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Background: Couples increasingly experience infertility and seek help from assisted reproductive techniques to become pregnant. However, 5%-15% of the couples that are selected for in vitro fertilisation (IVF) experience a total fertilisation failure (TFF), where no zygotes develop despite oocytes and semen parameters appear to be normal. We hypothesise that TFF during IVF could be related to improper membrane fusion of gametes., Objective: To investigate the membrane integrity and fusion proteins in spermatozoa from men in couples experiencing TFF., Materials and Methods: A total of 33 infertile couples, 17 of which experienced TFF during IVF and 16 matched control couples with normal IVF fertilisation rates, were selected and the men re-called to deliver an additional semen sample. Proteins involved in gamete membrane fusion on spermatozoa (IZUMO1, SPESP1 and Syncytin-1) as well as O-glycosylation patterns (Tn and GALNT3), were investigated by immunofluorescence. The DNA fragmentation index, acrosomal integrity and viability of spermatozoa were determined by flow and image cytometry., Results: No significant changes in the expression of GALNT3, Tn and Syncytin-1 were observed between the TFF and control groups. The fraction of spermatozoa expressing SPESP1, the median IZUMO1 staining intensity, and the percentage of viable acrosome-intact spermatozoa were significantly lower in the TFF group compared to controls. Furthermore, following progesterone-induced acrosomal exocytosis, a significant difference in the fraction of spermatozoa expressing SPESP1 and the median IZUMO1 staining intensity were observed between the control and TFF group., Discussion and Conclusion: Our results indicate that acrosomal exocytosis, IZUMO1 and SPESP1 expression in spermatozoa could play a crucial role in achieving fertilisation during IVF. However, the size of our cohort was quite small, and our results need to be validated with quantitative methods in larger cohorts., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

A severe decline in child births has occurred over the past half century, which will lead to considerable population declines, particularly in industrialized regions. A crucial question is whether this decline can be explained by economic and behavioural factors alone, as suggested by demographic reports, or to what degree biological factors are also involved. Here, we discuss data suggesting that human reproductive health is deteriorating in industrialized regions. Widespread infertility and the need for assisted reproduction due to poor semen quality and/or oocyte failure are now major health issues. Other indicators of declining reproductive health include a worldwide increasing incidence in testicular cancer among young men and alterations in twinning frequency. There is also evidence of a parallel decline in rates of legal abortions, revealing a deterioration in total conception rates. Subtle alterations in fertility rates were already visible around 1900, and most industrialized regions now have rates below levels required to sustain their populations. We hypothesize that these reproductive health problems are partially linked to increasing human exposures to chemicals originating directly or indirectly from fossil fuels. If the current infertility epidemic is indeed linked to such exposures, decisive regulatory action underpinned by unconventional, interdisciplinary research collaborations will be needed to reverse the trends., (© 2021. Springer Nature Limited.)

Background: Infertility affects 15%-25% of all couples during their reproductive life span. It is a significant societal and public health problem with potential psychological, social, and economic consequences. Furthermore, infertility has been linked to adverse long-term health outcomes. Despite the advanced diagnostic and therapeutic techniques available, approximately 30% of infertile couples do not obtain a live birth after fertility treatment. For these couples, there are no further options to increase their chances of a successful pregnancy and live birth., Objectives: Three overall questions will be studied: (1) What are the risk factors and natural life courses of infertility, early embryonic loss, and adverse pregnancy outcomes? (2) Can we develop new diagnostic and prognostic biomarkers for fecundity and treatment success? And (3) what are the health characteristics of women and men in infertile couples at the time of fertility treatment and during long-term follow-up?, Material and Methods: ReproUnion Biobank and Infertility Cohort (RUBIC) is established as an add-on to the routine fertility management at Copenhagen University Hospital Departments in the Capital Region of Denmark and Reproductive Medicine Centre at Skåne University Hospital in Sweden. The aim is to include a total of 5000 couples equally distributed between Denmark and Sweden. The first patients were enrolled in June 2020. All eligible infertile couples are prospectively asked to participate in the project. Participants complete an extensive questionnaire and undergo a physical examination and collection of biospecimens (blood, urine, hair, saliva, rectal swabs, feces, semen, endometrial biopsies, and vaginal swabs). After the cohort is established, the couples will be linked to the Danish and Swedish national registers to obtain information on parental, perinatal, childhood, and adult life histories, including disease and medication history. This will enable us to understand the causes of infertility and identify novel therapeutic options for this important societal problem., (© 2021 American Society of Andrology and European Academy of Andrology.)

