O-120 Unravelling reproductive failure by scrutinizing the male germline code

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Chung ◽  
S Cheung ◽  
Z Rosenwaks ◽  
G D Palermo

Abstract Study question Can whole exome sequencing (WES) of spermatozoal DNA provide insight into understanding the different steps that lead to inability of a couple to reproduce? Summary answer The identification of germline mutations can clarify different aspects of reproductive failure in couples with unexplained infertility. What is known already The limitation of a routine semen analysis in evaluating sperm characteristics, even according to the most stringent criteria, lies in its inability to provide substantial information on spermatozoa performance in ART. As a result, ancillary tests are being used to further assess the male gamete’s reproductive potential. More recently, WES of the male genome, carried out on somatic cells, has become a powerful technique that can potentially shed light on the genetic causes of infertility. Here, we aim to preferentially detect germline mutations by sequencing spermatozoal DNA to pinpoint genes related to different underlying etiologies for reproductive failure. Study design, size, duration In a 5-year period, 25 couples subdivided according to their ICSI outcomes were included in this study. Sperm aneuploidy assessment by fluorescent in situ hybridization (FISH) and copy number variant (CNV) analysis by WES were carried out on ejaculated specimens from consenting male partners. Following CNV analysis, gene mutation profiles were compared between the fertile (n = 10) and infertile cohorts (n = 15), as well as in relation to the reasons for reproductive failure. Participants/materials, setting, methods FISH was performed on at least 1,000 sperm cells with a threshold of > 1.6%. DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 605±137 ng/ul; quality, 1.7±0.1 nm) for CNV analysis by WES. Mutations corresponding to the CNV were annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was >1.5 or < 0.5 times the median read depth in the control. Main results and the role of chance Couples (n = 25) (maternal age, 38.6±3yrs; paternal age, 39.7±5yrs) had normal somatic karyotypes with normal semen parameters (59.2±30x106/mL concentration, 44.8±18% motility) by WHO standards. The fertile (n = 10) cohort underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate and 10/12 (83.3%) term pregnancies. The infertile cohort (n = 15) underwent 21 ICSI cycles, achieving a 66% (62/84) fertilization rate and 5/17 (29.4%) clinical pregnancies, all resulting in pregnancy loss. Sperm aneuploidy was consistently higher in the infertile (8.4%) versus fertile (4.0%) cohort (P<0.00001), as observed by FISH and DNA sequencing. For both cohorts, WES detected deletions responsible for sperm–egg fusion (ADAM3A) and acrosomal development (SPACA, SPATA), explaining the necessity for ICSI in these couples. The infertile cohort was characterized by 4 reasons for cycle failure: complete fertilization failure, poor embryo development, implantation failure, and pregnancy loss. Couples with complete fertilization failure (n = 4) had deletions (PLCZ1, PIWIL1) indicating a sperm-related oocyte-activating deficiency. Those with poor embryo development (n = 5) had mutations (HAUS1, KIF4A, XRN1) essential for centrosome integrity and spindle/microtubular stabilization. Couples who did not achieve pregnancy (n = 7) had a mutation (IL9R) in common related to cytokine constituents in the implantation pathway. Those with pregnancy losses (n = 5) had mutations (NLRP7, TP53) on post-implantation genes. Limitations, reasons for caution Several germline mutations, related to the different reasons for these couples’ reproductive failure, were identified. Although intriguing, these findings are still new and need to be validated in a larger study population. In addition, while maternal age was controlled for, we cannot definitively exclude other confounding female factors. Wider implications of the findings Additional screening methods for infertile couples, particularly those with unexplained infertility, can be used to clarify elusive factors underlying their reproductive ability. A genetic screening of spermatozoal DNA may therefore be considered a potential tool in precision medicine for the treatment of subtle male factor infertility. Trial registration number n/a

1974 ◽  
Vol 14 (71) ◽  
pp. 723 ◽  
Author(s):  
LP Cahill ◽  
RD Kearins ◽  
MAde B Blockey ◽  
BJ Restall

