A novel homozygous LRRC6 mutation causes male infertility with asthenozoospermia and primary ciliary dyskinesia in humans.

BACKGROUND: Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes. OBJECTIVES: To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved. MATERIALS AND METHODS: The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations. RESULTS: In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer dynein arms and inner dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband. DISCUSSION AND CONCLUSION: This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.

Bilateral versus unilateral orchidopexy: IVF/ICSI-ET outcomes.

Introduction: Cryptorchidism is a common genital disorder. Approximately 20% of azoospermic or infertile men reported having histories of cryptorchidism. Bilateral cryptorchidism may have been more condemned than unilateral cryptorchidism. Early treatment by orchidopexy is the definitive procedure for cryptorchid patients with cryptorchidism. However, fertility potency after orchidopexy may be adversely affected and assisted reproduction techniques will be required for infertile patients. Objective: To compare the reproductive outcomes between unilateral and bilateral orchidopexy groups. Methods: A retrospective cohort study at a tertiary hospital, including a total of 99 infertile men who underwent orchidopexy to treat cryptorchidism and subsequently underwent their first IVF/ICSI-ET cycle. Men were grouped according to the laterality of their cryptorchidism and orchidopexy surgeries they received. Fertilization rate and live birth rate were chosen as parameters for evaluating outcomes. Results: The sperm concentration and viability were significantly higher in unilateral orchidopexy group than in bilateral orchidopexy group (28.09 ± 27.99 vs 7.99 ± 14.68, P=0.001; 33.34 ± 22.52 vs 11.95 ± 17.85, P=0.001). Unilateral orchidopexy group showed lower demand for ICSI (66.07% vs 95.35%, P<0.001). Interestingly, both groups exhibited similar rates of fertilization, clinical pregnancy, live birth and birth defect. Boy birth ratio was lower in bilateral orchidopexy group as compared to unilateral orchidopexy group (27.27% vs 58.62%, P=0.026). Conclusion: A history of bilateral orchidopexy surgery correlates with a worsened sperm parameter and a higher demand for ICSI as compared to patients with history of unilateral orchidopexy. However, this does not influence the final live birth rate.

What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles among anovulatory women? A retrospective cohort study.

OBJECTIVE: Different endometrial preparation protocols are used prior to frozen-thawed embryo transfer (FET). It is not clear whether letrozole-stimulated cycles are beneficial for anovulatory women compared to artificial cycles (AC) in anovulatory women. Our objective was to investigate whether clinical outcomes following frozen-thawed embryo transfer in letrozole-stimulated cycles differ from artificial cycles in anovulatory women. STUDY DESIGN: This is a retrospective cohort study conducted in a public fertility center in China comparing letrozole-stimulated and artificial cycles during FET. A total of 5,322 anovulatory women undergoing the first cycle of FET between January 2020 and December 2021. The primary outcome was the live birth rate. The secondary outcomes included biochemical pregnancy, clinical pregnancy, ongoing pregnancy, miscarriage, and ectopic pregnancy. RESULTS: The live birth rates were significantly higher in the letrozole compared with AC (49.57 % vs. 45.22 %) both in the crude (odds ratio [OR] 1.19, 95 % confidence interval [CI] 1.01-1.40) and adjusted analysis (aOR 1.27, 95 %CI 1.08-1.50). After controlling for potential covariates, the adjusted probability of biochemical pregnancy (65.71 % vs. 63.25 %, aOR 1.20, 95 %CI: 1.01-1.43), clinical pregnancy (61.53 % vs. 58.79 %, aOR 1.20, 95 %CI: 1.02-1.43), and ongoing pregnancy (50.14 % vs. 45.66 %, aOR 1.28, 95 %CI: 1.08-1.51) were higher among those using letrozole-stimulated cycles than among those undergoing artificial cycles. The miscarriage rate was lower following letrozole compared with artificial cycles (aOR 0.59, 95 %CI: 0.43-0.80). The ectopic pregnancy in the letrozole group was similar to that in the artificial cycles. CONCLUSIONS: Letrozole-stimulated FET was significantly associated with higher rates of biochemical pregnancy, clinical pregnancy, ongoing pregnancy and live birth, and with a lower rate of miscarriage, compared with artificial cycles.

Luteal phase support of intramuscular progesterone associated with lower hypertensive disorders of pregnancy as compared to vaginal progesterone: A cohort study.