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P

Study Question: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)?Summary Answer: One in 28 NOA cases carried an identifiable, medically actionable SF.What Is Known Already: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited.Study Design, Size, Duration: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study.Participants/materials, Setting, Methods: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed.Main Results and the Role Of Chance: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population.Limitations, Reasons For Caution: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants.Wider Implications Of the Findings: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA.Study Funding/competing Interest(s): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is it possible, in an unbiased and clinical relevant way, to determine the number of viable acrosome-intact human spermatozoa in ejaculates and to use this as a measure of fertility chances?Summary Answer: Image cytometry enables easy and unbiased quantification of viable acrosome-intact spermatozoa and it correlates with semen quality and fertility status.What Is Known Already: The presence of the acrosome and its ability to respond to physiological inducers (e.g. progesterone) in the female reproductive tract at the appropriate time and place is required for fertilization. However, the available assays are labor intensive and therefore not used clinically.Study Design, Size, Duration: Washed semen samples and capacitated swim-up fractions from volunteers were used to develop the assay. Subsequently washed ejaculates from patients in fertility treatment (n = 156), proven fertile men (n = 54) and volunteers (n = 10) were assessed to evaluate the number of acrosome-intact spermatozoa in the ejaculate (acrosomal status) and compared to other semen parameters, fertility status, fertility treatments and pregnancy rates.Participants/materials, Setting, Methods: Image cytometry was used to assess the fluorescence intensity of Pisum sativum agglutinin and Propidium iodide.Main Results and the Role Of Chance: The assay was validated by inducing the acrosome reaction in swim-up-purified and capacitated spermatozoa with progesterone and ionomycin, and in repeated acrosomal status measurements of washed ejaculates a small coefficient of variation (3.7%) was observed. Men with poor semen quality had fewer viable acrosome-intact spermatozoa in the ejaculate (P = 0.0012; median 32.6% vs. 49.3%). A large proportion (44%) of normozoospermic men from infertile couples had less than the observed median fraction (46%) of viable acrosome-intact spermatozoa in the ejaculate. Furthermore, the total number of viable acrosome-intact spermatozoa was significantly lower among men with male factor infertility compared to fertile men (median 35 vs. 97 mill, P = 1 × 10-7). Men from couples going through one or more ICSI cycles had significant fewer viable acrosome-intact spermatozoa than men from couples who only underwent IUI (P = 0.002; 44.4% vs. 62.0%) and the fraction of viable acrosome-intact spermatozoa appeared better than classical semen parameters in classifying whether or not couples needed ICSI. A positive, although non-significant, tendency toward ongoing pregnancy with an increasing number of viable acrosome-intact spermatozoa was observed (P = 0.2).Large Scale Data: N/A.Limitations, Reasons For Caution: Even larger cohorts of infertile couples are needed to substantiate the clinical application of the assay in regard to estimation of fertility potential of an individual.Wider Implications Of the Findings: The presented assay makes it possible to measure the number of acrosome competent spermatozoa in an ejaculate in a standardized manner and hence may serve as a new biomarker for male fertility. Few spermatozoa in an ejaculate are acrosome competent and it might be a valuable measure when evaluating male reproductive function.Study Funding/competing Interest(s): This work was supported by grants from the Innovation Fund Denmark. M.G. and S.K. work at ChemoMetec, which produces the image cytometer used in the study, M.G. hold shares in the company. The other authors have no conflict of interest. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumours have a favourable prognosis if detected early, but are potentially lethal in a subset of patients. Multi-modality treatment is often necessary, thus the preferable time of diagnosis is at the pre-invasive, but unfortunately often asymptomatic precursor stage of carcinoma in situ (CIS). This review describes current possible approaches for the detection of CIS. At present, an open testicular biopsy is the only definitive way of establishing the presence of CIS. The tissue section should be of an adequate size, be properly fixed, and evaluation be supported by at least one solid immunohistochemical marker, for example PLAP, OCT-3/4 or AP-2γ. Determination of who should be offered testicular biopsies is based on clinical and ultrasonic examination along with the evaluation of risk factors. A surgical biopsy is an invasive procedure with potential complications, although rare. Therefore, a noninvasive and equally reliable method is needed. Testicular ultrasound is risk-free, painless and at present the only noninvasive method of aid for andrologists when CIS is suspected. The presence of testicular microlithiasis is, in some cases, indicative of pre-malignant changes, especially in males with additional risk factors. Promising results have recently been obtained with a novel noninvasive detection method based on immunocytological AP-2γ-staining of CIS cells in semen. This method could be a supporting method in andrology centres where careful follow-up is possible. In conclusion, one difficulty is to determine in which males CIS should be suspected; secondly, there does not as yet exist an optimal noninvasive method of diagnosis that is more acceptable than an open surgical biopsy. [ABSTRACT FROM AUTHOR]