Two experiments were conducted to investigate the level of fertilization failure that was attributed to oestrous ewes being mounted and marked by the harnessed ram but where service accompanied with ejaculation had not occurred. In ewes marked by harnessed rams, the occurrence of failure of service with ejaculation was determined by examining a 2 cm cube polyurethane sponge which was placed in the vagina before mating. The vaginal sponge was removed and examined at 0 to 24 hours after the onset of oestrus. In experiment 1, a second sponge was inserted at the time of removal of the first sponge and removed 24 hours later. The presence of spermatozoa in the vaginal sponge was taken as evidence of service. The service rate was determined by this method in one half of the flock. The fertilization rate (experiment 1) or the proportion of ewes that returned to oestrus following natural mating (experiment 2) was determined in the remainder of the ewes. In experiment 1 the service rate, as determined by examination of the sponges, was 86 per cent and the fertilization rate was 88 per cent. In experiment 2 the service rate was 94 per cent and the non-return to service rate was 94 per cent. The vaginal sponge reduced the number of 1 1/2 year old maiden ewes exhibiting oestrus but had no influence on the parous ewes. It was concluded that lack of service with ejaculation in oestrous ewes may be the major cause of reproductive failure in flocks where the levels of failure of fertilization are low.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Cheung ◽  
Z Rosenwaks ◽  
G D Palermo

Abstract Study question Can whole exome sequencing (WES) of spermatozoa from azoospermic men identify mutations related to the etiology of their infertility and ability to support a pregnancy? Summary answer Key de novo germline mutations that affect sperm production and/or embryo developmental competence may explain reproductive failure in azoospermic men, regardless of the etiology. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. While post-testicular azoospermia is mainly due to a mechanical obstruction, testicular azoospermia, the most severe form, is characterized by scattered functional germinal epithelia that strive to support the meiotic process during gamete development. To shed light on the etiology of this condition, genetic studies have been performed, albeit exclusively on peripheral blood. We chose to perform a genomic assessment of spermatozoa to preferentially detect germline mutations that may be passed to the progeny. Study design, size, duration In a 2-year period, we recruited infertile men undergoing epididymal aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular retrieval for nonobstructive azoospermia (NOA; n = 10). Four additional men were included as fertile controls. Following WES, copy number variants (CNVs) and gene mutation profiles were compared between the OA and NOA patients, and within those two categories, in relation to whether they generated a clinical pregnancy (fertile) or not (infertile). Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens of consenting men (DNA concentration, 762±492 ng/ul; quality, 1.7±0.1 nm). CNVs, gene mutations, duplications, and deletions were detected using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was >1.5 or < 0.5 times the median read depth in the control. Common mutations in the OA and NOA cohorts were assessed according to the couples’ clinical outcome. Main results and the role of chance Of 29 couples (maternal age, 41.9±7yrs; paternal age, 42.5±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility) while 10 NOA men underwent testicular biopsy (0.03±0.2x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.8%; NOA, 1.9%). In OA patients, only 3 genes were deleted, mainly housekeeping-related, while in the NOA cohort, 5 genes were deleted, involved in RNA transcription (POLR2L) and apoptosis (AP5M1), in addition to spermiogenic functions (AP1S2, AP1G2, APOE). OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles that resulted in a pregnancy and delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, a gene affecting only sperm production. The infertile individuals (n = 10) all had a deletion on PRB1, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 10 ICSI cycles, yielding a clinical pregnancy rate of 70% (7/10). The fertile men (n = 7) had a concurrent gene deletion involved in stem cell lineage differentiation (MPIG6B). Their infertile counterparts (n = 3) had deleted genes involved in spermato/spermio-genesis (n = 6) and, most importantly, in early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC9, REPIN1, MAPRE3, and ARL4C). Limitations, reasons for caution This is a novel study with limited observations. The presence of housekeeping-related mutations in fertile OA men as well as the DNA replication mutation in infertile OA patients, considering the acquired condition, remains puzzling. Although maternal age was controlled for, confounding factors related to the female partner cannot be excluded. Wider implications of the findings Screening men for germline mutations provides valuable information on their ability to reproduce, regardless of the etiology of azoospermia. Genome profiling was able to identify reasons for failed reproductive performance in azoospermic men, particularly those individuals with secretory azoospermia (NOA). Genomic profiling may identify gametes with retained embryo developmental competence. Trial registration number n/a


2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Anne C. Kotto-Kome ◽  
Celso Silva ◽  
Valerie Whiteman ◽  
Xiaoyuan Kong ◽  
Michael E. Fant

The aims of this study were to determine the prevalence of anti-PLAC1 antibodies in normal pregnant women and in women with infertility or recurrent pregnancy loss (RPL). Secondary outcomes were the development of complications associated with anti-PLAC1 seropositivity and the rate of seroconversion during pregnancy. Sera from 103 healthy pregnant women and 45 women with unexplained infertility or RPL were analyzed by ELISA. The prevalence of anti-PLAC1 antibodies was 2% in healthy pregnant women and 4.5% in women with unexplained infertility or RPL (P=0.355). There was no detectable association of seropositivity with increased risk of pregnancy complications. Finally, 2% of women seroconverted during pregnancy. The prevalence of anti-PLAC1 antibodies in women with unexplained infertility or RPL is not significantly higher than the prevalence in normal pregnant women. However, the sample size in this study was too small. The exposure to the PLAC1 antigen during pregnancy can lead to the spontaneous development of antibodies.


