 independent families (c.389+1G>T, c.412C>T [p.Arg138∗], c.282C>A [p.Cys94∗]; c.257dup [p.Tyr86∗]; and c.180del, [p.Ser61Profs∗30]). All the identified variants in LHX8 produced truncated LHX8 protein and resulted in loss of LHX8 nuclear localization in both HeLa cells and mouse oocytes. CONCLUSION: By combining genetic evidence and functional evaluations, we identified a novel pathogenic gene LHX8 and established the causative relationship between LHX8 haploinsufficiency and female infertility characterized by oocyte maturation arrest.

Gankyrin has a potential role in embryo implantation via activation of STAT3.

Embryo implantation, a critical step during the mammalian reproductive process, requires normal developing blastocysts and a receptive endometrium. Endometriosis, a common pathologically benign gynecological condition, is associated with decreased fertility and reduced endometrial receptivity. The oncoprotein, Gankyrin, has been associated with endometriosis and endometrial cancer. Here, we examined the role of Gankyrin during the process of embryo implantation and found that Gankyrin expression levels were significantly increased during the mid-secretory phase, but unaffected during the proliferative phase in the human endometrium. Using an in vitro cell adhesion assay to examine the cell adhesion rate of BeWo trophoblast spheroids to Gankyrin knockdown or overexpressing human endometrial carcinoma RL95-2 cells, we demonstrated that the adhesion rate was significantly reduced in Gankyrin-knockdown RL95-2 cells, while overexpression of Gankyrin promoted cell adhesion. Furthermore, we found that the downregulation of Gankyrin inhibited STAT3 activation and subsequent matrix metalloproteinase 2 (MMP2) expression, while overexpression led to STAT3 activation and MMP2 expression. In vivo, we found that Gankyrin expression was increased in the endometrium after conception but decreased with the prolongation of gestation time in female mice. siRNA-mediated knockdown of Gankyrin in the uterine horn led to a significant reduction in the number of implanted embryos 9 days post-gestation, which was associated with a decrease in p-STAT3 expression and MMP2 transcription. Taken together, our findings indicate that Gankryin has a potential role in embryo implantation via STAT3 activation.

Can the combination of time-lapse parameters and clinical features predict embryonic ploidy status or implantation?

RESEARCH QUESTION: Can models based on artificial intelligence predict embryonic ploidy status or implantation potential of euploid transferred embryos? Can the addition of clinical features into time-lapse monitoring (TLM) parameters as input data improve their predictive performance? DESIGN: A single academic fertility centre, retrospective cohort study. A total of 773 high-grade euploid and aneuploid blastocysts from 212 patients undergoing preimplantation genetic testing (PGT) between July 2016 and July 2021 were studied for ploidy prediction. Among them, 170 euploid embryos were single-transferred and included for implantation analysis. Five machine learning models and two types of deep learning networks were used to develop the predictive algorithms. The predictive performance was measured using the area under the receiver operating characteristic curve (AUC), in addition to accuracy, precision, recall and F1 score. RESULTS: The most predictive model for ploidy prediction had an AUC, accuracy, precision, recall and F1 score of 0.70, 0.64, 0.64, 0.50 and 0.56, respectively. The DNN-LSTM model showed the best predictive performance with an AUC of 0.78, accuracy of 0.77, precision of 0.79, recall of 0.86 and F1 score of 0.83. The predictive power was improved after the addition of clinical features for the algorithms in ploidy prediction and implantation prediction. CONCLUSION: Our findings emphasize that clinical features can largely improve embryo prediction performance, and their combination with TLM parameters is robust to predict high-grade euploid blastocysts. The models for ploidy prediction, however, were not highly predictive, suggesting they cannot replace preimplantation genetic testing currently.

Analysis of the risk of complications during pregnancy in pregnant women with assisted reproductive technology: a retrospective study using registry linkage from 2013 to 2018 in Shanghai, China.