OBJECTIVE: To compare the hypertensive disorders of pregnancy (HDP) risk between vaginal and intramuscular (IM) progesterone in programmed frozen-thawed embryo transfer (FET) cycles. METHODS: This was a retrospective cohort study at a tertiary hospital, and only women who achieved ongoing pregnancy after programmed FET between January 2018 and June 2022 were included. Women with chronic hypertension before pregnancy or with history of gestational hypertension or pre-eclampsia in previous pregnancies were excluded. All women were divided into IM progesterone or vaginal progesterone groups according to the route of progesterone supplementation. Follow-up information on obstetric complications and neonatal outcomes were obtained by telephonic interviews. The primary outcome was HDP. Association between routes of progesterone supplementation and HDP was assessed by subgroup analysis and multivariable logistic regression. RESULTS: A total of 5891 programmed FET cycles (3196 IM progesterone cycles and 2695 vaginal progesterone cycles) were included in the analysis. The prevalence of HDP in the IM progesterone group was significantly lower than that of the vaginal progesterone group (6.54% vs 9.17%, P < 0.001). Therein, the prevalence of gestational hypertension (3.57% vs 4.94%, P = 0.009) and pre-eclampsia (2.97% vs 4.23%, P = 0.009) in the IM progesterone group were all significantly lower as compared to the vaginal progesterone group. According to subgroup analysis, IM progesterone was associated with lower HDP risk in all subgroups. The logistic regression analysis showed that the IM progesterone cycles were associated with lower risk of HDP compared to vaginal progesterone cycles (adjusted odds ratio 0.66, 95% CI: 0.54-0.80, P < 0.001). CONCLUSION: Among women undergoing programmed FET cycles, progesterone supplementation with IM progesterone was associated with reduced HDP risk compared to vaginal progesterone.

Effectiveness of atosiban in women with previous single implantation failure undergoing frozen-thawed blastocyst transfer: study protocol for a randomised controlled trial.

BACKGROUND: Uterine contractions may interfere with embryo implantation in assisted reproductive technology. To reduce these contractions and improve success rates, the oxytocin antagonist atosiban has been suggested for administration during embryo transfer. The aim of this study is to evaluate the effectiveness of atosiban in increasing live birth rates among women who have previously experienced a single implantation failure and are scheduled for single blastocyst transfer. METHODS AND ANALYSIS: We conduct a single-centre randomised controlled study comparing atosiban and placebo in women undergoing a single blastocyst transfer with a previous failed blastocyst transfer. Women with endocrine or systemic illnesses, recurrent miscarriages, uterine malformations or fibroids, untreated hydrosalpinx, endometriosis (stage III or IV) or uterine fibroids, as well as women undergoing preimplantation genetic testing, are ineligible. The primary outcome is live birth resulting from the frozen-thawed embryo transfer. Secondary outcomes include biochemical/clinical pregnancy, miscarriage, ectopic pregnancy, multiple pregnancies as well as maternal and perinatal outcomes. We plan to recruit 1100 women (550 women per group). This will allow us to demonstrate or refute an increase in live birth rate from 40% to 50%. Data analysis will follow the intention-to-treat principle. We will measure patterns of uterine peristalsis which will allow subgroup analysis for women with or without uterine peristalsis. ETHICS AND DISSEMINATION: This study has been approved by the Institutional Review Board of Northwest Women's and Children's Hospital (No. SZ2019001). Written informed consent will be obtained from each participant before randomisation. The results of the trial will be presented at scientific meetings and reported in publications. TRIAL REGISTRATION NUMBER: ChiCTR1900022333.

The natural cycle protocol of endometrial preparation for frozen embryo transfer decreases the miscarriage rate in women with recurrent pregnancy loss.

OBJECTIVE: To investigate whether different endometrial preparation methods lead to different results. DESIGN: Retrospective cohort study. PATIENTS: Women with recurrent pregnancy loss undergoing frozen embryo transfer (FET). INTERVENTIONS: Natural cycle (NC) protocol (n = 111) with no drug or human chorionic gonadotropin (HCG) used for endometrial preparation, vs. the hormone replacement therapy (HRT) protocol (n = 797) with estrogen or gonadotropin releasing hormone agonist (GnRH-a) plus estrogen used for endometrial preparation. MAIN OUTCOME MEASURES: Miscarriage rate and live birth rate (LBR). RESULTS: Compared to women in the HRT protocol, women undergoing NCs had fewer previous FET cycles, lower antral follicle counts (AFCs), fewer oocytes retrieved and a thicker endometrium on the day of progesterone administration. Women in the HRT group had a higher miscarriage rate (29.4% vs. 17.2%) and a lower LBR (37% vs. 46.9%) than the rates of women in the NC group. Univariate analysis showed that female age also had a negative association with the miscarriage rate. Logistic regression indicated that endometrial preparation using the NC protocol was linked to a decreased likelihood of miscarriage. CONCLUSIONS: The NC protocol decreased the miscarriage rate and increased the LBR for patients with recurrent pregnancy loss compared with the HRT protocol.