Author(s):  
Waqas Ahmad ◽  
Shahid Bilal ◽  
Sarah Azhar ◽  
Muhammad Aitmaud Uddolah Khan ◽  
Nasima Iqbal ◽  
...  

Aims: As no data is available in Pakistan so the aim of current study is to find out the link of multiple risk factors with recurrent pregnancy loss (RPL) in Pakistan. Study Design: Case control study. Place and Duration of Study: Study conducted in Obstetrics and Gynecology Clinic of Benazir Bhutto Hospital, Holy Family Hospital Rawalpindi and Polyclinic Hospital Islamabad from November 2018 to April 2019. Methodology: Subjects were investigated on the basis of an in depth Performa. For data analysis Statistical package for social sciences version-20 was used. Beside this, height in cm, weight in kg and blood pressure in mmHg were recorded. All the statistical calculations were performed by using SPSS 20. For association analysis of qualitative variables Spearman bivariate correlation was calculated while for numerical variables ANOVA was applied. Multinomial logistic regression model was used and the odd ratio and relative risk were calculated. Results: Among cases 91.34% were having spontaneous miscarriage and majority (64.86%) were during first trimester. Spearman bivariate correlation reported a strong association of recurrent pregnancy loss with the risk factors including family history, smoking, obesity, history of hypertension and history of diabetes, having highly significant p-values, on the hand, significant association of maternal age with the frequency of recurrent pregnancy loss was found but not with the paternal age and parity. The multinomial logistic regression model showed that smokers were19.012 times more prone to develop recurrent pregnancy loss. Conclusion: The multiple risk factors including maternal age, obesity, smoking, family history, body mass index, hypertension and diabetes have a strong association with the recurrent pregnancy loss. So keeping these risk factors in mind a careful evaluation of each pregnancy is necessary to reduce the risk of recurrent pregnancy loss.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Seo. Pe. Yin

Abstract Study question Will artificial activation of oocytes alter the ploidy status of the resultant blastocysts? A sibling-oocytes pilot study Summary answer AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. What is known already Despite introducing ICSI as an aid to improve chances of fertilization, fertilization failure can still occur in 2–3% of ICSI cycles. Fertilization is a complex process triggered by a cascade of events following calcium (Ca2+) oscillations. Evidence suggests that the deficiency, localization or altered structure of the sperm-derived protein PLCζ in oocyte activation may be a reason for meiotic II arrest in the oocyte. Artificial oocyte activation has been proposed to compensate for the lack of calcium oscillation and resumes meiotic progression. There are however insufficient studies to determine its effect on the chromosomal status of the resultant blastocysts. Study design, size, duration This is a prospective, randomized study conducted at our Center from August-October 2020. A total of 20 couples intended for ICSI + Preimplatation Genetic Testing for Aneuploidy (PGT-A) cycles were recruited based on fulfilling one of the following criteria: 1) previous total fertilization failure (TFF), 2) history of low fertilization rate (<30%), 3) more than 2 cycles of failed IVF cycles (no implantation) 4) poor embryo development (no blastocysts formed) and 5) severe male factor. Participants/materials, setting, methods A total of 231 MII oocytes underwent randomization in a 1:1 ratio between AOA-ICSI and control group. All oocytes are subjected to ICSI treatment. Oocytes in the AOA-ICSI group are treated in 25μl droplets 10μM ready to use bicarbonate buffered calcium ionophore (Kitazato, Japan) for 15 minutes post-ICSI. The blastocysts were biopsied and subjected to PGT-A. Primary outcome was the aneuploidy rate and secondary outcomes were fertilization rate and blastocyst rate. Main results and the role of chance There were 11 out of 40 (27.5%) aneuploid blastocysts in the AOA-ICSI group and 7 out of 23 aneuploid blastocysts (30.4%) in the control group [odds ratio (OR) = 0.87; 95% confidence interval (CI) 0.28–2.68, p = 0.8040). There was no statistically significant difference between both groups. However, fertilization rate of the AOA- ICSI group was significantly higher than the fertilization rate in the control group (68.6% vs 49.6% respectively, OR = 2.22; 95% CI, 1.31–3.81, p = 0.0034). There were 40 blastocysts formed in the AOA-ICSI group and 23 blastocysts formed in the control group. It was found that the AOA-ICSI group yielded a higher blastocyst rate (49.4%) compared to the control group (41.1%) (OR = 1.40; 95% CI, 0.71 to 2.78, p = 0.3379) but the difference was not statistically significant. Limitations, reasons for caution The possibility of TE cells biopsied may not be representative of the whole blastocyst makes it possible to have false clinical data. The dosage and time were also not evaluated in this study as exposure time was found to be a critical factor of fertilization rate in a previous study. Wider implications of the findings: This study showed that AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. Additional studies involving a larger number of patients with more specific indication can further justify the benefits of AOA as a therapeutic application. Trial registration number NA


