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Fig. 27.8 Schematic of Oocyte Activation
of 3266 pg/mL, LH of 2.8 IU, and a lead follicle size of 22.5 mm. Her E2 the following day was 3845 pg/mL and her LH was 132 IU.
ICSI with AGA: A total of 14 oocytes were retrieved, of which 10 were mature. According to our proprietary protocol, prior to ICSI injection, ejaculated spermatozoa were briefy exposed to calcium ionophore in a drop on the ICSI dish. During the ICSI procedure, spermatozoa were aspirated individually from the drop containing calcium ionophore and immobilized in a separate PVP drop. Then, a small portion of calcium ionophore was aspirated into the micropipette and injected into the oocyte with the spermatozoa (Fig. 27.8). Post-ICSI oocytes were then exposed to calcium ionophore for a short period at 37°C, then washed and placed in IVF culture media.
Clinical Outcome: Fertilization was assessed 16–18 hours after ICSI. Four oocytes were successfully fertilized, con rmed by the appearance of two pronuclei and the extrusion of a second polar body. On day 3 post-ICSI, embryo cleavage was assessed. Two embryos (7-cell, 0% fragmentation; 8-cell, 7% fragmentation) were transferred. The other two embryos were maintained in culture, resulting in one arrested embryo and one average quality blastocyst that was cryopreserved. Approximately 2 weeks after embryo replacement, the patient’s βhCG level was 596 mIU/mL. Two fetal yolk sacs were observed at 5 weeks and 5 days, and two fetal heart beats were observed 1 week later. At a gestational age of 38 weeks and 3 days, 2 healthy female offspring, weighing 2353 g and 2807 g, were born by elective caesarean section for malpresentation. No prenatal or postnatal complications were reported.
Discussion
An infertile couple where the female partner presents with a negative workup may imply that the cause of infertility resides with the male partner. Semen analysis is the primary tool to assess the male reproductive pro le, however, it may not suf ce in providing information on the functional capacity of the male gamete. In this couple, we utilized several sperm function assays to evaluate the fertilization capacity of the spermatozoon as well as its embryo developmental competence.
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The protamine assay indicated an abnormal nuclear compaction and therefore a higher susceptibility of this individual’s spermatozoa to be damaged by reactive oxidative species within the male genital tract. Therefore, this protamine de ciency may lead to high DNA fragmentation, which we assessed by TUNEL that resulted in a borderline abnormal sperm chromatin fragmentation.
Prior to advising the couple to proceed with IVF, we decided to assess the chromosomal pro le of the male gamete in question, which fortunately resulted in only a borderline abnormal incidence of aneuploidy mainly characterized by autosomal and gonosomal disomies. This assay is important to measure the eventual contribution of the male partner to an increased incidence of embryo aneuploidy and higher pregnancy loss.
Another crucial aspect of the male gamete is the presence and integrity of the centrosome. Its presence in this particular individual is reassuring, as this indicates the ability of the spermatozoon to form the rst mitotic spindle and ordain a correct segregation of the chromosome at the rst cleavage division [3].
The ultrastructural analysis proved to be con rmatory in evidencing that a large majority of the cells had an absent acrosome, with almost nonexistent perinuclear theca, con rming abnormal compaction of the chromatin but also providing reassuring detail on the integrity of the midpiece with a normal structural appearance of the capitulum and the proximal centriole.
Needless to say, the most critical aspect is the assessment of the functional ability of the spermatozoon to activate the oocyte. A PLCζ assay con rmed that this labile protein, normally located in the perinuclear theca, was almost absent. The absence of this sperm-bound cytosolic factor undoubtedly proves the inability of the male gamete to activate an oocyte. The outcome of this assay represents the most important indication for assisted oocyte activation that has often been overlooked in the literature. Indeed, only about 20% of studies on this topic have carried out a functional male gamete assessment, such as the mouse oocyte activation test (MOAT) [1, 2].