BACKGROUND: To evaluate the differences in pregnancy outcomes between assisted reproductive technology (ART) patients and natural pregnant women in Shanghai, China in the past 6 years objectively. And to assess the feasibility of the research method of registry-database linkage in mainland China. METHODS: This retrospective study was conducted using registry-database linkage. A total of 8102 pregnancies with ART and 8096 parturients with spontaneous conception (SC) from 10 reproductive centers and 111 hospitals composed our retrospective study. The primary outcomes were the rates of obstetric complications (pregnancy-induced hypertention [PIH], gestational diabetes mellitus [GDM], placenta previa, mode of delivery, preterm birth [PTB], low birth weight [LBW], and macrosomia). The prenatal outcomes were compared between ART and SC parturients, frozen-thawed embryo transfer (FET) and fresh embryo transfer, and in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We calculated odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: The final matching rate of the target population was 92% by using registry linkage. ART resulted in a higher rate of multiple birth, PTB, LBW, cesarean section, placenta previa and GDM compared with SC in the singleton cohort. In ART patients, pregnant women with FET had a significantly higher risk of PIH than those with fresh embryo transfer (14.1% Vs 9.3%, AOR1.528, 95% CI 1.303-1.793), but there was no difference between IVF and ICSI. FET is also related to the severity of PIH. CONCLUSIONS: ART increased the rate of complications during pregnancy, the risk and severity of PIH in patients with FET was higher than that in patients with fresh embryo transfer. The registry-database linkage study is an objective and feasible research method in mainland China.

Investigation of the role of X chromosome inactivation and androgen receptor CAG repeat polymorphisms in patients with recurrent pregnancy loss: a prospective case-control study.

BACKGROUND: Previous research has revealed that skewed X chromosome inactivation (SXCI) and androgen receptor (AR) CAG polymorphisms are associated with increased risk of recurrent pregnancy loss (RPL); however, the results are conflicting, and the underlying mechanisms remain unclear. This study investigated the role of SXCI and AR CAG polymorphisms in patients with RPL and explored whether the underlying mechanisms were related to the ovarian reserve and preimplantation embryo aneuploidy. METHODS: This was a prospective case-control study carried out in a tertiary hospital-based reproductive medicine center. An external validation RPL cohort was recruited during the study period. Data on baseline and cycle characteristics were collected. X-chromosome inactivation (XCI) was measured using a human AR assay. AR polymorphisms were assessed using quantitative fluorescent polymerase chain reactions and direct sequencing. Blastocysts of the patients with RPL were tested by single nucleotide polymorphism microarray based preimplantation genetic testing for aneuploidy. RESULTS: In total, 131 patients with idiopathic RPL and 126 controls were included for the case-control study. Patients with RPL exhibited a significantly more skewed XCI distribution pattern (67.71 ± 10.50 vs. 64.22 ± 10.62, p = 0.011), as well as significantly shorter bi-allelic mean (18.56 ± 1.97 vs. 19.34 ± 2.38, p = 0.005) and X-weighted bi-allelic mean (18.46 ± 2.02 vs. 19.38 ± 2.53, p = 0.001) of AR CAG repeats. Multivariate logistic regression models indicated that CAG repeat < 20, SXCI, and duration of stimulation were independently associated with the risk of RPL. However, SXCI and AR CAG polymorphisms were not associated with ovarian reserve or preimplantation embryo aneuploidy in the RPL group, and the same results were attained in a separate validation cohort of 363 patients with RPL. CONCLUSION: SXCI and AR CAG polymorphisms are related to RPL; however, these two factors do not lead to RPL by affecting the ovarian reserve or increasing embryo aneuploidy. The roles of SXCI and AR CAG in RPL may involve other mechanisms that require further investigation. TRIAL REGISTRATION: NCT02504281, https://www. CLINICALTRIALS: gov (Date of registration, 21/07/2015; date of enrolment of the first subject, 30/07/2015).

The use of frozen embryos and frozen sperm have complementary IVF outcomes: a retrospective analysis in couples experiencing IVF/Donor and IVF/Husband.