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Lópe. Ruiz ◽  
C Olmed. Illueca ◽  
M Bare. Gómez ◽  
S Roy. Bolea ◽  
L Aba. d. Velasco ◽  
...  

Abstract Study question Does Calcymicin improve reproductive outcomes of ICSI cycles in cases of fertilization failure and/or embryo blockage indications? Summary answer The application of the Calcymicin after ICSI improves reproductive outcomes, especially in cases with clinical indication of fertilization failure. What is known already According to the bibliography, deficiencies in the oocyte activation process frequently lead to failed ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) in the oocyte using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. Ca2+ Ionophores have been shown to trigger an initial Ca2+ spike in the ooplasm that activates Ca2+/Calmodulin dependent protein kinase II, which initiates the cascade of cellular events leading to oocyte activation. Previous results suggest that Ca2+ ionophore treatment can give live offspring after failed ICSI cycles. Study design, size, duration 270 oocytes collected from 17 patients who presented cycles with low fertilization rates and/or embryo blockage or poor quality embryos (according to ASEBIR’s embryo classification criteria) were retrospectively analyzed. Oocytes were divided into two groups, a control group that underwent conventional IVF/ICSI and another group that underwent an ICSI cycle with AOA. Study groups were defined according to clinical indications and subgroups according to AOA or control. All data were collected from 2017 until 2020. Participants/materials, setting, methods Among the 270 oocytes of the study sample, 142 belonged to the control group and 128 belonged to the AOA group. The AOA group oocytes were activated for 15 minutes immediately after ICSI using a prepared solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization rate and type, blastocyst formation rate, blastocyst quality, embryo kinetics, and pregnancy rates were analyzed, all of them were compared to FIV/ICSI cycles without oocyte activation (control group). Main results and the role of chance In the analyses of the whole sample of oocytes, the AOA treatment gave a fertilization rate of 72.5%, which was significantly higher compared to 53.8% of the control cycles (p = 0.002). Good quality blastocysts and pregnancy rates were also significantly higher than the control (p = 0.01). In the group with an indication of fertilization failure, a significantly higher fertilization rate was recorded compared to the control (65% and 33%, respectively). A higher rate of abnormal embryos with three pronuclei was also found compared to the control (p < 0.001). There were no significant differences in blastocyst formation rates, quality, or embryo kinetics (p > 0.05). In the group with an indication of embryo blockage/poor embryo quality, a significantly higher rate of good quality blastocysts and lower blastulation time were recorded compared to the control (p < 0.05). Limitations, reasons for caution The safety of the AOA technique with Ca2+ ionophore has not been fully demonstrated. In our study, none of the newborns had malformations, and gestational weeks and birth weights were normal. However, further studies on the safety of this technique are needed to implement it routinely in human reproduction clinics. Wider implications of the findings: According to these findings, an increase in the initial levels of calcium in the oocyte through the application of the Ca2+ ionophore A23187 after ICSI improves the results of failed assisted reproduction cycles, especially in the case of those diagnosed with fertility failure, which is a clear indication for AOA. Trial registration number Not applicable


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Cahe. Peretz ◽  
J Haas ◽  
E Hadi ◽  
H Carp ◽  
A Hershk. Klement

