References

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16.\Meniru GI, Gorgy A, Batha J, Clarke RJ, Podsiadly BT, Craft IL. Studies of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection. Hum Reprod Update. 1998;4(10:57–71.

17.\Lin YM, Hsu CC, Kuo TC, Lin JS, Wang ST, Huang KE. Percutaneous epididymal sperm aspiration versus microsurgical epididymal sperm aspiration for irreparable obstructive azo- ospermia—experience with 100 cases. J Formos Med Assoc. 2000;99(6):459–65.

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reported that an unmarried cousin had 0% normal sperm morphology but did not provide further details. The patient’s prior semen analyses showed a concentration ranging from 9 to 35 × 106/mL, with 55–60% motility and consistently 0% normal sperm morphology which suggested globozoospermia. There was no evidence of Y microdeletions, and the patient’s hormonal pro le was normal.

The couple was diagnosed with severe male factor infertility and underwent 2 cycles of in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) at another center a few months prior to seeking treatment at our clinic. The rst stimulation cycle was with 187.5 IU of urinary menotropin and 150 IU of follitropin daily, with a GnRH antagonist, leading to nine retrieved oocytes, of which six were mature. All six were injected with ICSI, and in spite of their incubation in calcium ionophore for 15 min after insemination, none fertilized. The second ICSI cycle with the same superovulation protocol occurred shortly thereafter. A total of 19 oocytes were retrieved this time, yielding seven metaphase II oocytes that underwent ICSI. Once again, despite incubation in calcium ionophore, no fertilization was reported.

This history of failed IVF cycles with ICSI, in spite of arti cial oocyte activation (AOA), and the results of the previous semen analyses suggesting globozoospermia led us to suspect this diagnosis as well. After consultation with the reproductive endocrinologist, Andrology service and reproductive urologist, it was suggested to the couple that he should undergo a series of additional tests to assess sperm characteristics and function:

•\ Aniline blue to provide information on genome compaction during spermiogenesis

•\ TUNEL to assess the level of sperm DNA fragmentation

•\ Sperm aneuploidy assay by FISH to identify chromosomal abnormalities within the male gamete

•\ PLCζ assay to detect the presence of sperm cytosolic factor in the perinuclear theca of the sperm head to test the ability of the spermatozoa to activate the oocyte •\ Centrosome assessment to identify the presence and extrapolate the eventual

function of the centrosome

•\ Transmission Electron Microscopy to unequivocally con rm and identify the extent of globozoospermia, abnormal chromatin compaction, and absence of the acrosomal vesicle

•\ Genetic and epigenetic profling of the male gamete to assess mutations and expression levels of genes related to globozoospermia and to gain insight into the oocyte activating potential of the spermatozoa

Semen Analysis: Semen analysis was performed on a fresh ejaculate produced within 4 days of abstinence. Volume, concentration, motility, progressive motility, and morphology were assessed according to the most recent WHO guidelines. Following liquefaction, the semen analysis revealed a volume of 4.3 mL, a concentration of 50 × 106/mL, 45% motility, and 41% progressive motility, all within the normal threshold. However, there was 0% normal sperm morphology. All spermatozoa assessed demonstrated spherical heads with absent acrosomes. More than

negative supercoiling through a process that seals and repairs the DNA phosphate backbone. At the spermatid stage, single and double stranded DNA breaks, which wrap around a protamine core to form the toroid structure, are repaired by speci c enzymes such as topoisomerase II. This chromatin appears tightly condensed and transcriptionally inert; it is therefore highly resistant to digestion. However, histone-­ bound DNA persists in the linker region in between the toroid structures, representing at least 15–20% of the entire sperm genome. These areas, which are still very prone to DNA nicks and breaks due to reactive oxygen species present in the male genital tract, can be detected by sperm chromatin fragmentation (SCF) assays. SCF was assessed by labeling the 3′-OH ends using the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP-biotin nick-end labeling (TUNEL) assay. A minimum of 500 spermatozoa were scored using fuorescent microscopy (Fig. 27.3), and an SCF of ≤15% was considered normal. In this patient, the TUNEL assay evidenced a mild increase in sperm DNA fragmentation of 16.8%.

Sperm Aneuploidy Assessment: In cases of unexplained infertility in which the male partner has seemingly normal sperm parameters, the genetics of the male gamete should be explored. In spite of the fact that sperm morphology does not directly relate to the genetics of the sperm cell, globozoospermic men have been thought to have a higher rate of sperm aneuploidy. Fluorescent in situ hybridization (FISH), a cytogenetic technique that utilizes fuorescent probes that bind to speci c chromosomes, was applied to sperm cells to detect aneuploidy. This technique requires decondensation of the tightly compacted spermatozoal chromatin to access the DNA for probe hybridization. The multicolored labeled probe signals can then be visualized under fuorescent microscopy (Fig. 27.4). Nine chromosome (X, Y, 13, 15, 16, 17, 18, 21, 22) FISH was used to assess at least 1000 sperm cells for this case, with a normal threshold of 1.6%. The aneuploidy assessment by FISH for this patient revealed only a borderline increase of 1.9% total aneuploidy represented by gonosomal (XY, YY) and autosomal disomies for chromosomes 15, 16, and 17.