The time interval between laparoscopic tubal ligation and frozen-thawed embryo transfer does not affect the reproductive outcomes.

BACKGROUND: Hydrosalpinx may decrease implantation and pregnancy rates after embryo transfer. Laparoscopic tubal ligation after embryo freeze and before frozen-thawed embryo transfer (FET) is effective at improving reproductive outcomes for hydrosalpinx patients. This study is to find out the optimal interval between laparoscopic tubal ligation and FET. METHODS: We retrospectively analyzed 259 infertile women who performed laparoscopic tubal ligation for embryo freeze and FET. Participants were divided into three groups, based on the interval between laparoscopic tubal ligation and FET. Group I: <30 days; Group II: 31- 60 days; Group III: >60 days. Outcomes of cleavage-stage and blastocyst-stage embryo FET were analyzed respectively. RESULTS: There was no significant difference in clinical pregnancy rate, live birth rate, implantation rate, biochemical pregnancy rate, ectopic pregnancy rate, miscarriage rate and preterm birth rate among the three groups, in both cleavage-stage and blastocyst-stage embryo FET cycles. In cleavage-stage embryo FET cycle, singleton gestational age was significantly younger in group III (38.11 ± 2.28 weeks) compared with group I (39.29 ± 1.06 weeks, P = 0.001) and group II (38.96 ± 1.05, P = 0.026). Singleton birth weight was significantly heavier in group II (3.65 ± 0.32 Kg) compared with group I (3.38 ± 0.29 Kg, P = 0.001) and group III (3.35 ± 0.60 Kg, P = 0.004). Twin birth weight was significantly heavier in group III (2.72 ± 0.43 Kg) compared to group I (2.23 ± 0.67 Kg, P = 0.002). In blastocyst-stage embryo FET cycles, twin gestational age was significantly younger in group II (34.07 ± 3.18 weeks) compared with group I (35.56 ± 2.27 weeks, P = 0.049) and group III (36.50 ± 1.47 weeks, P = 0.005). Twin birth weight was significantly heavier in group III (2.71 ± 0.39 Kg) compared to group II (2.39 ± 0.67 Kg, P = 0.009). CONCLUSIONS: The duration of the interval between laparoscopic tubal ligation and FET does not affect the reproductive outcomes; however, it may affect the neonate outcomes to some extent.

SCM is potential resource for non-invasive preimplantation genetic testing based on human embryos single-cell sequencing.

The ongoing development of assisted reproductive technologies has provided hope to individuals struggling with infertility, promising the potential for a healthy pregnancy. One significant innovation in field of pre-implantation genetic screening (PGS) requires the biopsy of embryos or oocytes, which has potential implications for the health and development of the resultant offspring. Therefore, a non-invasive approach to preimplantation genetic screening is highly sought after. The clinical application of non-invasive preimplantation genetic testing (ni-PGT) is currently limited, with its sensitivity and specificity requiring further investigation. In this study, we used 218 human embryos for single-cell whole genome amplification (WGA), along with ni-PGT of blastocoele fluid (BF) and spent culture medium (SCM). Whole blastocyst (WB), trophectoderm biopsy (TB), and inner cell mass (ICM) from embryo biopsies were used as controls to track genomic signal alterations. Our results showed that the overall genome similarity between SCM and ICM was higher than that of BF. Apart from the Y chromosome, both SCM and ICM demonstrated numerous variant sites across other chromosomes.Further categorization of gene variants in these two sample types revealed that missense variants were the most prevalent, single nucleotide polymorphisms were more common than insertions or deletions, and C > T was the dominant single nucleotide variants in both ICM and SCM. Lastly, we found that the mutant genes in SCM and ICM had different biological functions and pathways. This study indicates that SCM provides a more effective source of embryonic DNA for preimplantation genetic screening, offering a novel reference point for genetic screening research.