Abstract Study question Is unexplained recurrent pregnancy loss (RPL) related to long term cancer morbidity? Summary answer Recurrent unexplained pregnancy loss patients showed lower cancer morbidity. This trend was significant in the secondary aborters and in a sub-analysis for gynecological cancers. What is known already The association between infertility and cancer was studied, but has scarcely been studied in RPL; One study reported a higher incidence of breast and uterine cancers, while another found no association. Immune dysfunction is a possible cause of ‘unexplained RPL’; RPL patients have an increased number of toxic natural killer cells (NKs) in both peripheral blood and decidua. The immune system is also involved in the recognition of cancer cells, potentially leading to effective killing. It is possible that the NK populations in RPL are capable of a better response towards cancer cells in the uterine environment and related organs. Study design, size, duration A retrospective cohort study comparing RPL patients and patients with normal deliveries presenting between 1990 –2010 and followed up until 2018. Participants/materials, setting, methods The RPL (exposed) group consisted of patients with 3 or more losses between 5–24 weeks. The comparison (unexposed) group included women who gave birth, and were not listed in the registry of RPL patients. Matching was based on maternal age and year of delivery, which was matched to the date of admission to the RPL clinic. Patients’ data were cross-linked to the national cancer registry. Kaplan-Meier survival curves were used to compare cancer incidence. Main results and the role of chance The RPL group comprised of 937 RPL patients, compared to 4685 patients with a live birth. The mean follow up time was 16.3 ±5.3 years for RPL cases and 15.9 ± 4.9 for the comparison group. Groups were compared in terms of lifetime risk, post-admission risk and according to cancer type. In a Univariate analysis, the life time risk for cancer was 5.3% (49/937) among RPL patients and 6.8% (317/4685) in the comparison group (p = 0.08). Survival analysis showed the same trend - a lower cancer morbidity in RPL patients (p = 0.06). The low cancer morbidity was more prominent, reaching statistical significance in secondary RPL patients (p = 0.05) , but not in primary RPL (p = 0.4). Breast cancer was the most common tumor, but was neither more nor less common in RPL than in the comparison group. Gynecological cancers, however, were significantly less common in RPL patients: 0.3% (3/937) compared to 1.3% (60/4685) in the comparison group (p = 0.01). After adjustment for maternal age the odds ratio for gynecological cancer was 0.247 (p = 0.018, 95% CI 0.077–0.791) and significantly represented in the survival analysis (p = 0.01). Limitations, reasons for caution There was no access to BMI and smoking status. Patients were followed for a mean period of 16 years; cancer may present later than 16 years. Wider implications of the findings: Unexplained RPL is assumed to have an immunological basis. Our study may provide an indirect support for hyper-responsive immunological mechanisms in RPL patients. Further research is needed to deepen our understanding of the underlying mechanisms and possibly to facilitate treatment options. Trial registration number Not applicable


2020 ◽  
Author(s):  
Ji Liu ◽  
Yan-Hua Zhou ◽  
Xiao-Xiao Wang ◽  
Ling-Xi Tong ◽  
Yan-Hong Li ◽  
...  

Abstract Background: Different types of incubators have been designed for gamete and embryo culture in the past few years. The main differences of these incubators are humidity, temperature and gas control system, which play important roles in regulating the steady state of culture media. The objective of this study was to compare the effects of different types of incubators (air jacket incubators and water jacket incubators) on embryo development and clinical outcomes in human in vitro fertilization (IVF).Methods: First, the physical performances of different incubators were tested by mimicking routine IVF procedures. After that, in a randomized controlled trial, 1013 cumulus oocyte complexes from 43 patients were equally divided into two groups, fertilized and cultured in two types of incubators to analyze the effects of different types of incubators on embryo development and clinical outcomes. Results: We found that temperature recovery time in the air jacket incubator was significantly shorter than that in water jacket incubator. Although the O2 recovering time was also significantly shorter in the air jacket incubator as compared with the water jacket incubator, no significant differences were observed in the CO2 recovering time between two groups, which was also verified by pH recovering time of culture media. Besides, the temperature of culture medium in the dish covered with oil recovered more quickly in the air jacket incubators than that in water jacket incubators. However, there were no significant differences observed in the fertilization rate, Day 3 high-quality embryo formation rate, blastocyst formation rate, good blastocyst rate and clinical outcomes between two groups.Conclusions: These results indicate that the microenvironment, especially the temperature, in air jacket incubator recover faster than that in conventional water jacket incubator, however, there were no significant differences in embryo development and clinical outcomes between two types of incubators.


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