The acrosome of the spermatozoa is required for fertilization and in the raw ejaculate the percentage of viable acrosome-intact spermatozoa, the acrosomal status, is higher among men with good semen quality. Here we investigated if the acrosomal status of the processed semen preparations used at a fertility clinic can also be informative and whether it is associated with fecundity. The acrosomal status was measured by image cytometry on purified semen samples from couples during in vitro fertilization (IVF) (n = 99) and intracytoplasmic sperm injection (ICSI) (n = 107) treatment. Purified frozen-thawed donor samples were also analyzed (n = 199). In purified semen preparations the acrosomal status was significantly higher among sperm donors (p = 5.3 × 10−8) and men from IVF couples (p = 2.2 × 10−5) when compared to men from ICSI couples. A significant difference was also found between female, male and mixed factor infertility (p = 0.003). No association with lifestyle factors was found. In frozen-thawed donor samples, a significant positive (r = 0.16, p = 0.025) association with the number of pregnancies per sold straw was observed together with an area under the curve of 75.3%, when comparing the top and bottom deciles. Our results indicate that the acrosomal status may be a valuable parameter for personalizing fertility treatments and might be a good predictor of pregnancy success among normozoospermic men. [ABSTRACT FROM AUTHOR]

Sperm concentration is an essential parameter in the diagnostic evaluation of men from infertile couples. It is usually determined by manual counting using a hemocytometer, and is therefore both laborious and subjective. We have earlier shown that a newly developed image cytometry (IC) method may be used to determine sperm concentration. Here we present a validation of the IC method by analysis of 4010 semen samples. There was high agreement between IC and manual counting at sperm concentrations above 3 mill/ml and in samples with concentrations above 12 mill/ml the two methods can be used interchangeable. However, we found substantial differences in samples below 3 mill/ml. We also assessed the accuracy of the two methods by repeated measurements of 248 samples, which revealed that IC measurements seemed more accurate. Moreover, based on ten samples counted by several operators the IC method had a lower coefficient of variation than the manual method (5% vs 10%), indicating a better precision of the IC method. In conclusion, measurement of sperm concentration by IC can be used at concentrations above 3 mill/ml and seems more accurate and precise than manual counting, making it an attractive option in the daily clinical practice. [ABSTRACT FROM AUTHOR]