Estradiol on trigger day: Irrelevant to live birth rates of fresh cycles but positively associated with cumulative live birth rates.

OBJECTIVE: To assess the effects of estradiol (E2) on trigger day on cumulative live birth rates (CLBRs), and pregnancy outcomes after fresh and frozen-thawed embryo transfer (FET). METHODS: This multicenter retrospective cohort study included 42 315 patients from five reproductive centers. Six subgroups were divided according to E2 on trigger day (<1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, >5000 pg/mL). Smooth curve fitting and nonlinear mixed-effects models were used. RESULTS: When E2 was <5500 pg/mL, the CLBR increased by 10% for every 1000 pg/mL increase in E2. When E2 was between 5500 and 13 281 pg/mL, CLBR increased by 1.8% for every 1000 pg/mL increase in E2. When E2 was >13 281 pg/mL, CLBR decreased by 3% for every 1000 pg/mL increase in E2. From group E2 < 1000 to group E2 > 5000 pg/mL, pregnancy and live birth rates in fresh cycles were not related to E2. The live birth rate after FET was higher in the E2 ≥ 5000 pg/mL group than in the E2 < 1000 pg/mL group (odds ratio [OR] 4.03, and 95% confidence interval [CI] 3.74-4.35; adjusted OR 1.20, 95% CI 1.05-1.37). CONCLUSION: CLBR is associated with E2 on trigger day in a segmented manner. Pregnancy and live birth rates in fresh cycles were not associated with E2. The live birth rate in FET cycles was highest when E2 ≥ 5000 pg/mL.

Effect modification by developmental stage of embryos on the association between late follicular phase progesterone elevation and live birth in fresh transfers.

BACKGROUND: Late follicular phase progesterone elevation (LFPE) during ovarian stimulation is associated with reduced live birth rates (LBRs) after cleavage-stage embryo transfer. However, due to better synchronization with a stimulated endometrium, prior studies shown that LFPE had no effect on transferring embryos at blastocyst stage. The study aim to exam whether the developmental stage of embryos and serum progesterone levels on the day of human chorionic gonadotropin (hCG) administration jointly affect the odds of live birth in fresh fresh IVF/intracytoplasmic sperm injection (ICSI) cycles.  METHODS: The single-center retrospective cohort study included a total of 4,471 fresh embryo transfer cycles with 2,342 at cleavage stage versus 2,129 at blastocyst stage. Patients underwent IVF/ICSI with ovarian stimulation in gonadotropin-releasing hormone antagonist protocol. The serum progesterone level was examined both as a continuous variable and as a categorical variable by quartiles. Analysis was performed using the generalized estimating equations framework and multivariate regression models. RESULTS: LBRs were inversely associated with progesterone as a continuous variable on the day of hCG in both the cleavage-stage (crude OR 0.87, 95%CI 0.73-1.03; adjusted OR 0.80, 95% CI 0.65-0.98) and the blastocyst-stage (crude OR 0.66, 95%CI 0.56-0,78; adjusted OR 0.61, 95%CI 0.50-0.73) groups. The interaction testing was highly significant (P = 0.018) indicating an effect modifying role of stage of embryos transferred on the association of pregesterone values with the LBRs in fresh cycles. A similar pattern for a greater reduction in ORs for live birth in cycles with blastocysts transfer was also observed when progesterone was analyzed by interquartile ranges. The findings remained unchanged in subgroup analysis stratified by types of ovarian response. CONCLUSIONS: In fresh cycles, detrimental effect of late follicular phase progesterone elevation on live birth was more prominent in blastocyst-stage group compared with that in clevaged-stage group.

Overweight and obesity affect the efficacy of vaginal vs. intramuscular progesterone for luteal-phase support in vitrified-warmed blastocyst transfer.

