A successful twin healthy live birth achieved from eggs with a rare perivitelline space abnormality: A case report

Abstract Background Phenotypic dysmorphism is not rare to be found in the human oocyte, especially in the perivitelline space, which are among the most important aberration of the extra cytoplasmic component. Case Presentation The case is of a 30-yr-old woman with no previous pregnancy, attempting an in vitro fertilization treatment for the first time. Given the extraordinary quantity of granular particles found in the perivitelline space, visible after the stripping procedure, it was not possible to establish the presence and position of the first polar body to appreciate the correct oocyte maturation (metaphase 2). Nevertheless, all the eggs were injected by the intracytoplasmic sperm injection. A time lapse incubator was used to perform the entire culture. Hence, a record of 6 days culture video was obtained. Only 2 eggs could fertilize correctly and reach the blastocyst stage on day 6. The embryos were frozen and subsequently transferred as frozen embryo transfer following the next menstrual cycle. Conclusion The exceptional presence of granular particles in the perivitelline space, which reminds us for aspects and behavior of the granulosa cells, seems to affect the fertilization but not the blastocysts quality. As a matter of fact, the woman, after the embryo transfer, achieved a successful twin live birth.


Introduction
Between 60-70% of the retrieved oocytes after pick-up has reported to exhibit one or more phenotypic dysmorphisms, normally classified as extra cytoplasmic or cytoplasmic (1,2).
Perivitelline space abnormalities are among the most important dysmorphism of the extra cytoplasmic component, but the fate of these anomalies remains unclear, giving contradictory results in the literature (2).
Here we report a successful case of a woman having a rare perivitelline space anomaly consistent in inclusion of what we believe to be granulosa cells, exhibited in most of the eggs, which, after intracytoplasmic sperm injection and frozen embryo transfer, the woman achieved a twin pregnancy and live births.
How to cite this article: Ferri D, Baldini D, Baldini GM. "A successful twin healthy live birth achieved from eggs with a rare perivitelline space abnormality:

Ethical considerations
The Local Ethics Committee of the MOMO' FERTILIFE Center has evaluated the present manuscriptand and deemed it ethically justifiable. The committee has recommended that certain conditions be documented and guaranteed, including that the couple participating in the study has given informed consent and that the consent was obtained from an adult who was aware and not subjected to any form of coercion. The committee discussed and approved the ethical aspects of the research project in their session on January 18 th , 2021.

Discussion
The oocyte and granulosa cells, secrets 4 glycoproteins (zona pellucida [ZP]1, ZP2, ZP3 and ZP4), which is known to form the pellucide zone during the folliculogenesis in human oocytes (4). In this specific case, we present a cohort of eggs where most of them have appeared, what we believe, to be granulosa cells trapped in the PVS, sometimes covering the egg completely and not allowing an easy assessment of oocyte quality and especially the presence and position of the polar body (which is particularly important to perform an intracytoplasmic sperm injection without damaging the meiotic spindle). Besides, due to the mucification reaction, the cumulus cells secrete a viscous extracellular matrix that dissociates them from one another (5). Furthermore, we noticed that the pressure generated by this phenomenon increases the PVS gradually during the early hours after the injection, stretching the ZP until it breaks, leaving the embryo to develop outside of the ZP. On this link (https://www.youtube.com/watch?v=vN-aPCFve8w), a full video is available, recorded through Geri TM time-lapse, showing the embryo developing of the egg n 14 ( Figure 1) after intracytoplasmic sperm injection.
It is difficult to explain the cause of this abnormality and to date, this particular behavior is not yet described in literature.
To the best of our knowledge, only one study conducted with mice knocked out for the ZP1 gene has shown an engulfment of cumulus cells in the PVS, and those trapped cells had undergone mucification. Subsequently, a granular matrix was observed surrounding them and infiltrating the zona matrix, the absence of the ZP1 protein has resulted in a thinner and more fragile ZP, which can lead to ectopic accumulation of granulosa cells within the PVS (2).
The time-lapse technology has allowed us to record the complete video of the embryonic development of those eggs and show us a similar behavior described in the murine oocytes experiment, which is still very unique in humans. Moreover, it allows us to differentiate it from coarse granulation or fragment accumulation in the PVS, which has a very different aspect in terms of size, color, and shape (Figure 2A and 2B shows granulosa cells line trapped in the PVS). Besides, ordinary fragments do not lead to any PVS expansion and it does not constantly move during the embryo development.
Since the lack of studies about these particular inclusions, no further comparison can be done.
However, as observed from the video, the absence of ZP did not compromise the development of the embryo, reaching the blastocyst stage at 135 hr from the injection.
In conclusion, this case report wanted to show the extraordinary case of this woman who successfully reached a healthy live birth of 2 baby girls despite the very distinctive oocyte anomaly in most of her eggs, which to date is still not described in the literature of human oocytes.