Discusion
Recent years, more and more stdudies about MMAF had been reported, and normaI fants were delivered following ICSI with ejaculated sperm[6,8,10-13,21,23,26,28,30,31].To our knowledge , the present case is the first report of Successful birth after injection of vitrified oocyte by immotile testicular spermatozo with totally MMAF. This report shows that even in case of MMAF patient,vitrified oocytes can be successfully fertilizated by ICSI ,and produce healthy offspring.Thus,Vitrification may serve as a useful tool in the preservation of oocytes for MMAF couples .
Oocyte cryopreservation has become an important part of infertility treatment for various reasons.In our case,vitrification of a part of oocyte cohort was proposed to save oocytes for a potential subsequent ICSI cycle in case of no embryos available in the first cycle. Oocyte survival at the MII stage after warming, for this case report, was 100% (12/12 oocytes),similar to the post-warming survival rates of 96.4% with 86.2% fertilization rate in 2020 from Deepa Talreja[10]. In R Azambuja’s report,it is 63% and 80% respectively[18]. While the fertilization rate in this report was 75% (9/12 surviving oocytes fertilized). This difference might be related to the different sperm origin, seriously impaired sperm motility of MMAF ;
According to the previos study, majority of the succesful MMAF cases are from the ejaculated sperm[1,3,5-8,14,15,22,23,25,27]. It is uncommon for MMAF patients that both the flagellar of the ejaculated sperm and testicular spermatoza are 100% abnormal and totally immotile.In Yang’s report, testicular spermatozoa were obtained by needle aspiration,which were also showed 100% immotile,but with a small portion of nomal flagella. And the sperm flagellar phenotype was in accordance with MMAF,showing absent 20.0%, short38.5%, coiled 30.5%, irregular 5.5%, and bent 5.5%,respectively[21]. Robert l showed a PCD patient that all testicular sperm were immotile,and the majority were structurally abnormal,being decapitated and with short thickened tails[19]. Similar to the above reports,both of the ejaculated and testicular spermatozoa were totally immotile and 100% abnomal flagella in our case(Figure1A). Different from them,Under light microscopy, 84% of the sperm flagellar phenotype were showing curled or bent back on itself at the end piece of the flagellar, but with normal head and middle piece percentage(Table1). To our knowledge, the case is the first report of MMAF that all the the morphology of the flagellar are abnormal,and majority of the end piece of the flagellar were cureled or bent.
The percentage of each abnormal sperm flagellar phenotypes was different in different MMAF patients.This difference might be related to the different disease-causing genes. Recent years, more and more new genes are reported to be associated with MMAF, including the latest DNAH8, DNAH17, CFAP61, QRICH2 and CFAP74[6,23,26,30,31]. These uncovered genes always lead to disrupted ultrastructural defects of the axoneme, which is the core structure of the sperm flagellar, several axonemal dyneins, radial spokes, nexin links and many other components. Any defects in these structures would result in abnormal sperm flagellar, showing a mosaic of morphological abnormalities, and further lead to severe asthenozoospermia or 100% sperm immotility.
The immotile spermatozoa in MMAF patients showed a viability of 9%–80%,and fertilization rate of 38.9%-75%[7,25 ]. These results are quite similar to those observed here. In the present case, with a Viability of 39%,the first ICSI cycle attempt with ejaculated sperm resulted in a 60% fertilization rate. While in the second cycle with fresh testicular spermatozoa,it was a better 75% fertilization rate.Yang’s report showed a 45.5% fertilization rate,when testicular spermatozoa from a MMAF patient were injected into the oocytes[21]. Twelve of 18 oocytes were fertilized in another MMAF-like PCD patient after testicular sperm extraction and intracytoplasmic sperm injection[19].Our results further demonstrated that testicular sperm with MMAF are capable of establishing a normal pregnancy,regardless of also being immotile and totally abnormal . Moreover, maybe immotile testicular sperm is better than immotile ejaculated sperm and has been recommended for use rather than completely immotile ejaculated sperm in MMAF patients.
Addationally,This difference of fertilization rate might be related to the selection of viable sperms. The hypo-osmotic swelling test to evaluate the vitality of the spermatozoa prior to ICSI was not possible because of the totally abnormal end piece of the flagella. All of the spermatozoa in the papient exhibit curlings of the flagellum that resemble HOS-positive spermatozoa. This especially lilmited the efficency of the HOS test ,and the result would be inaccurate. In our case, morphologically and physical characteristics (tail flaccidity) were used to select sperm,accurately,the curled flagellar with normal head(Figure1B). A similar fertilization rate (67%) was achieved.This demonstrates that a high fertilization rate can be obtained even if HOS is not used for MMAF patients.
Interestingly, it was reported that the success rates of ICSI may be correlated to the type of ultrastructural flagellar defect carried by the patients. Mitchelletal reported lower implantation(8%) and clinical pregnancy rates(15%) in patients without axonemal centralpair[15] . Unfortunately,In the patient reported here,TEM was not done on the sample , so the ultrastructure of the spermatozoa is unknown.
The is no doubt that the finding genes provided strong genetic evidence for MMAF[17,20,22,26-31]. Thus, genetic counseling is highly significant in helping evaluate and avoid the risk of transmission of genetic defects by ICSI in these patients. In the present report, the chances of genetic transmission were lessened by the fact that the new-born was a girl . However, the possibility that this girl may be a healthy carrier of the described variant of the immotile cilia syndrome should be discussed during genetic counseling . We are sure that genetic counseling and careful analysis using electron microscopy will make the course of MMAF treatment better .
In conclusion, we firstly reported a sucessful delivery after injection of vitrified oocyte with immotile testicular spermatozo with MMAF. Although more and more new genes were revealed in recent years,these can only explain 35%-60% of the MMAF cases[31]. Thus,more work will be done in the field of the genetics of MMAF. In our following study ,more MMAF patients will be collected to carrying out whole-exome sequencing and Transmission electron microscopy assessment.
Acknowledgments: The authors thank Bo Ma for assistance in preparing this manuscript
Disclosure of Interest: the authors declare no competing interests.
Ethics approval: This is a case study. The Ethics Committee has confirmed that no ethical approval is required.
Details of patient’s consent:  the patient’s permission to publication was obtained.
Contribution to authorship: Yuhu Li and Yuqun Huang was responsible for the literature search . Data interpretation and writing of initial draf twas by Liuguang Zhang . Manuscript writing and
approval was by Liuguang Zhang.
Funding: the research was supported by no funding