OBJECTIVE: To compare the difference in the live birth rates (LBRs) between vaginal progesterone and intramuscular progesterone as luteal-phase support in programmed vitrified-warmed blastocyst transfer cycles and determine whether the association was moderated by overweight/obesity. DESIGN: Retrospective cohort study. SETTING: Tertiary reproductive medicine center. PATIENT(S): Patients who underwent transfer of single vitrified-warmed blastocyst in a programmed cycle between January 2018 and June 2021. INTERVENTION(S): Vaginal or intramuscular progesterone as luteal-phase support. Analysis was performed using the generalized estimating equation framework and multivariate regression models. Interaction testing was used to determine whether overweight/obesity (body mass index of ≥25 kg/m2) moderated the association between progesterone replacement and LBRs. MAIN OUTCOME MEASURE(S): The primary outcome was live birth. The secondary outcomes were biochemical pregnancy, clinical pregnancy, miscarriage, and total pregnancy loss. RESULT(S): A total of 6,905 programmed cycles (4,616 with vaginal progesterone and 2,289 with intramuscular progesterone) were included in the analysis. In the general cohort who underwent cryopreserved blastocyst transfer, the LBRs were 46.23% and 48.62% in the vaginal and intramuscular progesterone groups, respectively (odds ratio [OR], 0.91; 95% confidence interval [CI], 0.82-1.01; adjusted OR [aOR], 0.89; 95% CI, 0.81-0.98), with a significantly increased rate of pregnancy losses in the vaginal progesterone group compared with that in the intramuscular progesterone group (22.22% vs. 18.90%; OR, 1.23; 95% CI, 1.08-1.39; aOR, 1.23; 95% CI, 1.08-1.40). Among normal-weight women, the LBR in the vaginal progesterone group was lower than that in the intramuscular progesterone group (aOR, 0.84; 95% CI, 0.75-0.95). On the other hand, among women with overweight/obesity, the LBRs were similar between the 2 groups of progesterone replacement (aOR, 1.06; 95% CI, 0.86-1.33). Interaction testing of the routes of progesterone administration and overweight/obesity was significant. CONCLUSION(S): Luteal-phase support with vaginal progesterone was associated with reduced LBRs compared with intramuscular progesterone for vitrified-warmed blastocyst transfer, and the association was modified by maternal overweight/obesity. Further research is needed to better understand the mechanisms behind the association.

Comparison of two gonadotropin-releasing hormone agonist suppression protocols for in vitro fertilization in young patients with low body mass index.

OBJECTIVE: To evaluate two different gonadotropin-releasing (pituitary downregulating) hormones used in in vitro fertilization (IVF) on the live birth rate in young patients with low body mass index (BMI) undergoing their first IVF cycle. METHODS: In a retrospective study in a single public medical center, 555 long gonadotropin-releasing hormone agonist (GnRH-a) protocols were compared with 431 prolonged GnRH-a protocols between 2016 and 2018. All analyses were performed using the SPSS version 22.0. The primary measured outcome was live birth rate. RESULTS: Compared with the long protocol, the prolonged protocol required more doses of gonadotropin and a longer duration of ovarian stimulation. Lower levels of serum luteinizing hormone and serum estrogen were detected on the day of chorionic gonadotropin administration, and a lower fertilization rate was found in the prolonged protocol. Although more oocytes were retrieved and more frozen embryos were recorded in the prolonged protocol, the live birth rate per fresh cycle was comparable between the two protocols (P = 0.057). The incidence of ovarian hyperstimulation syndrome was higher in the prolonged protocol group. In the subgroup of women with antral follicle count (AFC) of 12 or less, there was no difference in the live birth rate between the two protocols (P = 0.688). However, for women with AFC > greater than 12, the prolonged protocol was still a positive predictor of live birth rate. The odds ratio was 1.73 (95% confidence interval 1.04-2.89). CONCLUSION: The prolonged protocol might not increase the live birth rate in women with low BMI who are undergoing their first IVF cycle. However, for women with AFC greater than 12, a prolonged protocol could be a good choice to improve the live birth rate.

Comparison of pregnancy outcomes between GnRH antagonist protocol with freeze-all strategy and long-acting GnRH agonist protocol in women with adenomyosis undergoing IVF/ICSI: a propensity-score matching analysis.

