O-197 Correlation of intracellular pH of non-normozoospermic men with fertilization rates in assisted reproduction procedures (Pilot study)
Authors: Carrasquel Martínez G , Matamoros Volante A , Flores D  L , Torres Rodriguez P , Ramírez Dominguez L  B , Jiménez I , Villar L , Maldonado Rosas I , Treviño Santa Cruz C  L 
Published: July 2024

Abstract

Study question
Does measuring qualitative changes in the sperm pHi could predict the fertilizing potential of a non-normozoospermic patients under fertility treatment?

Summary answer
The increase of pHi correlates with fertilization rates.

What is known already
The intracellular pHi (pHi) is of great importance for a wide variety of sperm physiological processes, including its motility. The pHi regulates several key sperm proteins, such as the Ca2+ channel, CatSper (sperm cation channel) which is involved in sperm hyperactivation and the K+ channel SLO3.The latter participates in the regulation of membrane potential during capacitation. Interestingly, human sperm pHi has been found to positively correlate with both hyperactivated motility and in vitro fertilization (IVF) success in normozoospermic patients.

Study design, size, duration
This prospective study employed unidentified semen samples from 62 non-normozoospermic men (patients, age 38 ± 4 years old) undergoing fertility treatment from January to November 2023 at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 4 oocytes were excluded. The pHi of sperm samples was evaluated by time-lapse flow cytometry. For comparison, the pHi of 13 normozoospermic men (donors, age 28 + 6 years old) were also analyzed.

Participants/materials, setting, methods
Sperm were separated by density gradient and incubated in capacitating HTF media to be used for conventional IVF or ICSI. Cells were stained with vitality (propidium iodide) and pHi (BCECF-AM) sensitive fluorescent dyes. Fluorescence changes were evaluated using an AccuriC6 flow cytometer. For each pHi recording, 10 mM NH4Cl were used as an alkalinization positive control and for data normalization. Basal levels of sperm pHi and its correlation with fertilization rates, were performed using R.

Main results and the role of chance
The changes in pHi were recorded as changes in the BCECF fluorescence. The increase in fluorescence is proportional to an increase in pHi and vice versa. Interestingly, we observed that the increase in pHi in sperm used for ICSI but not for IVF procedures, which were paired with oocytes from female donors, are positively and significantly correlated with higher fertilization rates (R = 0.68; *p=0.02). This seems to be the same for ICSI treatment pairing sperm with oocyte from the female partner, although the correlation is not yet statistically significant (R = 0.44; p = 0.12). We did not find significant differences in the basal pHi values between sperm from donors and patients; however, we observed that sperm from patients, had higher pHi values (0.57 + 0.15 a.u.f) compared to sperm from donors (0.48 + 0.24 a.u.f). Our results suggest that sperm pHi may be an important regulator for the process of fusion and not only for fertilization. Qualitative sperm pHi values can contribute to predicting fertilization success in patients undergoing ICSI treatment.

Limitations, reasons for caution
The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples. Physiological responses may vary even among the same patient or cell’s donor. Our population-based analysis could be underestimating such single cell variations.

Wider implications of the findings
Current protocols for sperm analysis fail to successfully predict the fertilizing capacity of semen samplest.Measuring pHi in capacitated sperm, could be a promising tool to help clinicians choose the best fertilization technique for each couple. This protocol is easy to perform in a laboratory that has a flow cytometer.

Trial registration number
Not applicable