BACKGROUND: The cryopreservation of sperm or embryos has been an important strategy in the treatment of infertility. Recently studies have revealed the outcomes after IVF (in vitro fertilization) treatment for single-factor exposure either to frozen sperm or embryos. METHODS: This retrospective study was to uncover the exposure to both frozen sperm and embryo effects using IVF/H (in vitro fertilization using husbands' fresh sperm) or IVF/D (in vitro fertilization using donors' frozen sperm) treatment. RESULTS: The results showed the clinical pregnancy rate (CPR), live birth rate (LBR) and low birth weight rate (LBW) increased to 63.2% (or 68.1%), 61.1% (or 66.4%) and 15.8% (or 16.2%) after using frozen embryo transfer within Group IVF/H (or Group IVF/D). After using frozen sperm, the high-quality embryo rate (HER) increased to 52% and baby with birth defect rate (BDR) reduced to 0% in subgroup D/ET comparing to subgroup H/ET. While the fertilization rate (FER), cleavage rate (CLR), HER and multiple pregnancy rate (MUR) reduced to 75%, 71%, 45% and 9.2% in subgroup D/FET comparing to subgroup H/FET. Finally, our study found accumulative frozen gamete effects, including both sperm and embryos, led to the significantly increasing in the HER (p < 0.05), CPR (p < 0.001), LBR (p < 0.001) and LBW (p < 0.05) in subgroup D/FET comparing to subgroup H/ET. CONCLUSION: The use of frozen embryos and frozen sperm have complementary IVF outcomes. Our findings highlighted the parent's distinguished frozen effect not only for clinical studies but also for basic research on the mechanism of cellular response adaptations to cryopreservation.

Re-denudation of residual cumulus cells on day 3 increases the accuracy of cell-free DNA detection in spent embryo culture medium.

PURPOSE: To evaluate the effect of two-step denudation on maternal contamination, ploidy concordance between spent embryo culture medium (SCM) and trophectoderm, blastocyst formation, and clinical outcome. METHODS: Sibling embryos of the same couple were re-denuded (treatment) and not re-denuded (control) on day 3, and trophectoderm biopsy and SCM collection were performed on day 5/6. Sex chromosomes of 20 pairs of SCM and biopsy samples were analyzed to determine the reduction in maternal contamination. Blastocyst formation, implantation, and ongoing pregnancy rates were analyzed by recruiting 565 cleavage embryos on day 3. A total of 113 SCM samples and their corresponding trophectoderm results were collected for ploidy concordance analysis. RESULTS: The detection rate of XX between the treatment and control groups was significant (12/20 (60.0%) versus 19/20 (95.0%), p = 0.02). Concordance of sex chromosomes between the two groups was significant (17/20 (85.0%) versus 8/19 (42.1%), p = 0.003). There were no significant differences in blastocyst formation rate, implantation rate, and ongoing pregnancy rate between the two groups. Among the 113 pairs of SCM and its corresponding trophectoderm, 37 cases (33.33%) were completely concordant, 39 cases (35.14%) were partially concordant, and 35 cases (31.53%) were discordant. CONCLUSION: Our results suggest that re-denudation on day 3 reduces the influence of maternal contamination and improves the accuracy of cfDNA detection. Moreover, the procedure had no significant effect on blastocyst formation, implantation, and ongoing pregnancy rates. In addition, the ploidy concordance approached 70% compared with biopsy, which is acceptable but still not ideal.

Transfer of Fresh versus Frozen Embryos in Ovulatory Women.

BACKGROUND: Elective frozen-embryo transfer has been shown to result in a higher live-birth rate than fresh-embryo transfer among anovulatory women with the polycystic ovary syndrome. It is uncertain whether frozen-embryo transfer increases live-birth rates among ovulatory women with infertility. METHODS: In this multicenter, randomized trial, we randomly assigned 2157 women who were undergoing their first in vitro fertilization cycle to undergo either fresh-embryo transfer or embryo cryopreservation followed by frozen-embryo transfer. Up to two cleavage-stage embryos were transferred in each participant. The primary outcome was a live birth after the first embryo transfer. RESULTS: The live-birth rate did not differ significantly between the frozen-embryo group and the fresh-embryo group (48.7% and 50.2%, respectively; relative risk, 0.97; 95% confidence interval [CI], 0.89 to 1.06; P=0.50). There were also no significant between-group differences in the rates of implantation, clinical pregnancy, overall pregnancy loss, and ongoing pregnancy. Frozen-embryo transfer resulted in a significantly lower risk of the ovarian hyperstimulation syndrome than fresh-embryo transfer (0.6% vs. 2.0%; relative risk, 0.32; 95% CI, 0.14 to 0.74; P=0.005). The risks of obstetrical and neonatal complications and other adverse outcomes did not differ significantly between the two groups. CONCLUSIONS: The live-birth rate did not differ significantly between fresh-embryo transfer and frozen-embryo transfer among ovulatory women with infertility, but frozen-embryo transfer resulted in a lower risk of the ovarian hyperstimulation syndrome. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China; Chinese Clinical Trial Registry number, ChiCTR-IOR-14005406 .).