BACKGROUND: Plenty of studies explored the most optimal treatment protocol for infertile women with adenomyosis in in-vitro fertilization (IVF) /intracytoplasmic sperm injection (ICSI), however, there is still no consensus on which treatment protocol is ideal for these women at present. So, we conducted this study comparing the pregnancy outcomes in infertile women with ultrasound-diagnosed adenomyosis who underwent GnRH antagonist protocol with freeze-all strategy or long-acting GnRH agonist protocol. METHODS: This was a retrospective cohort study and a propensity-score matching (PSM) analysis including 282 women diagnosed with adenomyosis undergoing their first IVF/ICSI cycle from January 2016 to July 2021 at the Assisted Reproduction Center, Northwest Women's and Children's Hospital, China. The patients were divided into two groups: the GnRH antagonist protocol with freeze-all strategy (n = 168) and the long-acting GnRH agonist protocol with fresh embryo transfer (n = 114) according their treatment protocols. The primary outcome was live birth rate. Cumulative live birth rate was also calculated. RESULTS: After adjusting for confounders, clinical pregnancy rate (49.40% vs 64.04%; odds ratio (OR) 1.33; 95% confidence interval (CI) 0.70 to 2.37; P = 0.358), live birth rate (36.90% vs 45.61%; OR 1.10; 95% CI 0.61 to 2.00, P = 0.753) and cumulative live birth rate (51.79% vs 64.04%; OR 1.01; 95% CI 0.49 to 1.74, P = 0.796) were not significantly different between the GnRH antagonist protocol with freeze-all strategy and long-acting GnRH agonist protocol. Similar results were conducted in PSM analysis with clinical pregnancy rate (46.48% vs 60.56%; OR 1.33; 95% CI 0.76 to 2.34; P = 0.321), live birth rate (32.39% vs 45.07%; OR 1.31; 95% CI 0.63 to 2.72, P = 0.463) and cumulative live birth rate (54.90% vs 60.60%; OR 1.01; 95% CI 0.59 to 1.74, P = 0.958). CONCLUSIONS: For infertile women with adenomyosis, these two treatment protocols resulted in similar pregnancy outcomes. Larger, prospective studies are needed in the future.

Preimplantation genetic testing for recurrent autosomal dominant osteogenesis imperfecta associated with paternal gonosomal mosaicism.

Research Question: How to prevent the transfer of a mutation causing osteogenesis imperfecta (OI) to offspring in a couple with recurrent adverse pregnancy outcomes, when the male partner is a gonosomal mosaic carrier. Design: High-throughput sequencing and first-generation DNA sequencing were performed using the tissues from an aborted fetus and its parents. Regions 2 Mb upstream and downstream of the COL1A1 gene were subjected to multiplex PCR to identify single nucleotide polymorphisms (SNPs) and family haplotypes associated with the disease-causing mutation. Single-cell whole-genome amplification and sequencing were performed on trophoblasts cultured in vitro for 5-6 days to construct embryonic SNP haplotypes, and first-generation sequencing was used for pathogenic locus verification and aneuploidy screening. Preimplantation genetic testing for monogenic disorders (PGT-M) was also performed. Results: The aborted fetus was heterozygous for the COL1A1 mutation c.1454G>A (chr17-48272089, p.Gly485Asp) suspected to cause OI. The variant was also detected in the peripheral blood cells and sperm of the male partner, who appeared to be a gonosomal mosaic carrier of the mutation. Three morphologically usable blastocysts were obtained in vitro and successfully expanded after a trophectoderm biopsy. Two blastocysts were unusable owing to aneuploidy; however, one was euploid and did not carry the paternal mutation. Post-transfer gestation was confirmed by systematic B-scan ultrasound, and amniocentesis findings were consistent with the PGT-M results. Conclusion: Parental gonadal mosaicism was the cause of recurrent terminated pregnancies due to fetal skeletal dysplasia. Using PGT-M to select embryos without the paternal pathogenic mutation prevented the vertical transmission of OI in this family, and a successful pregnancy was achieved.

Embryo selection through non-invasive preimplantation genetic testing with cell-free DNA in spent culture media: a protocol for a multicentre, double-blind, randomised controlled trial.

INTRODUCTION: Morphological evaluation is used to select embryos for in vitro fertilisation. However, it does not fully reflect the implantation potential. Preimplantation genetic testing for aneuploidies (PGT-A) can detect embryonic aneuploidy, but biopsy procedure is invasive. Currently, a non-invasive PGT (ni-PGT) approach using spent medium is being evaluated. However, the clinical benefit of ni-PGT has not been clearly demonstrated. A multicentre randomised trial is needed to verify whether ni-PGT can be an new effective tool for evaluating embryos. METHODS AND ANALYSIS: Overall, 1148 couples aged 35~42 (women) receiving in vitro fertilization-intracytoplasmic sperm injection are planned to be enrolled. Couples will be digitally randomised to (1) ni-PGT and (2) conventional morphology groups at a 1:1 treatment ratio. The primary outcome will be the ongoing pregnancy rate related to the first transfer cycle within 6 months after oocyte retrieval. ETHICS AND DISSEMINATION: The study protocol is approved by the Ethics Committee of Peking University Third Hospital and the participating hospitals. The results will be disseminated through international conferences and scientific journals. TRIAL REGISTRATION NUMBER: NCT04339166.

