Blastocyst rate has fallen — separating patient factors from culture problems

Fertilisation is normal, day-3 embryos look average, and then too few make it to a usable blastocyst. Because the day 3 → day 5 step is where biology does its selecting, this is the KPI most easily blamed on patients — and the one where a culture-system fault does the most quiet damage. The job is to tell those apart.

Is it patients or is it the lab?

Ask the case log three questions:

1. Is the drop uniform? A culture problem hits everyone: young good responders and older patients alike. Patient mix hits the groups you'd expect. Split blastocyst rate by age band / AMH band for the last two months versus the previous six.
2. Where do embryos stop? Uniform arrest at 4–8 cells across many patients suggests a culture insult around the time of genome activation (day 2–3). Slowing and poor morphology from day 1 suggests an earlier problem (oil, medium, temperature). Fine embryos to day 3 that then fail to compact or cavitate suggests the day 3–5 medium, sequential-medium changeover, or incubator conditions.
3. Which incubator / operator / shelf? Compare. One incubator with a lower rate is a gift: you have your suspect.

Incubators (the usual culprit)

• Measured, not displayed: CO₂ with an independent analyser; temperature in a dish at the working position with a calibrated probe; O₂ if low-oxygen culture. Displays drift; sensors age; a recent service can leave a set-point wrong.
• Recovery after door opening — benchtop incubators recover in minutes, large box incubators can take 20–30. A busy week with more openings than usual, a new staff member who checks embryos more often, or one chamber shared by too many patients all reduce time at set-point.
• Humidity — dry incubators concentrate small drops over days; check osmolality of medium left in the incubator for the culture period.
• New incubator, or one back from service — validated? Run a mouse-embryo assay or at minimum sentinel culture before patient use.

Media and oil

• Lot change of the sequential day-3 medium or the single-step medium within a fortnight of the fall. Check dates. As with fertilisation, a lot that suffered a temperature excursion in transit or storage behaves like a bad lot.
• Oil — under-washed, oxidised, or an embryotoxic lot; a change in the oil volume-to-drop ratio; new oil brand. Oil affects late development strongly.
• Equilibration — dishes prepared too soon before use don't reach pH/temperature; prepared too long ahead lose water. Ask when dishes are actually poured versus when the SOP says.
• Medium changeover (sequential systems) — the day-3 move: how long out of the incubator, at what temperature, how many embryos per drop after the move.
• Drop volume and embryos per drop — very small drops or single culture in large volumes shift results in some systems; group culture in appropriate volumes tends to help.

Handling and environment

• Time out of the incubator for scoring — day-3 checks that stretch, or an extra look for training. Time-lapse systems remove this variable; if you have one, compare its rate with the conventional incubator.
• Stage temperature at the scoring microscope.
• VOCs — a fresh construction smell, new furniture, cleaning products, or an HVAC change. Blastocyst rate is more sensitive to air quality than fertilisation.
• Static or vibration — a compressor moved nearby, a fridge on the same bench.

Technique and policy

• Day-3 grading generosity — if more marginal embryos are being cultured on, the denominator grows and the rate falls without anything being wrong. Track usable blastocysts per 2PN alongside blastocyst rate.
• Biopsy or assisted hatching load — more manipulation, more time out.
• A change in extended-culture criteria by the clinicians.

Deciding and closing

Line up incubator readings, lot changes, staffing and building events against the fall and the recovery. Corrective action is usually one thing (that incubator, that oil lot). Preventive action is the QC habit that would have caught it: daily incubator QC with independent measurement, lot-change log, monthly KPI by incubator. Effectiveness: blastocyst rate back in range for one full month across all age bands.