In our genetic and epigenetic assessment, we identi ed a mutation of DPY19L by DNAseq, but when we epigenetically assessed the transcript by RNAseq, we found an additional imbalanced expression of PICK1. These are two of the most relevant genes involved in causing globozoospermia [5]. In addition, other genes (PIWIL1, BSX, NLRP5) involved in spermiogenesis and embryo development were imbalanced. Finally, signi cant underexpression of genes involved in oocyte activation by signaling calcium channel proteins (AHNAK2) and embryo development (MMP14) were identi ed. We therefore con rmed the genetic etiology of this man’s condition using DNA and RNAseq, which can be used to properly counsel the couple.
On the basis of our assessment with different sperm function assays, the utilization of ICSI supported by AGA proved to be appropriate [2]. The adoption of this treatment has also proven to be safe in generating healthy offspring [1].
Overall, these additional tests on the male gamete provided insightful information on the globozoospermic diagnosis and prompted us to utilize the proper course of action which, in this particular case, was ICSI with assisted gamete activation to compensate for the lack of sperm acrosome and cytosolic sperm activating factor.
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The utilization of advanced sperm function tests allowed this couple to achieve fertilization and successful embryo implantation, resulting in the live birth of two healthy baby girls.
References
1.\ Bonte D, Ferrer-Buitrago M, Dhaenens L, Popovic M, Thys V, De Croo I, et al. Assisted oocyte activation signi cantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study. Fertil Steril. 2019;112(2):266–74.
2.\ Neri QV, Lee B, Rosenwaks Z, Machaca K, Palermo GD. Understanding fertilization through intracytoplasmic sperm injection (ICSI). Cell Calcium. 2014;55(1):24–37.
3.\ Palermo GD, Colombero LT, Rosenwaks Z. The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev Reprod. 1997;2(1):19–27.
4.\ Cheung S, Parrella A, Rosenwaks Z, Palermo GD. Genetic and epigenetic pro ling of the infertile male. PLoS One. 2019;14(3):e0214275.
5.\ Modarres P, Tanhaei S, Tavalaee M, Ghaedi K, Deemeh MR, Nasr-Esfahani MH. Assessment of DPY19L2 deletion in familial and non-familial individuals with globozoospermia and DPY19L2 genotyping. Int J Fertil Steril. 2016;10(2):196–207.
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Chapter 28
Psychological Factors and Fertility
Counseling
Elizabeth Grill
Case
John (43) and Julie (39) have been married for 5 years and have tried unsuccessfully to start a family for the past 2 years. Julie wanted to start trying to conceive 1 year before John who wished to wait until he was promoted at work. They tried to conceive on their own for 1 year before seeking help from a reproductive endocrinologist (RE). After receiving a combined factor diagnosis, they attempted 3 cycles of intrauterine insemination (IUI) and 3 cycles of in vitro fertilization (IVF). They became pregnant during the last IVF cycle but miscarried at 8 weeks. They plan to do another treatment cycle but their RE advised them to start thinking about alternative family building options. Prior to starting a fourth IVF cycle, they decide to talk with a mental health professional who specializes in couple’s issues related to reproductive medicine.
The constant barrage of shots, blood tests, and surgical procedures left Julie feeling physically and emotionally exhausted. John found it diffcult to connect with Julie as she became more withdrawn, depressed, anxious, lethargic, and uninterested in the things they both used to enjoy doing together. John complained that he no longer recognized Julie and feared that he would never get the person he knew back. Over time, this couple began to feel that the very foundation of their relationship was shaken as they were challenged to cope with stress and vulnerability, the loss of a dream, a sense of powerlessness, and feelings of guilt as well as blame.
While their marital and sexual relationship were strong at the beginning of treatment, the cumulative stresses of the infertility experience started to take a toll on their marital and sexual satisfaction. Throughout the course of treatment, this couple
E. Grill (*)
Reproductive Medicine and Ob/Gyn, The Ronald Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Medical College of Cornell University, New York, NY, USA e-mail: eag2001@med.cornell.edu
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