Is duration of estrogen supplementation associated with clinical outcomes in frozen-thawed autologous single-blastocyst transfer cycles?

PURPOSE: To investigate the relationship between different duration of estrogen administration and live birth rate (LBR) after autologous single frozen blastocyst transfer with hormone replacement therapy. METHODS: A total of 2026 frozen blastocyst transfer cycles in the assisted reproductive center of northwest women and children's hospital from January, 2017, to August, 2020, were retrospectively analyzed. All the cycles were allocated into 3 groups according to the duration of estrogen administration: group A, 11-14 days (n = 346); group B, 15-18 days (n = 1191), and group C, ≥ 19 days (n = 489). Baseline data, clinical, and perinatal outcomes of the three groups were compared. A multivariate regression model was constructed to analyze the association between duration of estradiol administration and clinical outcomes. RESULTS: We did not observe a significant association between duration of estrogen supplementation and LBR in group B (adjusted odds ratio [aOR] 1.14; 95% confidence interval [CI], 0.89-1.45) or group C (aOR 1.16; 95% CI, 0.86-1.56) patients with group A as the reference group, through logistic regression analysis. No statistical differences were observed in perinatal outcomes among the three groups. CONCLUSION: The duration of estrogen administration was not associated with the likelihood of live birth in women undergoing frozen-thawed autologous single-blastocyst transfer.

Ploidy Testing of Blastocoel Fluid for Screening May Be Technically Challenging and More Invasive Than That of Spent Cell Culture Media.

Background: Recent studies have demonstrated that both blastocoel fluid (BF) and spent cell culture media (SCM) have potential as materials for non-invasive or less-invasive pre-implantation genetic analysis. BF may allow more opportunity to obtain cell-free DNA from the inner cell mass (ICM), and it has a lower risk of containing contaminant DNA from cumulus cells, sperm and culture media. There are no data regarding the ICM as a gold standard to evaluate the chromosome constitution of BF or SCM for embryo liquid biopsy. Methods: Two hundred eighteen donated human blastocysts were warmed and cultured in blastocyst culture media for 18-24 h. The corresponding SCM was collected, and only clear ICM was biopsied in blastocysts; otherwise, the whole blastocyst (WB) was biopsied. Quantitative PCR was performed to determine the DNA levels in the SCM and BF before and after amplification. ChromInst was used to amplify BF/SCM and blastocyst DNA before sequencing. Chromosomal copy number variation (CNV) was investigated to evaluate the chromosome constitution. Results: In total, 212 blastocysts were available for SCM and BF collection. The technical success rates (next-generation sequencing data) were 100 and 69.8% (148/212) for SCM and BF, respectively. Among the 148 blastocysts with both SCM and BF data, 101 were euploid and 47 were aneuploid based on ICM (n = 89) or WB (n = 59) analysis as the gold standard. Among all blastocysts, SCM was comparable to BF [specificity: 80.2 versus 61.4% (P = 0.005, χ2 test); sensitivity: 91.5 versus 87.2% (P = 0.738, χ2 test); negative predictive value (NPV): 95.3 versus 91.2% (P = 0.487, χ2 test); positive predictive value (PPV): 68.3% versus 51.3% (P = 0.042, χ2 test)]. The SCM and BF samples were 83.8% (124/148) and 69.6% (103/148) concordant with the corresponding ICM/WB samples when only two categories, euploid or aneuploid/mosaic, were grouped to calculate the concordance. Conclusions: Compared with BF, SCM has superior diagnostic performance, and it is non-invasive for embryos. Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR-BPD-17014087].

Gonadotropin-releasing hormone agonist downregulation combined with hormone replacement therapy improves the reproductive outcome in frozen-thawed embryo transfer cycles for patients of advanced reproductive age with idiopathic recurrent implantation failure.