Fig. 27.3  TUNEL on globozoospermic specimen

Fig. 27.5  PLCζ assessment on globozoospermic specimen

surrounded by brous pericentriolar material (PCM) that creates the aster and spindle bers. Human spermatozoa have two centrioles: one is distal and supports the fagellum, and the other is proximal and is delivered into the egg [3]. The centrosome, by anchoring the sperm aster, is involved in the migration and juxtaposition of the male and female pronuclei. Indeed, the spermatozoon forms an aster around the midpiece, a radial structure composed of microtubules anchored to the proximal centriole. While the spermatozoon decondenses, the dimension of the aster increases, jutting out from the male genome and adjoining the female pronucleus toward the center of the cell. The centrosome is also responsible for the regulation of cell polarity of the rst mitotic cell division. After syngamy, the centrioles duplicate into daughter centrioles that migrate to the opposite poles of the rst mitotic spindle during metaphase. Once the anaphase and telophase stages are complete, cell cleavage occurs, forming a 2-cell embryo that includes a set of two centrioles, pericentrin, centrin, and γ-tubulin, components of the centrosome that play a critical role during syngamy. Pericentrin is involved in centrosome and spindle organization, centrin is important for centriole function and centrosome duplication, and γ-tubulin is mainly associated with PCM and the initiation of microtubule nucleation. Since the centrosome is crucial for normal chromosomal segregation at the rst mitotic division, it seemed appropriate to perform centrosome assessment in the patient. To identify the human sperm centrosome, spermatozoa must be permeabilized and incubated with anti-centrin antibody followed by a secondary antibody. The specimen is then assessed under fuorescent microscopy, and the percentage of sperm cells exhibiting a centrosome is recorded (Fig. 27.6). In a normal sperm specimen, the percentage of sperm cells with a centrosome is expected to be between 60 and 80%. In this case, we detected a borderline normal presence of centrosome at 57%.

Transmission Electron Microscopy: To visually examine the ultrastructural aspect of the male gamete in this patient with faulty spermiogenesis, we performed transmission electron microscopy (TEM) to better assess sperm organelles such as the acrosome, nucleus, centrioles, and microtubular arrangement in fagella. The post-centrifugation sperm pellet was xed by glutaraldehyde and sliced by ultramicrotome to 100 nm slices. Sections were then viewed by an electron microscope (JEOL-1400, JEOL USA, Inc., Peabody, MA, USA) with a magni cation of 300,000×, where a 120-kV electron beam was transmitted through the specimen to

growing number of gene mutations speci cally related to male factor infertility, we have also begun to develop a method for infertility screening by assessing the genome of the male patient [4]. The current availability of more thorough molecular genetic techniques, such as Next Generation Sequencing (NGS), has made it possible to assess the entire genome of the infertile male. It is also possible to constrain NGS to speci c areas of interest, such as the exome, focusing on speci c genetic markers associated with infertility. While genetic assessments have more commonly been carried out on peripheral blood, which can only identify uninheritable somatic mutations, we perform NGS on spermatozoa to detect germline mutations that may be passed onto offspring and may possibly be missed in somatic mutation analysis. To perform NGS, DNA must rst be extracted and ampli ed, which can be achieved with the use of a commercially available kit. If the DNA is of adequate concentration and quality, it is submitted for sequencing. The resulting data can then be used to assess copy number variants (CNVs), which can be further annotated for the detection of gene mutations. Moreover, as it is not limited by the number of chromosomes that may be assessed, NGS can also be used for a more thorough evaluation of sperm aneuploidy. It is of paramount interest when assessing gene function to pro le the individual epigenetically. This can be done by sequencing RNA, also by NGS, which offers insight into the expression of the gene of interest. Our assessment of this globozoospermic case using NGS was carried out by sequencing DNA extracted from spermatozoa, yielding an overall aneuploidy of 8.2%. We found a mutation on the DPY19L gene, which is recognized as a major cause of globozoospermia, as well as mutations on SPATA16 and PICK1, which are also typical in globozoospermic men [5]. In addition, our NGS assessment revealed mutations on genes involved in spermiogenesis and embryo development, such as PIWIL1, BSX, and NLRP5. Our epigenetic analysis, carried out by RNAseq revealed that DPY19L2 and PICK1 were signi cantly overexpressed, con rming the malfunction of those two genes. Moreover, a signi cant underexpression of two other genes involved in oocyte activation by signaling calcium channel proteins (AHNAK2) and embryo development (MMP14) were also identi ed. Overall, these ndings con rmed a genetic etiology of globozoospermia due to speci c DNA sequencing mutations (DPY19L, SPATA16, PICK1), con rmed by gene expression pro ling regarding an impaired spermatozoa oocyte activating potential (AHNAK2).

The results of the advanced sperm function tests unanimously showed that the spermatozoa were clearly impaired in their ability to successfully activate the oocyte. Therefore, we opted to treat the couple with in vitro insemination by ICSI using a proprietary assisted gamete activation (AGA) protocol targeted toward the male gamete as well as the oocyte, justi ed by the remarkable lack of PLCζ observed.

Superovulation Protocol: On cycle day (CD) 2, the female partner had an FSH of 3.82 IU, LH of 4.17 IU, E2 of 134.6 pg/mL, and P4 of 0.35 ng/mL. The female was superovulated with E2 patch priming, 50 mg clomid, 225 units of follitropin and 75 units of hMG, with BID GnRH antagonist. The patient was triggered with 4 mg of Lupron and 1500 IU human chorionic gonadotropin (hCG) on CD15, with an E2