BACKGROUND: To determine whether gonadotropin-releasing hormone (GnRH) agonist downregulation combined with hormone replacement therapy (HRT) can improve the reproductive outcomes in frozen-thawed embryo transfer cycles for older patients (aged 36-43 years) with idiopathic recurrent implantation failure (RIF). METHODS: This retrospective cohort study involved 549 older patients undergoing their third cleavage-stage embryo or blastocyst transfer over a 5-year period (January 2015-December 2020) at Northwest Women's and Children's Hospital after in vitro fertilization/intracytoplasmic sperm injection cycles. Patients with known endometriosis or adenomyosis were excluded from the study. The patients were divided into three groups according to the endometrial preparation protocol: the natural cycle (NC) group (n = 65), the HRT group (n = 194), and the GnRH agonist downregulation combined with HRT cycle (GnRH agonist-HRT) group (n = 290). The primary outcome was the live birth rate, and the secondary outcomes were the clinical pregnancy, miscarriage, and ongoing pregnancy rates. RESULTS: The live birth rate in the GnRH agonist-HRT group (36.55%) was higher than that in the HRT group (22.16%) and NC group (16.92%) (P < 0.0001). Similarly, a logistic regression model adjusting for potential confounders showed that the live birth rate was higher in the GnRH agonist-HRT group than in the HRT group (odds ratio, 0.594; 95% confidence interval, 0.381-0.926; P = 0.021) and NC group (odds ratio, 0.380; 95% confidence interval, 0.181-0.796; P = 0.010). CONCLUSIONS: The GnRH agonist-HRT protocol improves the live birth rate in frozen-thawed embryo transfer cycles for patients of advanced reproductive age with RIF. We hypothesize that the GnRH agonist-HRT protocol enhances implantation-related factors and promotes optimal endometrial receptivity, leading to an improved live birth rate. These findings are also useful for further investigating the underlying mechanism of the GnRH agonist-HRT protocol in improving the reproductive outcomes for patients of advanced reproductive age with RIF. TRIAL REGISTRATION: This research protocol was approved by the hospital institutional ethics committee (No. 2021002).

Bi-allelic mutations in MOS cause female infertility characterized by preimplantation embryonic arrest.

STUDY QUESTION: Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women? SUMMARY ANSWER: We identified mutations in MOS that may cause human female infertility characterized by preimplantation embryonic arrest (PREMBA), and the effects of the mutations in human embryonic kidney 293T (HEK293T cells) and mouse oocytes provided evidence for a causal relation between MOS and female infertility. WHAT IS KNOWN ALREADY: MOS, an activator of mitogen-activated protein kinase, mediates germinal vesicle breakdown and metaphase II arrest. Female MOS knockout mice are viable but sterile. Thus, MOS seems to be an important part of the mammalian cell cycle mechanism that regulates female meiosis. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing, bioinformatics filtering analysis and genetic analysis were performed to identify two different biallelic mutations in MOS in two independent families. The infertile patients presenting with early embryonic arrest were recruited from October 2018 to June 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: The female patients diagnosed with primary infertility were recruited from the reproduction centres of local hospitals. Genomic DNA from the affected individuals, their family members and healthy controls was extracted from peripheral blood. We performed whole-exome sequencing in patients diagnosed with PREMBA. Functional effects of the mutations were investigated in HEK293T cells by western blotting and in mouse oocytes by microinjection and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: We identified the homozygous missense mutation c.285C>A (p.(Asn95Lys)) and the compound heterozygous mutations c.467delG (p.(Gly156Alafs*18)) and c.956G>A (p.(Arg319His)) in MOS in two independent patients. The mutations c.285C>A (p.(Asn95Lys)) and c.467delG (p.(Gly156Alafs*18)) reduced the protein level of MOS, and all mutations reduced the ability of MOS to phosphorylate its downstream target, extracellular signal-regulated kinase1/2. In addition, the identified mutations reduced the capacity of exogenous human MOS to rescue the metaphase II exit phenotype, and the F-actin cytoskeleton of mouse oocytes was affected by the patient-derived mutations. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from patient oocytes, the exact molecular mechanism affected by MOS mutations and leading to PREMBA is still unknown and should be further investigated using knock-out or knock-in mice. WIDER IMPLICATIONS OF THE FINDINGS: We identified recessive mutations in MOS in two independent patients with the PREMBA phenotype. Our findings reveal the important role of MOS during human oocyte meiosis and embryonic development and suggest that mutations in MOS may be precise diagnostic markers for clinical genetic counselling. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382,82001538 and 82071642), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500 and 21ZR1404800), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service and the collaborative innovation centre project construction for Shanghai Women and Children's Health. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.